Antibiotic Supplements in Primary Cell Culture: Weighing Necessity Against Unintended Consequences in Biomedical Research

Abstract

This article critically examines the widespread practice of antibiotic supplementation in primary cell culture, a cornerstone technique in biomedical research and drug development. It explores the foundational rationale for their use against a growing body of evidence revealing significant unintended effects, including altered cellular electrophysiology, gene expression, and genomic regulation. Methodological guidance for application and contamination control is provided, alongside robust troubleshooting and optimization strategies for transitioning to antibiotic-free cultures. The content further addresses the critical need for experimental validation to mitigate confounding results, synthesizing key takeaways to advocate for a more judicious, evidence-based paradigm that enhances data reproducibility and translational relevance.

The Double-Edged Sword: Understanding the Rationale and Hidden Costs of Antibiotics in Cell Culture

The routine inclusion of antibiotics in cell culture media is a practice deeply rooted in the history of in vitro biology. This application note explores the original justifications for this practice, the subsequent discovery of its unintended consequences on cellular systems, and the evolution of modern, evidence-based protocols. Understanding this rationale is crucial for primary cell culture research, where preserving native cell phenotypes is paramount for generating physiologically relevant data in drug development.

The practice originated from the convergence of two historical developments: the rise of antimicrobial chemotherapy in the mid-20th century and the concurrent establishment of continuous mammalian cell lines. Following the landmark discovery of penicillin by Alexander Fleming in 1928 and its mass distribution in 1945, antibiotics became widely available medical marvels [1]. This "Golden Age" of antibiotics created a paradigm where antimicrobial agents were viewed as simple solutions to bacterial contamination [2]. During this period, cell culture was transitioning from a specialized technique to a fundamental tool in biomedical research, creating a practical need to protect valuable cell lines from microbial contamination in increasingly busy laboratory environments.

Historical Justification for Antibiotic Use

The initial adoption of antibiotics in cell culture was driven by several compelling, practical advantages that addressed major technical challenges of the time.

Primary Drivers for Adoption

• Contamination Prevention: Cell culture media, rich in nutrients, provides an ideal growth environment not only for mammalian cells but also for accidental bacterial and fungal contaminants [3]. Antibiotics offered a simple, cost-effective preventive measure to safeguard cultures against potential losses from microbial contamination, which was a significant threat to research continuity and reproducibility [4].

• Aseptic Technique Support: In early laboratories, where specialized equipment like laminar flow hoods was not universally available, antibiotic supplements served as a critical safety net. They compensated for limitations in sterile technique, especially when culturing primary cells isolated non-sterically from animal or human tissues [4].

• Resource Protection: For irreplaceable cultures, including patient-derived primary cells or valuable genetically modified lines, antibiotics provided a sense of security. They were seen as a way to rescue contaminated cultures, a practice that remains relevant today when dealing with limited cell resources [4].

Commonly Adopted Antibiotic Formulations

Table 1: Traditional Antibiotic Supplements in Cell Culture

Antibiotic	Common Concentration	Spectrum of Activity	Historical Rationale
Penicillin-Streptomycin (PenStrep)	100 U/mL Penicillin, 100 µg/mL Streptomycin	Gram-positive (Penicillin) & broad-spectrum (Streptomycin)	Synergistic combination; cell wall synthesis inhibition facilitates entry of protein synthesis inhibitor [4].
Gentamicin	50 µg/mL	Broad-spectrum, including mycoplasma	Superior stability at 37°C and across pH ranges; effective against a wider range of contaminants [4].
Antimycotics (e.g., Amphotericin B)	Varies	Fungi & yeasts	Prevention of fungal overgrowth in rich media, often used in combination with antibiotics [5].

The penicillin-streptomycin combination emerged as the most popular supplement due to its perceived synergistic action and broad-spectrum coverage. The inhibition of bacterial cell wall synthesis by penicillin was thought to facilitate the entry of streptomycin, which then impaired bacterial protein synthesis [4]. This combination became a standard, almost reflexive, addition to culture media for decades.

The Paradigm Shift: Recognizing Unintended Consequences

Over time, a growing body of evidence revealed that antibiotics, far from being inert, exert significant and measurable effects on cultured mammalian cells. This prompted a critical re-evaluation of their routine use.

Documented Effects on Cellular Phenotypes

Research demonstrated that antibiotic supplements could induce a range of cytostatic and cytotoxic effects, altering fundamental cellular properties and potentially confounding experimental outcomes [4].

Table 2: Documented Cytostatic and Cytotoxic Effects of Common Antibiotics

Cell Type	Antibiotic	Observed Effect	Experimental Implication
Hippocampal Pyramidal Neurons (Primary Rat Culture)	Penicillin-Streptomycin	Depolarized RMP; ↑ AHP amplitude; ↓ firing frequency; ↑ AP duration [6].	Altered electrophysiology; invalidates studies on neuronal excitability and network activity.
Various Mammalian Cell Lines	Penicillin-Streptomycin	Altered gene expression (209 genes in HepG2 cells); ↑ ROS & DNA damage (with Gentamicin) [3].	Skewed transcriptomic data; potential masking of genuine experimental responses.
General Cell Cultures	Various (e.g., Tetracycline)	Inhibition of cell growth; morphological changes [4].	Reduced cell viability/proliferation; impacts data from proliferation and cytotoxicity assays.

A pivotal 2025 study demonstrated that antibiotic carryover is a significant confounding factor in research investigating the antimicrobial properties of cell-secreted products, such as extracellular vesicles (EVs) [3]. The study found that the observed bacteriostatic activity against Staphylococcus aureus in conditioned medium was due to residual penicillin retained on the tissue culture plastic and released by cells, rather than any inherent antimicrobial factor secreted by the cells themselves [3]. This finding underscores how antibiotic supplements can directly lead to false positive results and erroneous conclusions.

Additional Risks of Routine Antibiotic Use

• Masking Contamination: The continuous use of antibiotics can suppress but not eliminate low-level contaminants, leading to cryptic infections. Mycoplasma, in particular, can persist undetected for many passages, altering host cell biology without causing media turbidity [4] [5]. These cryptic infections can compromise the validity of countless experiments before being discovered.

• Promotion of Resistance: The long-term, sub-lethal exposure of environmental microbes to antibiotics in culture waste can contribute to the development of antibiotic-resistant strains, posing a broader laboratory and public health risk [4].

• Interference with Cellular Processes: Beyond the effects listed in Table 2, antibiotics can cross-react with cells and interfere with the specific cellular processes under investigation, a particular concern in studies of cellular metabolism, signaling, and drug mechanisms [5].

Modern Best Practices and Protocols

The historical rationale has been superseded by a more nuanced understanding, leading to updated guidelines centered on the principle of antibiotic-free culture as the gold standard for primary cell research.

Current Consensus and Rationale

Major institutions and cell culture experts now advise that antibiotics should not be used routinely [5]. Their continuous use is discouraged because it:

• Encourages the development of antibiotic-resistant strains.
• Allows low-level contamination to persist, which can develop into full-scale contamination once antibiotics are removed.
• Can mask mycoplasma infections and other cryptic contaminants.
• Risks cross-reacting with cells and interfering with the cellular processes under investigation [5].

The modern paradigm positions rigorous aseptic technique as the primary defense against contamination, with antibiotic use reserved for specific, justified short-term applications.

Experimental Workflow: Transitioning to Antibiotic-Free Culture

The following diagram and protocol outline the steps for establishing and maintaining primary cultures without routine antibiotics.

Diagram: Antibiotic-Free Cell Culture Workflow. This workflow prioritizes aseptic technique and defines antibiotics as a last resort.

Protocol: Decontamination of an Irreplaceable Culture

When a unique, irreplaceable primary culture becomes contaminated, the following validated protocol can be used to attempt rescue [5]. This should be considered a last resort.

Objective: To eliminate bacterial contamination from a valuable cell culture using a short, targeted course of antibiotics, minimizing cellular stress and toxicity.

Materials:

• High-concentration antibiotic/antimycotic (e.g., Penicillin-Streptomycin at 100-200X)
• Appropriate antibiotic-free growth medium
• Multi-well culture plate or small flasks
• Phosphate Buffered Saline (PBS), sterile
• Trypsin-EDTA or other dissociation reagent

Procedure:

• Diagnosis and Isolation: Confirm the type of bacterial contamination via microscopy and isolate the contaminated culture from all other cell lines immediately.
• Dose-Response Setup: a. Dissociate, count, and dilute the contaminated cells in antibiotic-free medium to the concentration used for regular passaging. b. Dispense the cell suspension into a multi-well plate or several small flasks. c. Add your chosen antibiotic to the wells/flasks in a range of concentrations (e.g., 0.5X, 1X, 2X the standard working concentration).
• Toxicity Assessment: Observe the cells daily for signs of toxicity, including sloughing, appearance of vacuoles, decrease in confluency, and rounding. The goal is to identify the concentration at which the antibiotic becomes toxic to the cells.
• Treatment Phase: Culture the cells for 2-3 passages using the antibiotic at a concentration one- to two-fold lower than the determined toxic concentration.
• Removal and Confirmation: a. Culture the cells for one passage in antibiotic-free media. b. Return the cells to the treatment concentration for one final passage. c. Finally, culture the cells in antibiotic-free medium for 4-6 passages to confirm that the contamination has been permanently eliminated.
• Quality Control: After confirmation of decontamination, perform a mycoplasma test and re-authenticate the cell line before returning it to your main cell bank.

The Scientist's Toolkit: Essential Reagents

Table 3: Key Reagents for Modern, Responsible Cell Culture Practice

Reagent / Solution	Function	Application Note
Penicillin-Streptomycin (PenStrep)	Dual-action antibiotic solution targeting a broad spectrum of bacteria.	For short-term use only. Unstable at 37°C; light-sensitive. Standard working concentration is 100 U/mL Penicillin, 100 µg/mL Streptomycin [4].
Gentamicin Sulfate	Aminoglycoside antibiotic, broad-spectrum, effective against mycoplasma.	More stable than PenStrep across pH and temperature variations. Standard working concentration is 50 µg/mL [4].
Antibiotic-Antimycotic (e.g., with Amphotericin B)	Combined solution to combat both bacterial and fungal/yeast contaminants.	Used when fungal contamination is a specific concern. Antimycotics can be toxic to some cell lines at high concentrations [5].
Mycoplasma Detection Kit (PCR-based)	Rapid and sensitive detection of mycoplasma contamination.	Essential for quarterly quality control, as mycoplasma does not cause media turbidity and can alter cell biology undetected [4] [5].
Cell Line Authentication Service (STR Profiling)	Validates cell line identity and detects cross-contamination.	A critical step in quality control, as cross-contamination is a widespread problem that can invalidate research [5].

The historical rationale for using antibiotics in cell culture media was rooted in practical necessity during the early development of cell culture techniques. However, modern research has clearly demonstrated that these supplements are not biologically inert and can significantly alter cellular physiology, gene expression, and experimental outcomes. The field is therefore undergoing a necessary paradigm shift away from routine antibiotic use and toward the gold standard of antibiotic-free culture maintained by rigorous aseptic technique.

For researchers in drug development and primary cell culture, this evolution in practice is not merely a technical detail but a fundamental requirement for data integrity. Adhering to modern best practices ensures that experimental results reflect genuine biological responses rather than artifacts induced by antibiotic supplements, thereby enhancing the reproducibility and translational value of preclinical research.

Antibiotic supplementation is a common practice in primary cell culture to prevent bacterial contamination, which can compromise experimental integrity and lead to significant data loss. This application note provides a detailed overview of commonly used antibiotic cocktails, their mechanisms of action, and practical protocols for their use in research settings. Within the context of a broader thesis on antibiotic supplementation in primary cell culture, it is crucial to recognize that while these agents are invaluable for maintaining sterility, a growing body of evidence indicates they may exert unintended effects on cellular physiology, gene expression, and experimental outcomes [7] [8]. This document aims to equip researchers, scientists, and drug development professionals with the knowledge to implement antibiotic regimens effectively while mitigating potential confounding variables.

Common Antibiotic Cocktails: Profiles and Mechanisms

The selection of an appropriate antibiotic regimen depends on the spectrum of activity required and the specific cell culture application. The table below summarizes key profiles of commonly used antibiotic solutions.

Table 1: Profiles of Common Cell Culture Antibiotics

Antibiotic Solution	Effective Against	Common Working Concentration	Mechanism of Action	Primary Application in Cell Culture
Penicillin-Streptomycin (PenStrep) [9] [10] [11]	Gram-positive & Gram-negative bacteria	50-100 U/mL Penicillin; 50-100 µg/mL Streptomycin [12]	Penicillin inhibits bacterial cell wall synthesis; Streptomycin inhibits bacterial protein synthesis by binding to the 30S ribosomal subunit [11].	General prevention of bacterial contamination; most widely used antibiotic in cell culture [12].
Gentamicin [10] [13]	Gram-positive & Gram-negative bacteria; some mycoplasma	0.5 - 50 µg/mL [13]	Broad-spectrum aminoglycoside that inhibits bacterial protein synthesis by binding to the 30S ribosomal subunit [13].	Prevention of bacterial contamination; often used as an alternative to PenStrep.
Antibiotic-Antimycotic [10]	Gram-positive & Gram-negative bacteria; yeasts; molds	1X concentration (typically 100 U/mL Penicillin, 100 µg/mL Streptomycin, 0.25 µg/mL Amphotericin B)	Combined action of antibiotics (Penicillin & Streptomycin) and an antimycotic (Amphotericin B) that targets fungal cell membranes.	Broad-spectrum protection against bacterial and fungal contamination.
Penicillin-Streptomycin-Neomycin [10]	Gram-positive & Gram-negative bacteria	Varies by formulation	Triple-antibiotic combination; Penicillin inhibits cell wall synthesis, while Streptomycin and Neomycin inhibit protein synthesis.	Enhanced protection against a wide range of bacteria.

The Scientist's Toolkit: Essential Research Reagent Solutions

The following table catalogs key reagents and their functions, crucial for implementing the protocols and understanding the studies cited in this note.

Table 2: Essential Research Reagents and Materials

Reagent/Material	Function/Description	Example Application
Penicillin-Streptomycin Solution [9] [11]	Ready-to-use sterile solution combining both antibiotics for convenience and consistency.	Supplementation of cell culture media for routine contamination prevention.
Gentamicin Solution [13]	A broad-spectrum, water-soluble antibiotic solution effective against many bacteria.	Used in cell culture media, particularly as an alternative to PenStrep or for specific bacterial threats.
Amphotericin B [10]	An antimycotic agent that acts on the fungal cell membrane.	Included in antibiotic-antimycotic cocktails to prevent and eliminate fungal and yeast contamination.
Cell-Free Gene Expression (CFE) System [14]	A synthetic biology tool for rapid, cell-free protein synthesis using engineered DNA templates.	Used for rapid production and screening of novel antimicrobial peptides like bacteriocins [14].
Gibco Penicillin-Streptomycin-GlutaMAX [10]	A specialized solution combining antibiotics with a stabilized form of L-glutamine.	Provides antibiotic protection while supplying a stable energy source, reducing ammonia buildup.
Dulbecco's Modified Eagle Medium (DMEM)	A widely used basal medium for supporting the growth of many mammalian cell types.	cited as a common medium supplemented with antibiotics like PenStrep [9].
Galleria mellonella Larvae [14] [15]	An invertebrate animal model used for in vivo efficacy and toxicity testing of antimicrobials.	Validating the therapeutic potential of bacteriocin cocktails or phage-antibiotic synergism [14] [15].

Experimental Protocols and Workflows

Protocol: Decontamination of Cell Cultures

When facing contamination in an irreplaceable culture, a systematic decontamination procedure can be attempted. The following protocol is adapted from manufacturer guidelines [11] [13].

• Identification and Isolation: Determine the nature of the contaminant (bacteria, fungus, yeast, mycoplasma). Immediately isolate the contaminated culture from all other cell lines.
• Environmental Decontamination: Thoroughly clean incubators, laminar flow hoods, and work surfaces with a laboratory disinfectant (e.g., Virkon, followed by 70% ethanol). Check HEPA filters [12].
• Dose-Response Toxicity Test:
  • Dissociate, count, and dilute the contaminated cells in antibiotic-free medium.
  • Dispense the cell suspension into a multiwell plate. Add the chosen antibiotic (e.g., Amphotericin B, Gentamicin) in a range of concentrations.
  • Observe cells daily for signs of toxicity (e.g., sloughing, vacuole appearance, decreased confluency, cell rounding).
• Decontamination Cycle:
  • Culture the cells for 2-3 passages using the antibiotic at a concentration one- to two-fold lower than the determined toxic level.
  • Culture the cells for one passage in antibiotic-free medium.
  • Repeat the antibiotic treatment cycle.
• Confirmation of Eradication: Culture the cells in antibiotic-free medium for 4-6 passages to verify that the contamination has been eliminated [11] [13].

Protocol: Assessing Impact of Antibiotics on 3D Cell Culture

This protocol is derived from research investigating the effects of Penicillin-Streptomycin (P/S) on the sphere-forming ability of cancer cells in suspension culture [8].

• Cell Preparation: Maintain at least two distinct cell lines (e.g., HT29 colon adenocarcinoma and A549 lung carcinoma) in monolayer culture for a minimum of two weeks in the presence or absence of 1% P/S.
• Monolayer Proliferation Assay:
  • Seed cells in adherent culture conditions with and without P/S.
  • Count cell numbers daily for 4 days to assess impact on proliferation in 2D culture.
  • Analyze cell cycle via propidium iodide staining by flow cytometry.
• Suspension Sphere-Formation Assay:
  • Harvest cells from monolayer culture and seed in serum-free, low-attachment plates at low density to promote sphere formation.
  • Culture cells in suspension with varying concentrations of P/S (e.g., 0%, 0.5%, 1%).
  • Incubate for the time required for sphere formation (e.g., 7-14 days).
• Analysis:
  • Quantify the number and size of spheres formed under each condition. A significant decrease in sphere count in P/S-treated groups indicates inhibition of sphere-forming ability.
  • To correlate with cancer stem cell pool, perform Aldehyde Dehydrogenase (ALDH) activity assay on cells from suspension culture using flow cytometry. A decrease in ALDH-positive cells suggests a reduction in tumor-initiating cells [8].

The logical workflow and key decision points for this experimental approach are summarized in the following diagram:

Diagram 1: Experimental workflow for assessing antibiotic effects in 2D vs. 3D culture.

Critical Considerations and Emerging Alternatives

Unintended Effects of Antibiotics on Cell Systems

A primary consideration for any thesis on antibiotic supplementation is their potential for off-target effects. Key evidence includes:

• Altered Gene Expression and Regulation: RNA-seq and ChIP-seq analyses of HepG2 liver cells cultured with PenStrep identified 209 differentially expressed genes and 9,514 differentially enriched H3K27ac peaks (a mark of active enhancers/promoters). Affected pathways included xenobiotic metabolism signaling and PXR/RXR activation, indicating that antibiotics can induce a systemic change in the cellular transcriptomic and epigenomic landscape [7].
• Inhibition of Sphere-Forming Ability: In suspension cultures—which enrich for tumor-initiating cells (TICs)—addition of P/S cocktail led to a dramatic, dose-dependent inhibition of sphere formation across six cancer cell lines. This effect was correlated with a significant reduction in ALDH-positive cells, suggesting a specific negative impact on the cancer stem cell population, crucial for in vivo tumorigenesis [8].
• Impact on Animal Models: In vivo, refined antibiotic cocktail regimens are used to create pseudo-germ-free (PGF) mouse models. Optimizing these regimens to balance effective gut microbiota depletion with minimized animal mortality is critical, as microbiota depletion has been shown to suppress tumor growth and alter chemotherapy efficacy in pancreatic cancer models [16].

Novel Antimicrobial Strategies

Research into alternatives to traditional antibiotics is advancing, offering new directions for contamination control and therapeutic intervention.

• Bacteriocin Cocktails: Bacteriocins are antimicrobial peptides of bacterial origin. Using cell-free gene expression (CFE) systems, cocktails of bacteriocins (e.g., ColM, SalE1B) can be rapidly synthesized. When cocktails are designed to target a pathogen via distinct cell envelope pathways, they can eradicate bacteria effectively while preventing the development of resistance [14].
• Phage-Antibiotic Synergism (PAS): Combining lytic bacteriophages (viruses that infect bacteria) with antibiotics presents a promising strategy against multidrug-resistant pathogens. For example, a cocktail of phages (KPKp and KSKp) synergized with ciprofloxacin (CIP), achieving over 90% inhibition of Klebsiella pneumoniae even at sub-lethal antibiotic doses, and significantly prolonging the survival of infected Galleria mellonella larvae [15].

The synergistic relationship between phages and antibiotics in combating bacterial resistance is illustrated below:

Diagram 2: Mechanism of Phage-Antibiotic Synergism (PAS) against resistant bacteria.

The use of antibiotic cocktails like Penicillin-Streptomycin and Gentamicin remains a cornerstone of contamination control in primary cell culture. However, researchers must be cognizant of their potential to alter fundamental cellular processes, which can confound experimental results, particularly in sensitive systems like 3D suspension cultures and genomic studies. The recommendation is to avoid the routine use of antibiotics for general cell culture maintenance, reserving them for specific applications such as primary culture establishment or decontamination procedures, and always prioritizing impeccable aseptic technique [12] [8]. Furthermore, emerging strategies such as bacteriocin cocktails and phage-antibiotic synergism represent promising frontiers not only for combating antimicrobial resistance in clinical settings but also potentially inspiring new, more selective approaches for safeguarding cell cultures in the future.

Unmasking Cytotoxic and Cytostatic Effects on Mammalian Cells

The routine supplementation of antibiotics in mammalian cell culture is a standard practice in laboratories worldwide, primarily as a preventive measure against bacterial contamination. However, a growing body of evidence indicates that these antibiotic supplements are not biologically inert and can exert significant, unintended effects on cultured cells [4]. This application note examines the cytotoxic and cytostatic properties of commonly used antibiotics in primary cell culture, providing researchers with critical insights and methodologies to identify and mitigate these effects within the context of rigorous scientific research.

The paradigm is shifting toward recognizing that customary antibiotic supplements in cell cultures exhibit cytotoxic and cytostatic activity at standard concentrations, while also altering the biological patterns of cultured mammalian cells [4]. Furthermore, antibiotics can induce genome-wide changes in gene expression and regulation, potentially confounding experimental outcomes in ways that are not immediately apparent [7]. This document synthesizes current research findings and provides standardized protocols to help researchers unmask these hidden effects, ensuring the reliability and reproducibility of cell-based research.

Key Findings: Documented Effects of Common Antibiotics

Cellular Viability and Metabolic Alterations

Antibiotic-induced effects on cell viability are complex, often dependent on specific compounds, their combinations, and exposure duration. Research on human adipose-derived stem cells (ADSCs) revealed that a penicillin-streptomycin mixture (PS), amphotericin B (AmB), and its complex with copper (II) ions (AmB-Cu²⁺) significantly affected cellular metabolic activity and viability in time-dependent and combination-specific manners [17].

Table 1: Effects of Antibiotics on Adipose-Derived Stem Cell Viability and Metabolism

Antibiotic Treatment	24-Hour Effects	48-Hour Effects	72-Hour Effects
Amphotericin B (AmB)	Significant ↓ viability vs. control [17]	Viability ↑ vs. PS-AmB-Cu²⁺ [17]	Significant ↑ mitochondrial activity vs. control [17]
AmB-Cu²⁺	Significant ↓ viability vs. control; ↑ mitochondrial activity vs. control [17]	Viability ↑ vs. control and all other treatments [17]	Significant ↓ viability vs. control [17]
PS-AmB	Significant ↓ viability vs. control [17]	Viability ↑ vs. AmB and PS-AmB-Cu²⁺ [17]	Significant ↑ mitochondrial activity vs. control [17]
PS-AmB-Cu²⁺	Significant ↓ viability vs. control [17]	-	Significant ↑ mitochondrial activity vs. control [17]

These findings demonstrate that antibiotics can alter fundamental cellular processes, with effects that may shift dramatically over time, underscoring the importance of longitudinal assessment in cytotoxicity studies.

Cell Type-Specific Responses

Different cell types exhibit varying susceptibility to antibiotic-induced effects:

• Stem Cells of Apical Papilla (SCAPs): Research on triple antibiotic paste (TAP) containing metronidazole, ciprofloxacin, and minocycline revealed a clear dose-dependent cytotoxicity. Concentrations of 10 and 25 μg/mL demonstrated higher cell viability across 1, 3, and 7 days, while 50 μg/mL showed the most pronounced cytotoxic effects [18].

• C2C12 Myoblasts: Streptomycin exposure did not impair myoblast proliferation but led to a ~40% reduction in myotube diameter and reduced protein synthesis rates during differentiation. Myotubes cultured with streptomycin showed a 25% lower differentiation and 60% lower fusion index, along with fragmentation of the mitochondrial network and a smaller mitochondrial footprint (-64%) [19].

• HepG2 Liver Cells: Penicillin-streptomycin treatment altered the expression of 209 genes, including transcription factors such as ATF3 that are likely to alter the regulation of other genes. Pathway analyses found significant enrichment for "xenobiotic metabolism signaling" and "PXR/RXR activation" pathways [7].

Gene Expression and Epigenetic Modifications

Beyond observable cytotoxic and cytostatic effects, antibiotics can induce profound changes at the molecular level. Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K27ac in HepG2 cells identified 9,514 peaks that were differentially enriched between PenStrep and control treatments [7]. These regulatory changes were enriched near genes functioning in:

• tRNA modification
• Regulation of nuclease activity
• Cellular response to misfolded protein
• Regulation of protein dephosphorylation [7]

These findings suggest that PenStrep treatment can significantly alter the epigenetic landscape in human cells, potentially affecting numerous cellular processes beyond the primary targets of investigation.

Experimental Protocols for Detection

Comprehensive Cytotoxicity Assessment

Objective: To evaluate the cytotoxic and cytostatic effects of antibiotic supplements on mammalian cells using multiple complementary assays.

Materials:

• Primary cells or cell lines of interest
• Standard culture media and sera
• Antibiotic stocks: penicillin-streptomycin, gentamicin, amphotericin B, etc.
• 96-well or 24-well tissue culture plates
• Phosphate-buffered saline (PBS)
• MTT reagent: 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
• SRB reagent: Sulforhodamine B
• LDH assay kit
• Fixation solution: Trichloroacetic acid (for SRB)
• Dissolution solution: Tris base (for SRB)
• Dye extraction buffer: 1% acetic acid, 40% methanol (optional, for NRU)
• Microplate reader

Methodology:

• Cell Seeding and Culture

  • Harvest exponentially growing cells and prepare a suspension of 5 × 10³ to 1 × 10⁴ cells/mL, depending on cell type and growth rate.
  • Seed cells in 96-well plates (100 μL/well for adherent cells, 200 μL/well for suspension cells).
  • Incubate for 24 hours at 37°C, 5% CO₂ to allow cell attachment and recovery.

• Antibiotic Treatment

  • Prepare serial dilutions of antibiotics in culture medium. Include a negative control (medium only) and positive control (e.g., 1% Triton X-100 for LDH assay).
  • Remove culture medium from wells and replace with 100-200 μL of antibiotic-containing medium.
  • Culture cells for 24, 48, and 72 hours to assess time-dependent effects.

• MTT Assay for Mitochondrial Activity [17] [20]

  • After treatment, add 10-20 μL of MTT solution (5 mg/mL in PBS) to each well.
  • Incubate for 2-4 hours at 37°C to allow formazan crystal formation.
  • Carefully remove medium and dissolve formed crystals in 100-200 μL of solvent (DMSO or isopropanol).
  • Measure absorbance at 570 nm with a reference wavelength of 630-690 nm.

• Sulforhodamine B (SRB) Assay for Cell Density [17]

  • After treatment, gently remove medium and fix cells with 100 μL of cold 10% trichloroacetic acid for 1 hour at 4°C.
  • Wash plates 5 times with tap water and air dry.
  • Stain cells with 100 μL of 0.4% SRB solution in 1% acetic acid for 30 minutes at room temperature.
  • Wash 4-5 times with 1% acetic acid to remove unbound dye and air dry.
  • Solubilize protein-bound dye with 100-200 μL of 10 mM Tris base.
  • Measure absorbance at 510-565 nm.

• Lactate Dehydrogenase (LDH) Assay for Membrane Integrity

  • After treatment, collect 50-100 μL of culture supernatant from each well.
  • Transfer to a new plate and add equal volume of LDH reaction mixture.
  • Incubate for 30 minutes at room temperature in the dark.
  • Measure absorbance at 490-500 nm with a reference wavelength of 680-690 nm.

• Data Analysis

  • Calculate percentage viability for each assay:
    • MTT: (Absorbance of treated cells / Absorbance of control) × 100
    • SRB: (Absorbance of treated cells / Absorbance of control) × 100
    • LDH: (Absorbance of treated cells / Absorbance of positive control) × 100
  • Use multiple assays in combination for comprehensive assessment of different aspects of cytotoxicity.

Assessment of Effects on Differentiation Potential

Objective: To evaluate the impact of antibiotics on stem cell differentiation capacity.

Materials:

• Mesenchymal stem cells (e.g., ADSCs, SCAPs)
• Osteogenic differentiation medium: DMEM with 10% FBS, 0.1 μM dexamethasone, 50 μM ascorbate-2-phosphate, 10 mM β-glycerophosphate
• Adipogenic differentiation medium: DMEM with 10% FBS, 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 μg/mL insulin, 200 μM indomethacin
• Fixation solution: 4% paraformaldehyde
• Staining solutions: Alizarin Red S (for mineralization), Oil Red O (for lipid droplets)
• RNA extraction kit
• RT-qPCR reagents and primers for lineage-specific markers

Methodology: [17]

• Cell Culture and Antibiotic Treatment

  • Culture stem cells in growth medium until 80-90% confluent.
  • Split cells and culture in medium with or without antibiotics for the desired duration.

• Induction of Differentiation

  • For osteogenic differentiation: Culture cells in osteogenic medium with or without antibiotics for 14-21 days, changing medium every 3-4 days.
  • For adipogenic differentiation: Culture cells in adipogenic medium with or without antibiotics for 14-21 days, changing medium every 3-4 days.

• Analysis of Differentiation

  • Alizarin Red Staining: Fix cells with 4% paraformaldehyde for 15 minutes, stain with 2% Alizarin Red S (pH 4.1-4.3) for 20-30 minutes, wash with distilled water, and visualize calcium deposits.
  • Oil Red O Staining: Fix cells with 4% paraformaldehyde for 15 minutes, stain with filtered Oil Red O working solution for 30 minutes, wash with distilled water, and visualize lipid droplets.
  • Gene Expression Analysis: Extract total RNA, synthesize cDNA, and perform RT-qPCR for osteogenic markers (e.g., osteocalcin, Runx2) and adipogenic markers (e.g., LEP, PPARγ).

Protocol for Antibiotic Carry-Over Assessment

Objective: To determine whether antimicrobial activity observed in conditioned medium is due to cell-secreted factors or residual antibiotics. [21]

Materials:

• Conditioned medium from cell cultures
• Penicillin-sensitive bacterial strain (e.g., S. aureus NCTC 6571)
• Penicillin-resistant bacterial strain (e.g., S. aureus 1061 A)
• Mueller-Hinton agar plates
• Sterile PBS
• Spectrophotometer

Methodology: [21]

• Conditioned Medium Collection

  • Culture cells in antibiotic-containing medium for 24-48 hours.
  • Replace with fresh, antibiotic-free medium and culture for an additional 24-72 hours.
  • Collect conditioned medium and centrifuge to remove cells and debris.

• Antibiotic Carry-Over Test

  • Prepare serial dilutions of conditioned medium in PBS or culture medium.
  • Inoculate penicillin-sensitive and penicillin-resistant bacterial strains in separate tubes.
  • Add diluted conditioned medium to bacterial cultures.
  • Incubate for 4-24 hours at 37°C with shaking.
  • Measure optical density at 600 nm to assess bacterial growth.

• Interpretation

  • If conditioned medium inhibits growth of penicillin-sensitive but not penicillin-resistant strains, the effect is likely due to antibiotic carry-over rather than genuine antimicrobial activity of cell-secreted factors.
  • Include controls with fresh medium that contained antibiotics and was then removed using the same protocol to account for residual antibiotics bound to plastic or carried over despite washing.

Visualization of Antibiotic Effects and Assessment Strategies

Molecular Mechanisms of Antibiotic-Induced Cellular Effects

Comprehensive Cytotoxicity Assessment Workflow

Research Reagent Solutions

Table 2: Essential Reagents for Cytotoxicity Assessment

Reagent/Chemical	Function/Application	Key Considerations
Penicillin-Streptomycin Solution	Most common antibiotic combination for preventing bacterial contamination [4]	Alters gene expression in HepG2 cells; may impair differentiation in stem cells [7]
Amphotericin B	Antifungal agent for preventing yeast and mold contamination [17]	Shows time-dependent effects on cell viability; copper complex may reduce toxicity [17]
Gentamicin	Aminoglycoside antibiotic with broad-spectrum activity [4]	Superior stability compared to PenStrep; less effect on cell morphology and metabolism at standard concentrations [4]
Triple Antibiotic Paste (TAP)	Metronidazole, ciprofloxacin, and minocycline combination for specialized applications [18]	Shows dose-dependent cytotoxicity; lower concentrations (10-25 μg/mL) recommended for cell viability [18]
MTT Reagent	Yellow tetrazolium salt converted to purple formazan by mitochondrial dehydrogenases [20]	Measures mitochondrial function; formazan crystals require solubilization before reading [17]
Sulforhodamine B (SRB)	Protein-binding dye that estimates cellular protein content [17]	Useful for measuring cell density; requires fixation step before staining [17]
Alizarin Red S	Calcium-binding dye that detects mineralization in osteogenic differentiation [17]	Essential for assessing effects on osteogenesis; quantitative extraction possible [17]
Oil Red O	Lipid-soluble dye that stains neutral lipids and adipocytes [17]	Critical for evaluating adipogenic differentiation; requires isopropanol for stock solution [17]

The evidence presented demonstrates that routine antibiotic supplementation in mammalian cell cultures can induce significant cytotoxic, cytostatic, and molecular changes that may compromise experimental outcomes. Based on these findings, the following recommendations are proposed:

• Critical Evaluation of Antibiotic Use: Researchers should carefully consider whether antibiotics are absolutely necessary for their specific experimental system. When possible, antibiotic-free cultures with strict aseptic technique should be implemented.

• Comprehensive Cytotoxicity Screening: Employ multiple complementary assays (MTT, SRB, LDH) at various time points to capture the full spectrum of antibiotic effects on different cellular processes.

• Functional Assessment: Evaluate antibiotic effects on specialized cell functions, particularly when working with stem cells or primary cells where differentiation capacity is crucial.

• Antibiotic Carry-Over Controls: Include appropriate controls to distinguish between genuine cellular effects and residual antibiotic activity in conditioned medium experiments.

• Concentration Optimization: When antibiotics are necessary, utilize the lowest effective concentration and consider alternatives with fewer off-target effects, such as gentamicin for bacterial control.

By implementing these practices, researchers can unmask the hidden effects of antibiotic supplements, leading to more reliable and physiologically relevant cell culture models and ultimately enhancing the validity of scientific discoveries.

The use of antibiotics as a standard supplementation in primary cell culture represents a common but critically significant variable in biomedical research. While employed to prevent microbial contamination, a growing body of evidence demonstrates that antibiotics directly and functionally impact fundamental cellular processes in mammalian cells, including primary neurons and neural precursors. This Application Note delineates the specific mechanisms through which antibiotic exposure alters neuronal excitability and compromises genomic integrity, providing researchers with quantitative frameworks and standardized protocols to identify, quantify, and mitigate these confounding effects in experimental models. The documented impacts extend beyond generalized cytotoxicity to include specific disruption of neurotransmitter systems, induction of oxidative stress, and interference with DNA repair mechanisms—all critical parameters in neuroscience research and neuropharmacological development [22] [7] [23].

Quantitative Data Synthesis of Antibiotic-Induced Cellular Impacts

Table 1: Documented Impacts of Antibiotic Exposure on Key Neuronal and Cellular Parameters

Antibiotic Class	Concentration Tested	Experimental Model	Key Measured Impact	Magnitude of Effect	Citation
Quinolones (Levofloxacin)	20-80 μg/mL	Human Sinonasal Epithelial Cells (SNECs)	↑ Caspase-3 Activity (Apoptosis)	5.9-fold increase	[24]
Quinolones (Levofloxacin)	20-80 μg/mL	Human SNECs	↑ Reactive Oxygen Species (ROS)	1.2-1.8-fold increase	[24]
Penicillin-Streptomycin	1% (Standard culture)	HepG2 Liver Cells	Differentially Expressed Genes	209 genes altered	[7]
Penicillin-Streptomycin	1% (Standard culture)	HepG2 Liver Cells	Altered H3K27ac peaks (Regulatory regions)	9,514 peaks changed	[7]
Amoxicillin	9.62 mg/kg	Young Male Mice (in vivo)	↓ Brain Glutathione (GSH)	Significant decrease	[25]
Amoxicillin + Cotrimoxazole	Combined exposure	Young Male Mice (in vivo)	↑ Lipid Peroxidation (MDA)	Significant increase	[25]

Table 2: Efficacy of Potential Protective/Intervention Strategies

Intervention Strategy	Antibiotic Challenge	Experimental Model	Protective Effect	Efficacy	Citation
Sulforaphane (Nrf2 activator)	Levofloxacin (20-80 μg/mL)	Human SNECs	Suppressed Caspase-3 activation	Reduced from 5.9 to 1.9-fold	[24]
Probiotic Administration	Amoxicillin + Cotrimoxazole	Young Male Mice	Normalized hematological indices, reduced oxidative stress	Significant mitigation of damage	[25]
Periodic Dosing (Computational Model)	Simulated treatment	Bacterial Biofilms (in silico)	Reduced required antibiotic dose	Up to 77% reduction	[26]

Experimental Protocols for Assessing Antibiotic Impacts

Protocol: Quantification of Antibiotic-Induced ROS and Apoptosis in Primary Cells

Purpose: To quantitatively measure the production of reactive oxygen species (ROS) and activation of apoptotic pathways in primary cell cultures following antibiotic exposure.

Materials:

• Primary cells (e.g., human sinonasal epithelial cells, neuronal precursors)
• Complete cell culture medium
• Antibiotic stock solutions (e.g., levofloxacin, penicillin-streptomycin)
• H2-DCFDA (2',7'-dichlorofluorescin diacetate) fluorescent probe
• Caspase-3 colorimetric assay kit (e.g., R&D Systems)
• TUNEL assay kit (e.g., TACS 2 TdT fluorescein kit, Trevigen)
• Plate reader capable of fluorescence and absorbance measurements
• Lysis buffer compatible with caspase assay

Methodology:

• Cell Culture and Differentiation: Culture primary cells at air-liquid interface (ALI) for at least 3 weeks to achieve proper differentiation [24].
• Antibiotic Exposure:
  • Prepare serial dilutions of antibiotics in culture medium (e.g., levofloxacin: 0, 1, 10, 20, 40, 80 μg/mL).
  • Apply antibiotic treatments to the basal chamber of ALI cultures.
  • Include pretreatment arms with potential protective compounds (e.g., 10 μM sulforaphane for 72 hours prior to antibiotic exposure).
• ROS Quantification:
  • Load cells with 20 μM H2-DCFDA for 45 minutes.
  • Wash once with 1x PBS to remove excess probe.
  • Measure fluorescence using plate reader (excitation: 485 nm, emission: 528 nm).
  • Re-dose with H2-DCFDA and antibiotics every 24 hours for time-course studies.
• Apoptosis Assessment:
  • Caspase-3 Activity: Collect cell lysates 48 hours post-stimulation. Incubate on ice for 30 minutes, centrifuge at 15,000xg for 2 minutes. Assay supernatants per manufacturer's protocol.
  • DNA Fragmentation: Perform TUNEL assay in situ according to manufacturer's instructions to detect apoptotic nuclei.
• Data Analysis:
  • Normalize ROS fluorescence to untreated controls.
  • Express caspase-3 activity as fold-change relative to control.
  • Quantify TUNEL-positive cells per field of view.

Notes: Maintain consistent cell passage numbers and differentiation status across experiments. Include vehicle controls (e.g., DMSO) matched to compound pretreatment groups [24].

Protocol: Genome-Wide Identification of Antibiotic-Induced Changes in Gene Expression

Purpose: To comprehensively identify alterations in gene expression and regulatory elements in primary cells resulting from standard antibiotic supplementation.

Materials:

• Primary cells of interest (e.g., hepatic, neuronal)
• Culture media with and without antibiotic supplementation (e.g., 1% penicillin-streptomycin)
• RNA extraction kit (e.g., column-based with DNase treatment)
• RNA-seq library preparation kit
• ChIP-seq validated antibodies (e.g., H3K27ac)
• Next-generation sequencing platform access
• Bioinformatics analysis tools (DESeq2, GREAT, DAVID)

Methodology:

• Experimental Design:
  • Culture cells in parallel with media supplemented with 1% penicillin-streptomycin or vehicle control.
  • Maintain cultures for equivalent passages (minimum 3 replicates per condition).
  • Passage cells at identical densities and intervals.
• RNA-seq:
  • Extract high-quality RNA (RIN > 8.0) from both treatment conditions.
  • Prepare stranded RNA-seq libraries following manufacturer's protocols.
  • Sequence to minimum depth of 30 million paired-end reads per sample.
• ChIP-seq for H3K27ac:
  • Cross-link cells with 1% formaldehyde for 10 minutes at room temperature.
  • Quench cross-linking with 125 mM glycine.
  • Sonicate chromatin to 200-500 bp fragments.
  • Immunoprecipitate with validated H3K27ac antibody.
  • Prepare sequencing libraries from immunoprecipitated DNA.
• Bioinformatic Analysis:
  • Align sequences to appropriate reference genome (e.g., GRCh38).
  • Identify differentially expressed genes using DESeq2 with FDR-adjusted p-value ≤ 0.1.
  • Call differential H3K27ac peaks using appropriate peak callers with FDR ≤ 0.1.
  • Perform pathway enrichment analysis (DAVID, IPA) and regulatory region annotation (GREAT).

Notes: This protocol adapted from [7] revealed 209 differentially expressed genes and 9,514 altered H3K27ac regions in HepG2 cells with standard antibiotic use, demonstrating the profound impact of routine supplementation.

Signaling Pathways and Molecular Mechanisms

Antibiotic-Induced Oxidative Stress and Apoptosis Signaling

Diagram: Antibiotic-induced oxidative stress and apoptosis signaling pathway.

The molecular pathway illustrated above demonstrates how bactericidal antibiotics like levofloxacin trigger mitochondrial dysfunction, leading to increased reactive oxygen species (ROS) production [24]. ROS subsequently activates both pro-apoptotic pathways (via Caspase-3) and protective antioxidant responses through the Nrf2 transcription factor. The natural compound sulforaphane enhances Nrf2-mediated protection by promoting its dissociation from the Keap1 repressor protein [24] [27]. This pathway underscores the dual oxidative/pro-apoptotic impact of antibiotics and identifies potential intervention points for mitigation.

Microbiota-Gut-Brain Axis Disruption by Antibiotics

Diagram: Gut-brain axis disruption mechanisms by antibiotic exposure.

Antibiotic exposure induces gut dysbiosis, which disrupts the production of key microbial metabolites including short-chain fatty acids (SCFAs), serotonin precursors, and GABAergic modulators [28] [29]. These alterations impair intestinal barrier function, promote neuroinflammation, and directly impact neuronal excitability through multiple neurotransmitter systems. This pathway explains how antibiotics can indirectly influence central nervous system function despite potentially limited direct penetration, highlighting the importance of considering microbiome interactions in neuropharmacological studies.

The Scientist's Toolkit: Essential Research Reagents and Solutions

Table 3: Key Research Reagents for Investigating Antibiotic Impacts in Neural Systems

Reagent/Category	Specific Examples	Function/Application	Considerations for Use
Nrf2 Activators	Sulforaphane, Curcuminoids, FA-97	Mitigate antibiotic-induced oxidative stress by enhancing antioxidant gene expression	Pretreatment (72h) required for maximal efficacy; dose-dependent effects observed [24] [27]
ROS Detection Probes	H2-DCFDA, MitoSOX Red	Quantitative measurement of general and mitochondrial-specific ROS production	Load for 45 minutes; requires careful standardization between experiments [24]
Apoptosis Assays	Caspase-3 Colorimetric Kits, TUNEL Assays	Quantify apoptotic activation via caspase activity and DNA fragmentation	Combine multiple methods for confirmation; time-course recommended (24-72h) [24]
Probiotics	Various Lactobacillus and Bifidobacterium strains	Restore microbial balance after antibiotic exposure in gut-brain axis studies	Strain-specific effects; administer during/following antibiotic treatment [25]
Epigenetic Tools	H3K27ac Antibodies, ATAC-seq Kits	Map antibiotic-induced changes in regulatory elements and chromatin accessibility	Requires validated antibodies; include input controls for ChIP-seq [7]
Neurotransmitter Analytes	GABA, Serotonin, Glutamate ELISA Kits	Quantify neurotransmitter alterations in antibiotic-exposed models	Consider regional brain differences; collect samples rapidly to prevent degradation [28] [29]

The compiled evidence demonstrates that routine antibiotic supplementation in primary cell culture systems exerts direct functional impacts on neuronal excitability and genomic integrity through multiple mechanisms including oxidative stress induction, apoptotic pathway activation, and epigenetic remodeling. Researchers working with primary neuronal cultures or investigating neuropharmacological mechanisms should implement the following evidence-based practices:

• Validate Antibiotic Necessity: Conduct preliminary studies to determine if antibiotic-free culture is feasible with proper aseptic technique.
• Implement Mitigation Strategies: When antibiotics are required, include appropriate protective compounds (e.g., Nrf2 activators) as pretreatment controls.
• Standardize Reporting: Document antibiotic lot numbers, concentrations, and exposure durations in all methodological descriptions.
• Include Antibiotic Controls: Always include parallel cultures without antibiotics or with vehicle controls to identify antibiotic-specific effects.

These protocols and analytical frameworks provide researchers with standardized approaches to quantify and account for antibiotic-mediated effects, thereby improving the translational validity of primary cell culture models in neuroscience and drug development research.

The routine supplementation of cell culture media with antibiotics is a standard practice in many laboratories working with primary cells. While intended to prevent bacterial contamination, this practice introduces a significant, often overlooked risk: the masking of low-level mycoplasma contamination. Mycoplasmas, the smallest self-replicating organisms, lack a cell wall, rendering common antibiotics like penicillin largely ineffective [30]. Their covert presence can persist for extended periods, subtly compromising experimental integrity without causing overt turbidity in the culture medium [30] [31].

This Application Note details the mechanisms by which antibiotics conceal mycoplasma contamination, outlines the profound consequences for primary cell culture research, and provides validated protocols for the detection, eradication, and prevention of this silent threat, framed within the context of responsible antibiotic use.

The Masking Mechanism and Its Consequences

How Antibiotics Facilitate Covert Mycoplasma Contamination

The masking of mycoplasma occurs through a process of selective pressure. Standard cell culture antibiotic cocktails (e.g., penicillin-streptomycin) target essential bacterial structures, primarily cell wall synthesis. Mycoplasmas, belonging to the class Mollicutes (literally "soft skin"), naturally lack a cell wall, making them intrinsically resistant to these classes of antibiotics [30] [31]. The addition of antibiotics to culture media eliminates competing, antibiotic-sensitive bacteria, thereby creating an uncontested niche for mycoplasma to proliferate. Furthermore, some mycoplasma species, such as Mycoplasmopsis fermentans, can actively invade eukaryotic cells, finding a protected reservoir safe from even those antibiotics effective against mycoplasma in the medium [31].

Impact on Primary Cell Physiology and Data Integrity

The consequences of undetected mycoplasma contamination are far-reaching and can invalidate research data. Mycoplasmas adhere to host cell membranes, competing for essential nutrients and altering the cell's metabolic profile [30]. Key physiological parameters affected include:

• Cell Growth and Viability: Contaminated cultures may exhibit slowed proliferation or halted growth [30].
• Gene Expression and Signaling: Mycoplasma infection can induce significant alterations in host cell gene expression and interfere with signal transduction pathways [31].
• Cellular Metabolism: Disruption of glycolysis and amino acid metabolism rates is common [30].
• Genomic Stability: Contamination can lead to chromosomal abnormalities and instability [30] [31].

These changes are often nonspecific and develop gradually, leading researchers to attribute aberrant results to other experimental variables rather than contamination.

Essential Methodologies for Detection and Validation

Comprehensive Mycoplasma Detection Strategies

Relying on visual inspection for mycoplasma detection is futile due to their small size (0.1–0.3 µm) [30]. Robust, routine testing is essential. The table below summarizes the most common detection methods:

Table 1: Comparison of Mycoplasma Detection Methods

Method	Principle	Time to Result	Sensitivity	Key Advantage	Key Limitation
Culture Method	Growth on specialized agar and broth [30].	1-4 weeks [31]	High (Gold Standard) [30]	High specificity.	Time-consuming; cannot detect non-cultivable species [31].
DNA Fluorochrome Staining	Stains extraneous mycoplasma DNA with Hoechst dye [30].	1-2 days	Moderate	Visually demonstrates contamination on cells.	Subjective; lower sensitivity [31].
qPCR	Amplifies mycoplasma-specific DNA sequences [30].	Several hours	Very High	Rapid, highly sensitive, and specific [30] [31].	Requires specific equipment; does not distinguish viable/dead cells.
Universal PCR Protocol	Uses ultra-conserved primers covering >90% of Mollicutes [31].	Several hours	Very High	Cost-effective, broad-spectrum detection suitable as a common standard [31].	Requires PCR expertise and equipment.

Recommended Protocol: Universal PCR for Mycoplasma Detection

The following protocol, adapted from a 2023 study, provides a reliable and cost-effective method for routine screening [31].

Principle: A four-primer PCR that simultaneously amplifies a conserved region of the mycoplasma 16S rRNA gene and a eukaryotic housekeeping gene, which serves as an internal control for DNA extraction quality and PCR inhibition [31].

Workflow Diagram:

Materials & Reagents:

• Sample: 1 mL of cell culture supernatant (preferred) or trypsinized cell pellet.
• DNA Extraction Kit: e.g., QIAamp DNA Micro Kit or equivalent.
• PCR Reagents: Ultra-pure water, PCR buffer, dNTPs, Taq DNA polymerase.
• Primers:
  • Mycoplasma-specific primer pair (e.g., from [31]).
  • Eukaryotic internal control primer pair (e.g., targeting a human genomic sequence).
• Equipment: Thermal cycler, agarose gel electrophoresis system, gel documentation unit.

Procedure:

• Sample Preparation: Centrifuge 1 mL of cell culture supernatant at 12,000 × g for 10 minutes. Use the pellet for DNA extraction. Alternatively, use a trypsinized cell pellet.
• DNA Extraction: Extract total DNA from the sample according to the manufacturer's instructions for your DNA extraction kit. Elute in 50-100 µL of elution buffer.
• PCR Setup: Prepare a master mix for the number of reactions needed (include a no-template control and a positive mycoplasma control if available).
  • Per reaction: 12.5 µL of 2X PCR master mix, 1 µL of each primer (mycoplasma forward and reverse), 1 µL of eukaryotic control primers, 5 µL of DNA template, and nuclease-free water to a final volume of 25 µL.
• PCR Amplification: Run the PCR using the following cycling conditions:
  • Initial Denaturation: 95°C for 5 minutes.
  • 35-40 Cycles of:
    • Denaturation: 95°C for 30 seconds.
    • Annealing: 55-60°C for 30 seconds.
    • Extension: 72°C for 1 minute.
  • Final Extension: 72°C for 7 minutes.
  • Hold at 4°C.
• Analysis: Resolve 10 µL of the PCR product on a 2% agarose gel. Visualize under UV light.

Interpretation:

• Negative Result: A single band corresponding to the eukaryotic internal control. No mycoplasma-specific band.
• Positive Result: Two bands: the eukaryotic control band and the mycoplasma-specific band (166-191 bp, depending on the species).
• Invalid Result: If the eukaryotic control band is absent, the test is invalid. Repeat the test, optimizing DNA extraction and PCR conditions.

Eradication and Decontamination Strategies

Mycoplasma Contamination Treatment Options

Once contamination is confirmed, the optimal action is to discard the contaminated cells and thaw a new, clean aliquot. If the cell line is irreplaceable, the following eradication strategies can be attempted.

Table 2: Mycoplasma Eradication Strategies

Method	Protocol Summary	Efficacy & Considerations
Discard and Replace	Autoclave contaminated cultures and replace with a validated, mycoplasma-free stock.	Most Reliable. The gold-standard approach to ensure data integrity [30].
Antibiotic Treatment	Passaging cells multiple times in medium supplemented with mycoplasma-specific antibiotics (e.g., doxycycline, ciprofloxacin) [30].	Variable Efficacy. Risk of generating antibiotic resistance; can be toxic to host cells; requires confirmation of clearance [30].
Heat Treatment	Incubating infected cultures at 41°C for 5-18 hours [30].	Limited Applicability. Many primary cell lines are sensitive to heat stress, which can damage viability [30].
Commercial Reagents	Application of combination reagents (e.g., Pricella Anti-Mycoplasma Treatment Reagent) per manufacturer's instructions [30].	Recommended Approach for Valuable Cultures. Often include membrane-disrupting agents and antibiotics; designed to be less toxic to host cells [30].

Recommended Protocol: Antibiotic Eradication with Combination Reagents

Principle: Use of a formulated reagent containing a combination of antibiotics and membrane-disrupting agents to penetrate the mycoplasma membrane and inhibit its DNA replication and metabolism [30].

Materials & Reagents:

• Anti-Mycoplasma Reagent: e.g., Pricella Anti-Mycoplasma Treatment Reagent (Cat. No.: P-CMR-001) or equivalent.
• Mycoplasma-Free Cell Culture Media (without standard antibiotics).
• Phosphate Buffered Saline (PBS).
• Validated Mycoplasma Detection Kit (e.g., qPCR kit).

Procedure:

• Preparation: Ensure the contaminated culture is in a log phase of growth. Wash cells with PBS to remove any residual standard antibiotics.
• Treatment: Add the recommended volume of the anti-mycoplasma reagent directly to the fresh culture medium (without standard antibiotics). The typical treatment concentration is 1X.
• Incubation: Incubate the cells for the recommended duration, usually 3-7 days, with regular medium changes containing the eradication reagent. Monitor cell health and morphology closely.
• Recovery: After the treatment period, wash the cells with PBS and culture them in standard medium (without the eradication reagent) for at least one week.
• Validation: Test the cells for mycoplasma contamination using a highly sensitive method like qPCR at least two weeks after the cessation of treatment. Perform at least two consecutive tests with a one-week interval to confirm complete eradication.

Proactive Prevention and Best Practices

The most effective strategy is to prevent mycoplasma contamination entirely. A robust prevention framework is outlined below.

Diagram: A Proactive Framework for Mycoplasma Control

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Key Reagents for Mycoplasma Management

Reagent / Solution	Function & Rationale
Mycoplasma Detection qPCR Kit	Validated kits provide sensitive and specific detection of a broad range of mycoplasma species, essential for routine screening [31].
Anti-Mycoplasma Eradication Reagent	Commercial formulations combine multiple active agents to treat contaminated cultures effectively while minimizing toxicity to primary cells [30].
Mycoplasma-Free Fetal Bovine Serum (FBS)	FBS is a historical source of mycoplasma; using gamma-irradiated, certified mycoplasma-free serum is critical for prevention [31].
Cell Culture Media without Antibiotics	For maintaining critical stock cultures and for use post-eradication to prevent masking and monitor aseptic technique.
DNA Extraction Kit	For preparing high-quality template DNA from cell culture supernatants or pellets for PCR-based detection methods [31] [32].

The routine use of antibiotics in primary cell culture creates a dangerous illusion of security, providing a veil behind which mycoplasma contamination can thrive and fundamentally skew research data. Adherence to a disciplined culture practice—centered on the avoidance of prophylactic antibiotics, implementation of rigorous, routine detection protocols, and meticulous aseptic technique—is paramount. By acknowledging this silent threat and adopting the detailed methodologies outlined in this Application Note, researchers can safeguard the authenticity of their cellular models and ensure the reliability of their scientific findings in drug development and basic research.

Best Practices for Application: Protocol Development, Dosing, and Contamination Control

The use of antibiotics in primary cell culture represents a critical decision point with far-reaching implications for research integrity. While these supplements offer apparent protection against microbial contamination, a growing body of evidence reveals their profound, often unrecognized effects on cellular physiology and experimental outcomes. The strategic application of antibiotics requires a nuanced approach that carefully balances risk mitigation with the preservation of biological fidelity. This application note establishes evidence-based guidelines for the judicious use of antibiotic supplements, differentiating between short-term stabilization needs and the requirements for long-term culture maintenance. Within the broader thesis of antibiotic supplementation in primary cell culture research, we posit that routine antibiotic use represents a significant confounding variable, and their application should be restricted to specific, justified circumstances with full awareness of their potential consequences on biological systems.

The Impact of Antibiotics on Cellular Systems

Antibiotics, once considered benign additives for contamination control, exert multiple, measurable effects on mammalian cells that can compromise experimental data.

Documented Effects on Gene Expression and Epigenetics

Groundbreaking research has demonstrated that standard antibiotic concentrations can significantly alter transcriptional and epigenetic landscapes. A comprehensive RNA-seq and ChIP-seq study revealed that penicillin-streptomycin (PenStrep) treatment in HepG2 cells altered the expression of 209 genes and modified the enrichment of 9,514 H3K27ac peaks, an epigenetic mark associated with active enhancers and promoters [7]. These changes are not random; pathway analysis identified significant enrichment for "xenobiotic metabolism signaling" and "PXR/RXR activation" pathways, indicating that cells mount a coordinated drug response even to common antibiotics [7]. This finding fundamentally challenges the assumption that antibiotics are biologically neutral in cell culture systems.

Consequences for Experimental Outcomes

The biological changes induced by antibiotics translate into direct experimental confounders:

• Masked Contaminations: Antibiotics can suppress but not eliminate contaminants like mycoplasma, leading to persistent, low-grade infections that alter cell behavior without visible signs of contamination [33]. One study found that approximately 19% of cell lines tested positive for mycoplasma contamination, despite antibiotic use [33].
• Altered Cellular Phenotypes: Antibiotics can influence critical cellular processes including metabolism, proliferation, and stress response, potentially skewing data from high-content screening, toxicology studies, and phenotypic assays [33] [4].
• Carry-Over Effects: Recent investigations have documented that residual antibiotics can persist in conditioned media and extracellular vesicle preparations, leading to false conclusions about antimicrobial properties of cellular secretions [21].

Table 1: Documented Effects of Common Antibiotics on Mammalian Cell Cultures

Antibiotic	Reported Cellular Effects	Impact on Experimental Data
Penicillin-Streptomycin	Alters expression of 209 genes; modifies H3K27ac epigenetic marks; induces stress response pathways [7]	Confounds gene expression studies; affects drug metabolism research; alters cellular stress responses
Gentamicin	Increases reactive oxygen species; causes DNA damage in some cell lines; potential cytotoxicity at higher concentrations [33] [4]	Interferes with oxidative stress assays; compromises DNA damage studies; affects viability measurements
Amphotericin B	Can damage mammalian cell membranes at higher concentrations; light-sensitive [33]	Alters membrane permeability studies; requires careful handling protocols

Guidelines for Strategic Antibiotic Use

The decision to incorporate antibiotics into cell culture protocols should be guided by specific experimental contexts and risk-benefit analysis.

When to Consider Antibiotic Supplementation

Antibiotics may be appropriate in specific circumstances where the risk of contamination outweighs the potential for experimental confounding:

• Initial Primary Culture Establishment: During the isolation and initial expansion of primary cells, which are particularly vulnerable to contamination [33].
• Thawing and Recovery of Cryopreserved Cells: Cells are stressed during thawing and may benefit from temporary protection [33].
• High-Risk Manipulations: During extended procedures or when working in shared laboratory spaces with elevated contamination risk [33].
• Irreplaceable Cell Resources: When working with unique or valuable primary cell isolates that cannot be easily replaced [5].

When to Avoid Antibiotics

Antibiotic-free cultures represent the gold standard for many experimental contexts:

• Long-Term Culture Maintenance: Continuous antibiotic use promotes resistant contaminants and can mask low-level infections [5] [33].
• Gene Expression and Epigenetic Studies: Given their documented impact on transcription and chromatin landscape, antibiotics should be excluded [7].
• Stem Cell and Primary Cell Differentiation Studies: Antibiotics may alter differentiation potential and cell fate decisions [33].
• Drug Metabolism and Toxicology Studies: Antibiotic-induced changes to xenobiotic metabolism pathways can confound results [7].
• Any Study Requiring Authentic Cellular Physiology: When the goal is to approximate in vivo conditions without pharmacological influences [4].

Table 2: Decision Framework for Antibiotic Use in Different Culture Scenarios

Culture Scenario	Recommended Approach	Rationale	Alternative Strategies
Primary cell isolation and early passages	Short-term antibiotic use (1-2 passages)	Reduces risk of losing valuable primary material to contamination	Enhanced aseptic technique; regular contamination screening
Long-term maintenance of established cultures	Antibiotic-free medium	Prevents development of resistant strains; avoids cellular adaptations to antibiotics	Rigorous aseptic technique; segregated culture areas; regular authentication
Sensitive assays (transcriptomics, metabolomics)	Antibiotic-free medium for at least 2 passages prior to assay	Eliminates antibiotic-induced changes to gene expression and metabolism	Plan antibiotic-free culture into experimental timeline
Shared or multi-user facility	Consider short-term use during training periods	Provides safety net during high-risk periods	Intensive training in aseptic technique; clear signage and protocols

Experimental Protocols

Protocol: Transitioning from Antibiotic-Containing to Antibiotic-Free Cultures

Purpose: To safely adapt cells to antibiotic-free conditions while monitoring for previously suppressed contaminants.

Materials:

• Primary cells of interest
• Complete growth medium with antibiotics
• Complete growth medium without antibiotics
• Multi-well culture plates or small flasks
• Mycoplasma detection kit (PCR-based)

Procedure:

• Preparation: Ensure all culture vessels, media, and supplements are sterile. Confirm that biological safety cabinet is properly certified.
• Initial Passage: Subculture cells as usual, but split into two parallel culture conditions: (1) medium with antibiotics, and (2) medium without antibiotics.
• Monitoring: Observe both cultures daily for signs of contamination (turbidity, pH changes, unusual morphology) and cellular health.
• Contamination Testing: After the first passage in antibiotic-free medium, test for mycoplasma and other contaminants using appropriate methods.
• Continued Culture: If no contamination is detected after one passage, continue culturing in antibiotic-free medium. Maintain the antibiotic-containing parallel culture for 2-3 passages as a backup.
• Documentation: Record cell morphology, doubling time, and any changes in phenotypic characteristics.

Troubleshooting:

• If contamination appears in antibiotic-free cultures, investigate source and discard affected cultures. The parallel antibiotic-containing culture may be used for decontamination protocols if irreplaceable.
• If cells show altered morphology or growth in antibiotic-free conditions, allow several passages for adaptation to the new medium.

Protocol: Rescue of Contaminated Irreplaceable Cultures

Purpose: To eliminate microbial contamination from valuable primary cell cultures when replacement is not possible.

Materials:

• Contaminated cell culture
• Appropriate antibiotics at various concentrations
• Multi-well culture plates
• Cell dissociation reagent
• Hemocytometer or automated cell counter

Procedure:

• Contaminant Identification: Determine the identity of the contaminant (bacteria, fungus, yeast) through microscopy and possibly microbial testing [5].
• Antibiotic Selection: Choose an appropriate antibiotic based on the contaminant identified. Consult Table 1 for guidance.
• Toxicity Test:
  • Dissociate, count, and dilute cells in antibiotic-free medium.
  • Dispense cell suspension into a multi-well plate.
  • Add the selected antibiotic to wells in a range of concentrations.
  • Observe cells daily for signs of toxicity (sloughing, vacuolization, decreased confluency, rounding).
  • Identify the concentration that is toxic to the cells [5].
• Decontamination:
  • Culture cells for 2-3 passages using the antibiotic at a concentration one- to two-fold lower than the toxic concentration.
  • Culture the cells for one passage in antibiotic-free media.
  • Repeat the antibiotic treatment for another 2-3 passages.
  • Culture in antibiotic-free medium for 4-6 passages to confirm elimination of contamination [5].
• Validation: Confirm successful decontamination through microbial testing and re-authentication of cell identity.

Visualization of Strategic Decision Pathways

Decision Pathway for Antibiotic Use in Cell Culture This workflow illustrates the strategic decision-making process for antibiotic supplementation in primary cell culture, emphasizing key considerations at different experimental stages.

Essential Research Reagent Solutions

Table 3: Key Reagents for Antibiotic Management and Contamination Control

Reagent/Category	Function/Purpose	Application Notes
Penicillin-Streptomycin (100X)	Broad-spectrum antibacterial against Gram-positive and Gram-negative bacteria	Common default choice; working concentration 100 U/mL penicillin, 100 µg/mL streptomycin; water-soluble; store at -20°C [33]
Gentamicin Sulfate (50 mg/mL)	Broad-spectrum aminoglycoside antibiotic	Effective against mycoplasma; working concentration 10-50 µg/mL; monitor for cytotoxicity in sensitive cells [33] [4]
Antibiotic-Antimycotic Solutions	Combined antibacterial and antifungal protection	Typically contains penicillin, streptomycin, and amphotericin B; convenient for suspected mixed contamination [33]
Amphotericin B	Antifungal agent targeting yeast and mold contamination	Working concentration 0.25-2.5 µg/mL; light-sensitive; higher concentrations may impact mammalian cell viability [33]
Mycoplasma Removal Reagents	Targeted elimination of mycoplasma contamination	Essential for resolving mycoplasma infections; used according to manufacturer protocols; not a substitute for routine testing [33]
PCR-Based Mycoplasma Detection Kits	Sensitive identification of mycoplasma contamination	Critical for routine screening; detects non-visible contaminants that may persist despite antibiotic use [5] [33]

The strategic use of antibiotics in primary cell culture requires moving beyond routine supplementation toward intentional, context-dependent application. The evidence clearly indicates that antibiotic-free culture should be the standard for most experimental applications, particularly those investigating unperturbed cellular physiology. Short-term antibiotic use remains justified in specific high-risk situations, but should be implemented with clear exit strategies and awareness of potential confounding effects. Ultimately, robust aseptic technique, regular contamination monitoring, and systematic quality control represent more sustainable approaches to cell culture integrity than reliance on pharmacological supplements. By adopting these evidence-based guidelines, researchers can better preserve the biological relevance of their primary cell systems while maintaining necessary safeguards against catastrophic culture loss.

Optimal Concentrations and Stability: Navigating pH, Temperature, and Serum Effects

Antibiotic supplementation is a common practice in primary cell culture to prevent the loss of valuable cells and reagents to microbial contamination [34] [33]. However, the efficacy and stability of these antibiotics are not absolute; they are significantly influenced by the physicochemical environment, including pH, temperature, and the presence of serum components [35] [36] [37]. A lack of careful consideration of these factors can lead to incomplete protection or, conversely, induce subtle cellular changes that compromise experimental integrity [33] [21]. For researchers in drug development, understanding and controlling these parameters is crucial for ensuring the reliability and reproducibility of cell-based assays. This application note provides a structured overview and practical protocols to navigate the complex effects of pH, temperature, and serum on antibiotic performance in primary cell culture systems.

Effects of pH, Temperature, and Serum on Antibiotics

The activity and stability of antibiotics are critically dependent on their surrounding environment. The table below summarizes the key effects of pH, temperature, and serum on antibiotic efficacy.

Table 1: Environmental Effects on Antibiotic Efficacy and Stability

Environmental Factor	Observed Effect on Antibiotics	Key Findings & Impact on Cell Culture
pH	Alters antibiotic activity and production by microorganisms [35].	A two-stage pH shift (pH 6.5 to 7.5) significantly enhanced antibiotic activity in Xenorhabdus nematophila fermentation, increasing yield from 100.0 U/mL to 185.0 U/mL [35].
Temperature	Influences antimicrobial potency and pharmacokinetics [36] [37].	Increasing temperature from 35°C to 45°C enhanced the activity of daptomycin, vancomycin, and other antibiotics against staphylococcal biofilms [36]. In Atlantic salmon, higher water temperatures (12°C vs. 20°C) led to more rapid clearance of tetracycline and florfenicol, reducing their concentrations in plasma and muscle [37].
Serum	Induces broad antibiotic tolerance in bacteria [38].	Incubation of Staphylococcus aureus in human serum triggered high-level tolerance to daptomycin, vancomycin, and gentamicin, reducing killing efficacy dramatically. This was linked to host defense peptides and membrane lipid changes [38].

The following diagram illustrates the logical relationship and combined impact of these three key environmental factors on antibiotic performance in cell culture.

Figure 1: Interplay of Environmental Factors on Antibiotic Efficacy. This diagram outlines how pH, temperature, and serum components independently and collectively influence antibiotic performance, leading to significant outcomes for cell culture systems.

Essential Reagents and Research Solutions

Successful management of antibiotic stability requires the use of specific, high-quality reagents. The following table details essential materials for related experiments.

Table 2: Research Reagent Solutions for Antibiotic Studies

Reagent/Solution	Primary Function in Context	Example Use Case
Penicillin-Streptomycin (Pen-Strep) [34] [33]	A synergistic combination targeting a broad range of Gram-positive and Gram-negative bacteria.	Routine supplement in primary cell culture medium to prevent bacterial contamination [34] [12].
Antibiotic-Antimycotic Solution [33]	A combination of Pen-Strep and Amphotericin B to provide broad coverage against bacteria and fungi.	Used when handling primary cells or in shared incubators where risk of mixed contamination is high [33].
Amphotericin B [33]	An antifungal agent that targets fungal and yeast contaminants.	Added to culture medium to specifically prevent or suppress fungal overgrowth. It is light-sensitive and requires storage at -20°C [33].
Dulbecco's Modified Eagle Medium (DMEM) [39]	A common standard cell culture medium containing salts, vitamins, amino acids, and a buffer.	Serves as the basal nutrient medium for culturing a wide variety of primary and immortalized cells [39].
Normal Human Serum [38]	A model host environment containing innate immune factors and nutrients.	Used in ex vivo experiments to study the induction of antibiotic tolerance in bacteria like S. aureus under host-mimicking conditions [38].
Mycoplasma Removal Reagents [33]	Targeted agents specifically formulated to eliminate mycoplasma contamination.	Used to eradicate mycoplasma, which is resistant to standard antibiotics due to its lack of a cell wall [33].

Detailed Experimental Protocols

Protocol: Evaluating the Impact of a Two-Stage pH Shift on Antibiotic Production

This protocol is adapted from a study that significantly enhanced antibiotic production in Xenorhabdus nematophila using a controlled pH strategy [35].

Workflow Overview

Figure 2: Workflow for pH-Shift Antibiotic Production Experiment.

Materials

• Microorganism: Xenorhabdus nematophila YL001 (or relevant antibiotic-producing strain) [35].
• Culture Media:
  • Seed Medium: Nutrient Broth (NB), pH 7.2 [35].
  • Fermentation Medium (per liter): Glucose (6.13 g), Peptone (21.29 g), MgSO₄·7H₂O (1.50 g), (NH₄)₂SO₄ (2.46 g), KH₂PO₄ (0.86 g), K₂HPO₄ (1.11 g), Na₂SO₄ (1.72 g) [35].
• Equipment: 5-L Bioreactor with probes for online monitoring of pH, dissolved oxygen (DO), and temperature; shaking incubator; centrifuge; lyophilizer [35].

Procedure

• Inoculum Preparation: Inoculate a loopful of the bacteria from a fresh NBTA plate into a 250 mL flask containing 100 mL of sterile NB medium. Incubate in darkness at 28°C on a rotary shaker at 150 rpm for 16–24 hours until the optical density at 600 nm (OD₆₀₀) reaches 1.50–2.00 [35].
• Bioreactor Setup: Transfer 3.5 L of fermentation medium into a 5-L bioreactor. Inoculate with the seed culture at a 10% (v/v) ratio. Set the initial process parameters: aeration rate at 2.5 L/min, agitation speed at 300 rpm, and temperature at 28°C [35].
• Two-Stage pH Fermentation:
  • First Stage (0–24 hours): Maintain the culture pH at 6.5 by automatically titrating with 2.0 M NaOH or 2.0 M HCl [35].
  • Second Stage (24–72 hours): At the 24-hour mark, switch the pH setpoint to 7.5 and maintain it for the remainder of the fermentation [35].
• Sampling: Aseptically withdraw approximately 30 mL samples from the bioreactor every 6 hours. Record OD₆₀₀ (for biomass) and process samples immediately for antibiotic activity analysis [35].
• Antibiotic Activity Assay:
  • Centrifuge 20 mL of the sample broth at 22,400 × g for 20 minutes at 4°C to separate cells [35].
  • Subject the supernatant to ammonium sulfate precipitation to remove proteins. Filter the mixture [35].
  • Load the filtrate onto a column filled with activated D101 macroporous adsorption resin. Wash with distilled water and elute the antibiotic compound with methanol [35].
  • Lyophilize the eluent and redissolve in a known volume of H₂O. Determine antibiotic activity using an agar diffusion plate assay against a sensitive indicator strain like Bacillus subtilis. Express activity in Units per mL (U/mL) [35].

Protocol: Assessing Serum-Induced Antibiotic Tolerance

This protocol describes a method to investigate how host-mimicking conditions, such as adaptation to human serum, can induce tolerance to antibiotics in bacteria, a critical consideration for validating the antimicrobial properties of novel therapeutics [38].

Materials

• Bacterial Strain: Staphylococcus aureus (e.g., NCTC 6571, a penicillin-sensitive strain) [38].
• Culture Media: Tryptic Soy Broth (TSB), Normal Human Serum [38].
• Antibiotics: Daptomycin, Vancomycin, Gentamicin, etc., prepared at clinically relevant concentrations [38].
• Equipment: Biological safety cabinet, shaking incubator, spectrophotometer (for OD measurement), equipment for colony counting.

Procedure

• Culture Preparation:
  • TSB-Grown Control: Grow S. aureus to mid-exponential phase in TSB [38].
  • Serum-Adapted Group: Harvest TSB-grown cells, wash, and resuspend in normal human serum to a high density (e.g., 2 × 10⁸ CFU/mL). Incubate for 16 hours without shaking [38].
• Antibiotic Challenge:
  • For both TSB-grown and serum-adapted bacteria, perform survival assays directly in serum to control for serum protein binding effects [38].
  • Expose bacterial cultures to a range of antibiotic concentrations (e.g., 0, 20, 40, 80 µg/mL for daptomycin). Incubate at 37°C with shaking [38].
• Viability Assessment:
  • At predetermined time points (e.g., 0, 3, 6, 24 hours), remove aliquots, perform serial dilutions in PBS, and plate on non-selective agar plates [38].
  • Count colony-forming units (CFU) after overnight incubation. Calculate bacterial survival as the percentage of the initial inoculum [38].
• Analysis: Compare the time- and dose-dependent killing curves of TSB-grown versus serum-adapted bacteria. A significant reduction in killing efficiency against serum-adapted bacteria indicates serum-induced antibiotic tolerance [38].

Data Presentation and Analysis

Table 3: Quantitative Data on Environmental Effects on Antibiotics

Source	Antibiotic / Agent	Test System	Key Parameter Change	Quantitative Effect on Efficacy/Concentration
pH-shift [35]	Cyclo(2-Me-BABA-Gly)	X. nematophila fermentation	pH shift from 6.5 (24h) to 7.5 (48h)	Antibiotic Activity: 100.0 U/mL (constant pH) → 185.0 U/mL (pH-shift). Productivity: 1.39 U/mL/h → 4.41 U/mL/h.
Temperature [36]	Daptomycin	Staphylococcal biofilms	Temperature increase from 35°C to 45°C	Significant reduction in biofilm biomass at 45°C compared to 35°C (P < 0.05).
Temperature [37]	Tetracycline	Atlantic salmon (in vivo)	Water temperature: 12°C vs 20°C	Peak Plasma Concentration: ~ 22 µg/mL (12°C) vs ~ 9 µg/mL (20°C). Faster clearance at higher temperature.
Temperature [37]	Florfenicol	Atlantic salmon (in vivo)	Water temperature: 12°C vs 20°C	Peak Plasma Concentration: ~ 35 µg/mL (12°C) vs ~ 19 µg/mL (20°C). Faster clearance at higher temperature.
Serum [38]	Daptomycin (80 µg/mL)	S. aureus survival	Adaptation in human serum for 16h	Bacterial Survival (6h): < 0.0001% (TSB-grown) vs ~17% (Serum-adapted). >200,000-fold increase in survival.

Within the broader context of antibiotic supplementation in primary cell culture research, the integration of antibiotics during the initial cell isolation phase represents a critical foundation for successful in vitro experiments. Primary cells, directly isolated from tissue, are exceptionally vulnerable to microbial contamination due to the non-sterile nature of source tissues and the extensive manipulation required during processing. The strategic incorporation of antimicrobial agents aims to preserve culture integrity while minimizing potential cytotoxic effects that could compromise downstream experimental validity. This application note provides a detailed, step-by-step methodology for effectively integrating antibiotic supplementation into primary cell isolation workflows, ensuring both aseptic conditions and optimal cell viability for researchers, scientists, and drug development professionals.

Antibiotic supplementation requires careful consideration of multiple factors, including tissue source, isolation duration, agent spectrum, and concentration. Different antimicrobial cocktails target distinct contaminant profiles. This protocol outlines the preparation of effective antibiotic solutions, their integration into dissociation media and washing buffers, and a subsequent withdrawal strategy to minimize long-term effects on cell physiology. The procedures described herein are designed to be adaptable across various primary cell types, providing a robust framework for contamination control without inducing significant antibiotic stress on the isolated cells.

Materials and Reagents

Research Reagent Solutions

The following table details the essential materials and their specific functions within the antibiotic-integrated isolation protocol:

Table 1: Essential Research Reagents for Antibiotic-Integrated Cell Isolation

Reagent/Material	Function/Application in Protocol
Antibiotic-Antimycotic Cocktails (e.g., Penicillin-Streptomycin-Amphotericin B)	Provides broad-spectrum prophylaxis against gram-positive and gram-negative bacteria, and fungi during the initial isolation phase.
Dissociation Enzymes (e.g., Collagenase, Trypsin)	Facilitates tissue disintegration and release of individual cells; antibiotic co-administration prevents introduced contamination.
Basal Cell Culture Media (e.g., DMEM, RPMI-1640)	Serves as the base for creating antibiotic-supplemented washing and holding media.
Phosphate-Buffered Saline (PBS)	Used for tissue washing and dilution of antibiotic stocks; the isotonic solution maintains cell osmolarity.
Heat-Inactivated Fetal Bovine Serum (FBS)	Quenches enzymatic dissociation activity and provides nutrients; used in antibiotic-containing media.
Cell Strainers (70 µm, 100 µm)	Removes tissue debris from the cell suspension after dissociation in antibiotic-supplemented solution.

Antibiotic Working Solution Preparation

Prepare stock solutions according to manufacturer instructions. The following table summarizes standard working concentrations for common agents in isolation protocols:

Table 2: Standard Antibiotic Working Concentrations for Primary Cell Isolation

Antibiotic Agent	Typical Stock Concentration	Working Concentration in Isolation Media	Target Contaminants
Penicillin G	10,000 U/mL	100 - 200 U/mL	Gram-positive bacteria
Streptomycin Sulfate	10,000 µg/mL	100 - 200 µg/mL	Gram-negative bacteria
Amphotericin B	250 µg/mL	2.5 µg/mL	Fungi, yeasts
Gentamicin Sulfate	50 mg/mL	50 µg/mL	Broad-spectrum bacteria
Primocin	50 mg/mL	100 µg/mL	Broad-spectrum bacteria & mycoplasma

Step-by-Step Protocol

The following diagram illustrates the complete workflow for primary cell isolation with integrated antibiotic use, from tissue collection to the establishment of a pure culture.

Detailed Experimental Methodology

Step 1: Pre-Isolation Preparation

• Prepare Antibiotic-Supplemented Media: Aseptically add the selected antibiotic or antibiotic-antimycotic cocktail to the basal isolation medium. For instance, add 1% (v/v) Penicillin-Streptomycin and 0.5% (v/v) Amphotericin B to cold Dulbecco's Modified Eagle Medium (DMEM). Use the working concentrations detailed in Table 2.
• Prepare Enzyme Solution: Dissolve the required dissociation enzymes (e.g., 2 mg/mL Collagenase IV, 0.25% Trypsin-EDTA) in the antibiotic-supplemented medium. Sterilize by filtration (0.22 µm pore size) if not performed aseptically.
• Chill all Solutions: Keep the antibiotic-wash media on ice to slow metabolic activity and protect cells during initial processing.

Step 2: Tissue Processing and Dissociation

• Initial Rinse: Upon receipt, transfer the tissue to a sterile petri dish. Wash it three times with a generous volume (e.g., 10-15 mL per wash) of cold, antibiotic-supplemented PBS to reduce surface contaminants.
• Mechanical Disruption: Mince the tissue into 1-2 mm³ fragments using sterile scalpels or scissors in a small volume of antibiotic-supplemented media to keep the tissue moist.
• Enzymatic Digestion: Transfer the minced tissue to a sterile tube containing the pre-warmed (37°C) enzyme-antibiotic solution. Use a volume 5-10 times the tissue volume.
• Incubate: Place the tube in a shaking water bath or on an orbital shaker at 37°C for 30-90 minutes. Duration depends on tissue type and enzyme activity.

Step 3: Cell Harvesting and Washing

• Neutralization: After digestion, add a volume of complete growth medium (containing FBS) equal to the enzyme solution volume to neutralize trypsin or other serine proteases.
• Filtration: Pass the cell suspension through a sterile cell strainer (70-100 µm) into a new 50 mL conical tube to remove undigested tissue fragments and debris.
• Centrifugation: Centrifuge the filtered suspension at 300-400 x g for 5-10 minutes at 4°C. Carefully decant the supernatant.
• Wash: Resuspend the cell pellet in 10-20 mL of cold, antibiotic-supplemented wash media. Repeat the centrifugation step. This wash is critical for removing enzymes, dead cells, and residual non-sterile components.

Step 4: Plating and Initial Culture

• Resuspension and Counting: Resuspend the final cell pellet in an appropriate volume of complete primary cell culture medium, which also contains antibiotics at the standard working concentration. Perform a cell count and viability assessment using Trypan Blue exclusion.
• Plate Cells: Seed the cells at the recommended density for the specific cell type into culture vessels pre-coated with the appropriate substrate (e.g., Collagen, Poly-L-Lysine).
• Incubate: Place the culture vessels in a humidified 37°C incubator with 5% CO₂.

Step 5: Antibiotic Withdrawal and Monitoring

• Monitor for Contamination: Check cultures daily under a microscope for signs of bacterial or fungal contamination.
• Media Change: After 24-48 hours, perform a complete medium change to remove non-adherent cells, debris, and any residual components from the isolation process. The new medium should still contain antibiotics.
• Strategic Withdrawal: Once the primary cells have adhered and begun to proliferate (typically after 2-4 days), and after confirming no contamination is present, gradually withdraw antibiotics. This can be done by switching to antibiotic-free media in subsequent feeds. This step is crucial for preventing long-term, low-level antibiotic effects on cell metabolism and gene expression [40].

Results and Data Interpretation

Expected Outcomes and Quality Control

Successful execution of this protocol should yield a viable, sterile primary cell culture ready for experimental use. Key quantitative and qualitative metrics for evaluation include:

Table 3: Post-Isolation Quality Control Metrics

Parameter	Expected Outcome	Acceptance Criterion
Cell Viability (Trypan Blue)	High proportion of live, intact cells	>85% viability post-isolation
Microbial Sterility	No visible cloudiness or fungal growth under microscope	No signs of contamination in culture
Cell Adherence & Morphology	Cells adhere and display characteristic morphology within 24-48 hrs	Cell-type specific, healthy morphology
PCR-based Mycoplasma Detection	Negative for mycoplasma contamination	Negative test result [40]

Troubleshooting Common Issues

The following flowchart outlines a systematic approach for diagnosing and resolving common problems encountered during antibiotic-integrated isolations.

Discussion

The integration of antibiotics into primary cell isolation protocols is a foundational practice for ensuring culture purity, yet it demands a balanced and critical approach. While their use is highly effective for preventing microbial contamination from non-sterile starting materials, researchers must remain cognizant of potential subtle effects on cell biology. Certain antibiotics, particularly at high concentrations or with prolonged exposure, can influence metabolic pathways, induce low-level cellular stress, or alter gene expression profiles, which could confound sensitive experimental outcomes [40]. The withdrawal of antibiotics after the initial establishment of a contamination-free culture, as emphasized in this protocol, is therefore a critical step for studies requiring a pristine physiological baseline.

The methodology described aligns with quality control principles observed in other sensitive molecular biology techniques. For instance, the use of standardized master mixes and rigorous quality control in qPCR applications, such as microbial detection and BCR-ABL fusion gene quantification, ensures reliable and reproducible results [40] [41]. Similarly, the preparation of in-house quality control materials, as demonstrated in the BCR-ABL (P210) monitoring study, underscores the importance of internal validation for any laboratory-developed protocol [41]. The step-by-step guide and troubleshooting framework provided here are designed to offer this same level of reliability and adaptability for researchers working across diverse primary cell systems. Ultimately, the goal is to empower scientists to make informed decisions regarding antibiotic use, optimizing for both sterility and the physiological relevance of their primary cell models.

Effective Decontamination Procedures for Irreplaceable Cultures

Cell culture contamination is one of the most common setbacks in biological research laboratories, sometimes with very serious consequences for irreplaceable primary cultures [5]. Biological contaminants include bacteria, molds, yeasts, viruses, and mycoplasma, while chemical contaminants can include impurities in media, sera, and water [5]. While it is impossible to eliminate contamination entirely, researchers can reduce its frequency and seriousness by understanding contamination sources and following proper aseptic technique [5].

The use of antibiotic supplementation in cell culture presents a complex dilemma for researchers. While antibiotics can provide a safety net against contamination, particularly for valuable or irreplaceable cultures, their continuous use encourages the development of antibiotic-resistant strains, allows low-level contamination to persist, and may interfere with cellular processes under investigation [5]. This application note provides structured protocols for rescuing contaminated irreplaceable cultures while contextualizing the role of antibiotics within a broader research framework.

Contamination Identification and Assessment

Classifying Common Contaminants

Before attempting decontamination, accurate identification of the contaminant is essential. The table below summarizes common contaminants and their characteristic indicators.

Table 1: Identification of Common Cell Culture Contaminants

Contaminant Type	Visual/Microscopic Indicators	Medium Changes	Additional Notes
Bacteria	Tiny, moving granules between cells; rods, spheres, or spirals visible under high magnification [5].	Turbidity (cloudiness); sudden drop in pH [5].	One of the most common contaminants due to ubiquity and fast growth [5].
Yeast	Individual ovoid or spherical particles that may bud off smaller particles [5].	Turbidity; pH usually remains stable initially, then increases with heavy contamination [5].	Unicellular eukaryotic microorganisms [5].
Mold	Thin, wisp-like filaments (hyphae) or denser clumps of spores [5].	Turbidity; stable pH initially, then rapid increase with heavy contamination [5].	Spores can survive harsh conditions and activate in favorable environments [5].
Mycoplasma	No visible changes; requires specialized detection methods [33].	No typical changes; can persist covertly [33].	Lacks a cell wall, making it resistant to standard antibiotics like penicillin-streptomycin [33].

Decision Framework for Decontamination

The following workflow outlines the critical decision points when contamination is suspected in an irreplaceable culture. This process helps determine whether decontamination should be attempted and selects the appropriate strategy.

Decontamination Protocols

Pre-Decontamination Procedures

When contamination is confirmed in an irreplaceable culture, immediate isolation and cleaning protocols must be implemented:

• Isolate the contaminated culture from other cell lines immediately to prevent cross-contamination [5].
• Clean incubators and laminar flow hoods thoroughly with a laboratory disinfectant, and check HEPA filters to ensure they are functioning properly [5].
• Decontaminate work areas before and after handling contaminated cultures.

Empirical Toxicity Testing for Antibiotics

Before applying antibiotics to valuable cultures, determining the maximum non-toxic concentration is crucial. The following protocol outlines this empirical testing process.

Table 2: Antibiotic Stock and Working Concentrations

Antibiotic	Common Stock Concentration	Typical Working Concentration	Cytotoxicity Concerns
Penicillin-Streptomycin (Pen-Strep)	100× (10,000 U/mL Penicillin, 10 mg/mL Streptomycin) [33]	1× (100 U/mL Penicillin, 100 µg/mL Streptomycin) [33]	Can alter gene expression; may cross-react with cells [5] [7].
Gentamicin	50 mg/mL [33]	10–50 µg/mL [33]	Higher doses can impair membrane function and slow proliferation [33].
Amphotericin B	250 µg/mL [33]	0.25–2.5 µg/mL [33]	Higher concentrations can damage mammalian cells; light-sensitive [33].

Step-by-Step Protocol:

• Dissociate, count, and dilute the cells from the contaminated culture in antibiotic-free medium. Dilute the cells to the concentration used for regular cell passage [5].
• Dispense the cell suspension into a multi-well culture plate or several small flasks [5].
• Add the antibiotic of choice to each well in a range of concentrations. Include an antibiotic-free control well [5].
• Observe the cells daily for signs of toxicity such as sloughing, appearance of vacuoles, decrease in confluency, and rounding [5].
• Record the toxic concentration where significant cytotoxicity is observed. The working decontamination concentration should be one- to two-fold lower than this toxic level [5].

Antibiotic-Based Decontamination Procedure

Once the appropriate antibiotic concentration has been determined, proceed with the full decontamination protocol:

• Culture the cells for two to three passages using the antibiotic at the determined, non-toxic concentration [5].
• Culture the cells for one passage in antibiotic-free media to assess whether the contamination has been suppressed [5].
• Repeat the antibiotic treatment for another two to three passages if contamination reappears [5].
• Finally, culture the cells in antibiotic-free medium for 4 to 6 passages to confirm that the contamination has been completely eliminated [5].

Specialized Contaminant Scenarios

Mycoplasma Decontamination

Mycoplasma requires specialized approaches due to its lack of a cell wall, which makes it naturally resistant to standard antibiotics like penicillin and streptomycin [33].

• Use targeted mycoplasma removal reagents according to the manufacturer's instructions, as these are specifically formulated to eliminate this contaminant [33].
• Confirm elimination with PCR-based detection methods after treatment, as mycoplasma is difficult to detect by microscopy alone [33].

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Reagents for Cell Culture Decontamination

Reagent Solution	Primary Function	Application Notes
Penicillin-Streptomycin (Pen-Strep)	Broad-spectrum antibiotic combination against Gram-positive and Gram-negative bacteria [33].	Synergistic effect; standard in most labs. Low cytotoxicity at 1× concentration [33].
Antibiotic-Antimycotic Solution	Combined formulation to combat both bacterial and fungal contaminants [33].	Typically contains Pen-Strep and Amphotericin B. Convenient for suspected mixed contamination [33].
Gentamicin Sulfate	Broad-spectrum antibiotic, particularly effective against Gram-negative bacteria [33].	Used at 10-50 µg/mL. Monitor for dose-dependent cytotoxicity [33].
Amphotericin B	Antifungal agent targeting yeast and mold contaminants [33].	Light-sensitive. Use at 0.25-2.5 µg/mL; higher doses may impact mammalian cell viability [33].
Mycoplasma Removal Reagent	Specifically formulated to eliminate mycoplasma contamination [33].	Essential for treating this resistant contaminant. Not a substitute for routine mycoplasma testing [33].

Molecular and Functional Impacts of Antibiotics

Understanding the scientific basis for cautious antibiotic use is essential for proper experimental design. The following diagram illustrates the documented cellular effects of penicillin-streptomycin exposure in mammalian cell cultures.

Research has demonstrated that antibiotic exposure can induce significant molecular changes in cell cultures. A genome-wide study on HepG2 cells cultured with penicillin-streptomycin identified 209 differentially expressed genes compared to antibiotic-free controls [7]. These included transcription factors such as ATF3, SOX4, and FOXO4 that are known to play significant roles in drug and stress response [7]. Pathway analysis revealed significant enrichment for "xenobiotic metabolism signaling" and "PXR/RXR activation" pathways, indicating that the cells are mounting a chemical defense response against the antibiotics themselves [7]. Furthermore, chromatin immunoprecipitation sequencing (ChIP-seq) for H3K27ac, a mark of active enhancers and promoters, identified 9,514 genomic regions that were differentially enriched following antibiotic treatment [7]. These findings provide a molecular basis for why antibiotic treatment should be considered a significant variable in experimental design.

The decision to use antibiotics for decontaminating irreplaceable cultures requires careful consideration of risks and benefits. Based on current evidence, the following best practices are recommended:

• Prioritize Aseptic Technique: Strict adherence to aseptic technique remains the most reliable long-term defense against contamination and avoids the confounding effects of antibiotics on cellular physiology [5] [33].

• Use Antibiotics Strategically: Reserve antibiotics for specific situations such as thawing frozen stocks, working with primary cultures in early passages, or attempting to rescue contaminated irreplaceable cultures [33].

• Validate Decontamination Success: Always confirm the elimination of contaminants by maintaining cells in antibiotic-free conditions for multiple passages after treatment [5].

• Implement Routine Monitoring: Establish regular screening protocols for microbial contaminants, particularly for mycoplasma, which requires specialized detection methods [33].

When dealing with irreplaceable cultures, a methodical approach to decontamination—beginning with accurate contaminant identification, followed by empirical toxicity testing, and culminating in a targeted treatment protocol—maximizes the chances of successful culture recovery while minimizing the potential for antibiotic-induced artifacts in future experiments.

Mastering Aseptic Technique as the Primary Line of Defense

In primary cell culture research, the practice of aseptic technique represents the fundamental barrier between reliable scientific data and compromised experimental outcomes. While antibiotic supplementation has been historically utilized to control microbial contamination, a paradigm shift is underway within advanced research communities. Aseptic technique refers to the set of practices and procedures performed under controlled conditions to prevent contamination from microorganisms, whereas sterilization is an absolute process that destroys or eliminates all forms of microbial life [42]. This distinction is crucial: sterilization creates the contamination-free starting environment, while aseptic technique maintains it throughout the research process [42].

The growing body of evidence questioning routine antibiotic use in cell culture has elevated the importance of mastering aseptic principles. Recent findings demonstrate that antibiotics like penicillin and streptomycin can persist in culture systems through carry-over effects, potentially confounding experimental results by altering cellular phenotypes and gene expression profiles [21] [3]. Furthermore, continuous antibiotic use encourages the development of antibiotic-resistant strains and allows low-level contamination to persist, which can develop into full-scale contamination once the antibiotic is removed [5]. For researchers engaged in drug development and primary cell culture, mastering aseptic technique is no longer merely a best practice—it has become an essential component of experimental validity.

Core Principles: Aseptic Technique as the Foundation

The Essential Framework

Successful aseptic technique rests upon several interconnected principles that together create a comprehensive contamination control strategy. First, the creation and maintenance of a sterile field, typically within a biological safety cabinet (BSC), provides the physical environment for contamination-free work [42]. This requires proper airflow management and meticulous disinfection protocols. Second, the sterile handling of all culture components—including media, reagents, and vessels—ensures that these critical elements remain free from contaminants throughout experimental procedures [43] [42].

Third, researchers must maintain constant self-awareness of their potential as contamination vectors, utilizing appropriate personal protective equipment (PPE) and proper movement control within the BSC [42]. Finally, a proactive approach to monitoring and validation through regular contamination screening ensures early detection of any breaches in technique [5]. Together, these principles form a defensive framework that minimizes reliance on antibiotic supplementation.

Equipment and Workspace Fundamentals

The physical environment and equipment form the first line of defense in aseptic technique. Proper laboratory design begins with a separate enclosed room or laboratory with a single entry/exit point to control traffic flow, with hand washing sinks readily available for cleaning on entry and exit [43]. Dedicated cell culture lab coats and safety goggles should be stored at the lab entrance, and the laminar flow hood and incubators should be positioned away from the entrance to minimize contamination risk [43].

The biosafety cabinet (BSC) serves as the centerpiece of aseptic operations, creating a sterile working environment by continuously filtering air through a HEPA filter [42]. Proper BSC practice includes:

• Allowing at least 15 minutes of operation before beginning work to stabilize airflow
• Strategic placement of materials to avoid blocking airflow grilles
• Thorough disinfection of interior surfaces with 70% ethanol before and after each session [42]

Essential personal protective equipment includes a clean lab coat, sterile gloves, and safety glasses, all of which protect both the researcher and the cultures [43] [42]. Additional critical materials include Bunsen burners or alcohol lamps for flaming vessel openings, sterile pipettes and tips, and properly sterilized media and reagents [42].

Table 1: Essential Equipment for Aseptic Cell Culture

Equipment Category	Specific Items	Critical Function
Primary Workspace	Biosafety Cabinet (BSC)	Creates HEPA-filtered sterile work environment
Sterilization Equipment	Autoclave, 70% ethanol spray bottles, aspirator pump	Sterilizes equipment and surfaces; removes media
Consumables	Sterile pipettes and tips, culture vessels, syringes, needles, forceps	Maintain sterility during handling procedures
Environmental Control	CO₂ incubator, timers, tube racks	Maintains optimal culture conditions (35–37°C, 5–10% CO₂) [43]
Monitoring Tools	Inverted microscope, permanent markers, waste bins	Allows regular culture observation and labeling

Experimental Evidence: Antibiotic Supplementation Versus Aseptic Technique

The Antibiotic Carry-Over Effect

Recent investigations have revealed significant methodological concerns regarding antibiotic supplementation in cell culture systems. A 2025 study demonstrated that conditioned medium (CM) collected from various cell lines exhibited bacteriostatic effects against penicillin-sensitive Staphylococcus aureus NCTC 6571, but not against penicillin-resistant S. aureus 1061A [21] [3]. This selective inhibition was traced not to cell-secreted factors, but to residual antibiotics that persisted in the culture system despite medium changes [21].

The research identified that the antimicrobial activity was due to the retention and release of penicillin to tissue culture plastic surfaces, creating a reservoir of antibiotic that could subsequently leach into fresh medium [3]. This carry-over effect was sufficiently potent to inhibit growth of sensitive bacterial strains, potentially leading researchers to falsely attribute antimicrobial properties to their cell-derived products [21]. These findings have profound implications for research studying antimicrobial mechanisms of cell-based therapeutic applications.

Impact on Cellular Phenotypes

Beyond the direct carry-over effects, antibiotic supplementation has been demonstrated to alter fundamental cellular characteristics across multiple cell types. Transcriptomic analysis of HepG2 liver cells revealed that 209 genes were differentially expressed in the presence of penicillin-streptomycin (PenStrep), including several transcription factors suggesting widespread transcriptional alterations [21] [3]. Functional studies have confirmed physiological impacts, including:

• Altered action and field potential of cardiomyocytes [3]
• Modified electrophysiological properties of hippocampal pyramidal neurons [3]
• Increased production of reactive oxygen species and subsequent DNA damage in breast cancer cell lines with gentamicin exposure [3]

These phenotypic changes introduce significant confounding variables that can compromise experimental validity, particularly in drug development research where subtle cellular responses are critically important.

Comparative Effectiveness of Training Methodologies

The development of proficiency in aseptic technique requires effective training methodologies. A 2025 quasi-experimental study compared video-assisted teaching versus traditional skill demonstration for teaching surgical aseptic skills to nursing students [44]. While both groups showed high post-test knowledge scores, the video-assisted group demonstrated significantly higher psychomotor skill levels in gown and glove-wearing techniques [44]. This finding underscores the value of innovative teaching strategies that provide consistent content delivery and allow for self-paced review of complex techniques.

Figure 1: Decision Workflow for Antibiotic Use in Cell Culture. This workflow guides researchers in determining when antibiotic supplementation may be appropriate and emphasizes the primary role of aseptic technique in maintaining culture integrity.

Protocols: Implementing Robust Aseptic Technique

Comprehensive Aseptic Procedure for Cell Culture

The following step-by-step protocol provides a standardized approach to aseptic technique for routine cell culture procedures, emphasizing practices that minimize reliance on antibiotic supplementation.

Preparation Phase

• Personal Preparation: Tie back long hair, remove jewelry, and don appropriate PPE (lab coat, gloves, safety goggles) before entering the cell culture room [42].
• BSC Preparation: Turn on the biosafety cabinet for at least 15 minutes before beginning work to allow airflow stabilization. Thoroughly disinfect all interior surfaces (side walls, back panel, work surface) with 70% ethanol and allow to air dry [42].
• Material Gathering: Organize all required media, reagents, and culture vessels within the BSC, ensuring they are placed at least six inches from the front grille to maintain proper airflow. Avoid overcrowding the work surface [42].

Execution Phase

• Flaming Technique: Flame the necks of all bottles and flasks before opening and again before closing to create an upward convection current that prevents airborne contamination [42].
• Sterile Handling: Work over the clean surface of the BSC when pipetting, never allowing the non-sterile end of the pipette to contact any surface. Keep lids and caps facing downward when placed on the work surface [42].
• Time Management: Minimize the duration that culture vessels remain open to the environment. Perform procedures efficiently but methodically to reduce contamination risk [42].
• Cross-Contamination Prevention: Never use the same pipette for different cell lines without changing tips. Clean the pipette shaft with 70% ethanol if accidental contact occurs [5].

Completion Phase

• Proper Disposal: Discard all used consumables in appropriate biohazard waste containers immediately after use.
• Surface Decontamination: Thoroughly disinfect all BSC surfaces with 70% ethanol after completing work sessions.
• Documentation: Record culture manipulations and observations promptly to maintain accurate experimental records.

Protocol for Validating Aseptic Technique

Regular validation of aseptic technique is essential for maintaining contamination-free cultures. The following protocol provides a framework for assessing technique proficiency:

• Media Incubation Control: Incubate a sample of complete cell culture media under standard culture conditions (37°C, 5% CO₂) for 7-14 days without cells present. Monitor daily for signs of contamination including cloudiness, pH changes, or fungal growth [5].

• Procedural Mock-Through: Perform a complete cell culture procedure (media change, passaging, etc.) using dye-colored sterile PBS instead of actual media. After the procedure, plate the PBS on nutrient agar and incubate for 48 hours to detect any introduced contaminants [45].

• Environmental Monitoring: Place open agar plates in the BSC during a mock procedure to assess airborne contamination levels. Expose for 30 minutes, then cover and incubate for 48 hours [45].

• Technique Assessment: Utilize standardized checklists to evaluate adherence to aseptic principles, similar to those validated in surgical aseptic skills assessment [46].

Table 2: Contamination Identification Guide

Contaminant Type	Visual Indicators	Microscopic Appearance	Medium Changes
Bacteria	Cloudy/turbid medium; thin surface film [5]	Tiny, moving granules between cells [5]	Rapid pH drop; turbidity within 24-48 hours [5]
Yeast	Turbid medium, especially in advanced stages [5]	Individual ovoid or spherical particles that may bud [5]	pH usually increases with heavy contamination [5]
Mold	Fuzzy, off-white or black surface growth [5]	Thin, wisp-like filaments (hyphae); denser spore clumps [5]	pH stable initially, then increases with heavy growth [5]
Mycoplasma	No visible change; subtle effects on cell growth [5]	Not detectable by standard microscopy [5]	No turbidity; requires specialized testing [5]

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Essential Reagents for Aseptic Cell Culture

Reagent Category	Specific Examples	Function in Aseptic Culture
Basal Media	Neurobasal Plus Medium, DMEM, HBSS	Provides nutritional foundation; formulation affects cell health and contamination susceptibility [47]
Serum & Supplements	Fetal Bovine Serum (FBS), B-27 Plus Supplement	Supports cell growth and specialization; quality control is essential for contamination prevention [47]
Detaching Agents	Trypsin-EDTA solutions	Enables subculturing of adherent cells; must be sterile-filtered and aliquoted to prevent contamination [43]
Buffered Solutions	Phosphate-Buffered Saline (PBS)	Used for washing cells and dilutions; potential contamination source if not properly sterilized [43] [47]
Cryoprotective Agents	Dimethylsulfoxide (DMSO)	Enables cell preservation; must be maintained sterile and properly stored [43]
Disinfection Solutions	70% Ethanol	Primary surface decontaminant; concentration critical for efficacy (higher evaporates too quickly) [43] [42]

Quality Control: Monitoring and Validation Parameters

Method Validation Framework

For laboratories implementing aseptic technique as their primary contamination control strategy, regular validation of methods is essential. The following parameters provide a framework for quality assurance:

• Specificity: The ability of monitoring methods to detect a range of microorganisms. For general cell culture, this should include bacteria, fungi, and mycoplasma [45].
• Accuracy: Determined by comparing contamination detection rates between established and new monitoring methods. Recovery rates of 50-200% are generally considered acceptable [45].
• Precision: Assessed through repeatability (same technician, same reagents) and intermediate precision (different technicians, different days) testing [45].
• Limit of Detection: The lowest number of microorganisms that can be reliably detected. For most applications, this should be below 100 CFU [45].

Contamination Response Protocol

When contamination occurs despite preventive measures, a systematic response is crucial:

• Immediate Isolation: Quarantine contaminated cultures from other cell lines to prevent cross-contamination [5].
• Identification: Determine the contaminant type (bacteria, fungus, yeast, mycoplasma) through morphological assessment and specialized testing if needed [5].
• Decontamination Assessment: For irreplaceable cultures, evaluate potential decontamination strategies using dose-response tests to determine antibiotic toxicity levels before treatment [5].
• Root Cause Analysis: Review all procedures, reagents, and equipment to identify the contamination source and implement corrective actions.

Mastering aseptic technique represents more than just a laboratory skill—it embodies a fundamental philosophy of proactive contamination prevention that is increasingly essential in an era of heightened awareness about antibiotic limitations. The evidence clearly demonstrates that technical proficiency in aseptic methods provides superior protection compared to reliance on chemical supplements, while simultaneously avoiding the confounding variables introduced by antibiotic carry-over and cellular effects [21] [3].

For the research and drug development community, the integration of rigorous aseptic technique represents an investment in data integrity and reproducibility. By establishing comprehensive training programs, implementing validated protocols, and maintaining continuous quality monitoring, laboratories can achieve the contamination-free cultures essential for reliable scientific advancement. In this framework, antibiotic supplementation becomes a carefully considered exception rather than a standard practice, reserved for specific justified circumstances rather than serving as a substitute for technical excellence.

Troubleshooting Common Pitfalls and Implementing Antibiotic-Free Culture Systems

Identifying and Resolving Fibroblast Overgrowth in Primary Cultures

The uncontrolled proliferation of fibroblasts presents a significant and common challenge in primary cell culture, often leading to the overgrowth of the culture and the loss of the target cell population. This issue is particularly acute in research contexts where preserving the unique phenotypic characteristics of primary cells is paramount for experimental validity. Within the broader thesis investigating the implications of antibiotic supplementation in primary cell culture, it is crucial to recognize that standard practices, such as the use of penicillin-streptomycin solutions, can have unintended consequences on cellular dynamics [3]. Recent evidence suggests that residual antibiotic carry-over from culture reagents can not only confound antimicrobial studies but may also influence the growth dynamics of different cell types, potentially exacerbating fibroblast overgrowth issues [3]. This application note provides a detailed framework for identifying, managing, and preventing fibroblast overgrowth, with specific consideration of the role of antibiotic supplementation.

The Challenge of Fibroblast Contamination

Underlying Causes and Identification

Fibroblast overgrowth is a frequent obstacle in primary culture, notably during the isolation of cancer cells from tumour tissue [48]. The resilience and rapid proliferation rate of fibroblasts allow them to quickly outcompete more fastidious primary cells. The problem is often rooted in the initial isolation method; protocols that rely on prolonged enzymatic digestion or insufficient mechanical dissociation can inadvertently favour fibroblast survival and expansion [48]. Visually, fibroblasts are typically recognized by their elongated, spindle-shaped morphology and their tendency to form swirling patterns in culture. Beyond morphology, the definitive hallmark of activated myofibroblasts is the organization of alpha-smooth muscle actin (αSMA) into stress fibers, a key indicator of a pathological fibroblast phenotype that can be detected via immunostaining [49].

Impact of Antibiotic Supplementation

The routine inclusion of antibiotics like penicillin-streptomycin (PenStrep) or combinations with antimycotics (e.g., amphotericin B) is standard in many laboratories to prevent microbial contamination. However, a 2025 study demonstrated that these antibiotics can bind to tissue culture plasticware and be gradually released into the conditioned medium, creating a persistent carry-over effect [3]. This residual antibiotic activity is a significant confounding factor in studies investigating the intrinsic antimicrobial properties of cell secretions or extracellular vesicles. Furthermore, transcriptomic analyses have shown that PenStrep can alter the expression of hundreds of genes in cultured cells, including transcription factors, which may indirectly influence the competitive balance between fibroblasts and other co-cultured primary cells [3]. Therefore, the very reagents used to protect the culture can become unseen variables that skew experimental outcomes.

Quantitative Analysis of Fibroblast Behaviour

Understanding the behavioural characteristics of fibroblasts from different microenvironments is essential for diagnosing overgrowth. The table below summarizes quantitative data on the migratory capacity of fibroblasts isolated from distinct regions of chronic venous ulcers, revealing critical phenotypic differences maintained in vitro.

Table 1: Functional Heterogeneity of Fibroblasts from Chronic Wounds

Fibroblast Isolation Location	Migratory Capacity (Relative to Normal Dermal Fibroblasts)	Response to GM-CSF (100 ng/ml)
Non-Healing Edge	Almost none	No response
Wound Base	Intermediate	Not Specified
Healing Edge	Similar to normal	Not Specified

Data adapted from primary culture studies of patient-derived venous ulcers [50]. This functional heterogeneity underscores that not all fibroblasts are equal, and overgrowth by a specific, potentially dysfunctional subpopulation can halt the expansion of other cells.

Established Protocols for Fibroblast Isolation and Culture

A variety of enzymatic compositions and mechanical techniques are employed for primary tissue dissociation. The choice of method profoundly impacts the initial cellular population and the subsequent risk of fibroblast overgrowth. The following table compares several key protocols from the literature.

Table 2: Comparison of Primary Cell Isolation Methods and Their Efficacy

Method Reference	Key Enzymatic Composition	Key Mechanical Steps	Reported Efficacy & Notes
Cancer Cell Isolation (Method 5) [48]	Collagenase IV (1.6 mg/mL) + Hyaluronidase (0.14 mg/mL)	Overnight digestion, followed by centrifugation	Highly effective for primary breast cancer cultures; yielded primary cell line BC160.
Cardiac Fibroblasts [51]	Collagenase Type II + Trypsin	Tissue chunks digested, centrifugation, plating on Poly-L-Lysine	Simplified protocol for neonatal murine hearts; used to study TGF-β1-induced transdifferentiation.
Lung Fibroblasts [52]	Collagenase/Dispase (1 mg/mL)	Fine mincing of tissue, overnight digestion with shaking	Optimized for Pogona vitticeps lung tissue; culture maintained at 31°C.
Wound Fibroblasts [53]	Not Specified in Detail	Explant culture (1mm fragments, dried 1-2 min)	Standardized for human acute/chronic wounds; explant size and drying time are crucial.

Detailed Protocol: Isolation of Primary Cardiac Fibroblasts

This protocol, adapted from Bio-Protocol, provides a robust method for obtaining cardiac fibroblasts, which can be adapted to other tissues with optimization [51].

Materials and Reagents

• PBS with D-glucose (PBSG): Provides energy and osmotic balance during tissue processing.
• Digestion Mixture (DM): A blend of 0.25% Trypsin and Collagenase Type II in PBSG to dissociate the extracellular matrix.
• Complete DMEM: Dulbecco's Modified Eagle Medium supplemented with 10% Fetal Bovine Serum (FBS) and 1x antibiotic-antimycotic mix.
• Poly-L-Lysine: Coating agent for culture surfaces to enhance fibroblast attachment.

Procedure

• Heart Tissue Harvesting: Euthanize 0-2 day old murine pups. Excise the hearts under sterile conditions and immediately place them in ice-cold PBSG. Remove the atria and any attached tissues or blood clots.
• Tissue Digestion: Transfer hearts to a sterile microcentrifuge tube containing ice-cold DM. Mince the tissue into ~1 mm³ pieces using a sterile surgical blade. Incubate the tube in a shaker incubator at 37°C for 10-15 minutes.
• Cell Collection: After digestion, vortex the tube vigorously for 10-15 seconds. Allow large tissue debris to settle, then collect the supernatant containing dissociated cells and transfer it to a new tube containing horse serum to inhibit further trypsin activity.
• Centrifugation and Seeding: Centrifuge the cell suspension at 1000 rpm for 5 minutes. Discard the supernatant, resuspend the cell pellet in complete DMEM, and seed the cells onto poly-L-lysine coated tissue culture plates.
• Culture Maintenance: Incubate cultures at 37°C with 5% CO₂. Refresh the medium every 2-3 days. Fibroblasts will attach and proliferate, forming a monolayer typically within a week.

Strategies to Combat Fibroblast Overgrowth

Procedural and Technical Solutions

• Optimized Mechanical Dissociation: The choice of isolation method is critical. For breast cancer biopsies, a protocol combining mechanical disaggregation with enzymatic digestion using collagenase and hyaluronidase (Method 5) proved more effective than other tested methods [48].
• Exploit Differential Adhesion: A common technique to separate fibroblasts from other cells, like epithelial cells, involves leveraging their faster attachment rate. Briefly incubating the mixed cell suspension on a culture surface allows fibroblasts to attach first. The supernatant, enriched with other cell types, can then be transferred to a new vessel [48].
• Minimize Antibiotic Carry-Over: To prevent the confounding effects of residual antibiotics, a pre-washing step of the cell monolayer before experiments is highly effective. Research shows that even a single wash with sterile PBS can remove antimicrobial activity derived from prior antibiotic supplementation [3]. Furthermore, using the lowest effective antibiotic concentration during routine culture and opting for antibiotic-free media during conditioning phases for experiments is recommended.

Advanced Phenotyping and Monitoring

Advanced image analysis can be employed to objectively identify and quantify fibroblast activation states, providing a more precise understanding of culture composition. Mathematical descriptors of cell morphology and intracellular structures, such as Fourier analysis of cell shape and quantification of αSMA stress fiber organization, can be used to distinguish quiescent fibroblasts from activated myofibroblasts in a high-throughput manner [49]. This moves beyond subjective morphological assessment to a quantitative and automated grading system.

The Scientist's Toolkit: Essential Reagents

Table 3: Key Research Reagents for Fibroblast Management

Reagent / Tool	Function / Purpose	Example Context
Collagenase Type II/IV	Enzymatic digestion of collagen in the extracellular matrix to dissociate tissues.	Isolation of cardiac fibroblasts [51] and breast cancer cells [48].
Hyaluronidase	Degrades hyaluronic acid, another major component of the ECM, often used with collagenase.	Part of the optimized digestion cocktail for breast cancer biopsies [48].
Poly-L-Lysine	A charged polymer coating for culture surfaces that enhances cell attachment.	Used to improve the adherence of primary cardiac fibroblasts [51].
TGF-β1	Cytokine used to experimentally induce transdifferentiation of fibroblasts into myofibroblasts.	In vitro modelling of fibrosis and fibroblast activation [51].
Antibiotic-Antimycotic (e.g., PenStrep)	Prevents bacterial and fungal contamination in primary cultures.	Routine culture supplement, but requires wash-out to prevent carry-over [3].
GM-CSF	Growth factor used in functional assays to test migratory response of fibroblasts.	Identified non-responsive fibroblast phenotypes in chronic wounds [50].

Workflow and Signaling Pathways

Experimental Workflow for Managing Fibroblast Overgrowth

The following diagram outlines a logical workflow for identifying and resolving fibroblast overgrowth, integrating the strategies discussed in this note.

Key Signaling in Fibroblast Activation

The transition of fibroblasts to an activated, pro-fibrotic state is a central event in overgrowth and dysfunction. This process is driven by specific signaling pathways, primarily the TGF-β/SMAD axis, which can be studied in vitro using primary cultures.

Successfully managing fibroblast overgrowth requires a multifaceted strategy that begins with an optimized isolation protocol, includes vigilant monitoring using both traditional and advanced quantitative methods, and incorporates specific techniques to selectively inhibit fibroblast expansion. Critically, researchers must account for the potential impact of standard laboratory supplements, particularly antibiotics, on their culture systems. By integrating these evidence-based practices—ranging from simple pre-wash steps to remove antibiotic carry-over to the application of sophisticated image analysis for phenotyping—scientists can significantly improve the purity, reliability, and physiological relevance of their primary cell cultures. This, in turn, enhances the validity of downstream research in drug development and disease modeling.

The routine inclusion of antibiotics in cell culture media is a standard practice in many laboratories to prevent bacterial contamination. However, a critical and often overlooked consequence of this practice is the antibiotic carry-over effect, where residual antibiotics from the culture process are transferred into subsequent analytical assays. This phenomenon can confound experimental results, particularly in research investigating the intrinsic antimicrobial properties of biological samples, such as conditioned medium (CM) or extracellular vesicles (EVs) [3]. Within the broader context of antibiotic supplementation in primary cell culture, this carry-over effect presents a significant challenge to data integrity, potentially leading to the false attribution of antimicrobial activity to cell-secreted factors [3] [54]. This application note details the scope of the problem, provides quantitative evidence of its effects, and outlines robust protocols to identify and mitigate antibiotic carry-over in downstream applications.

Evidence and Quantification of the Carry-Over Effect

Key Findings from Experimental Data

Recent investigations have demonstrated that antibiotic carry-over is a potent confounding factor. A pivotal 2025 study showed that conditioned medium (CM) collected from various human cell lines—including dermal fibroblasts and keratinocytes—exhibited significant bacteriostatic effects against penicillin-sensitive Staphylococcus aureus (NCTC 6571), but not against a penicillin-resistant strain (1061 A) [3]. This selective activity was a primary indicator that the observed effect was due to residual penicillin rather than innate antimicrobial factors secreted by the cells.

Critically, the study identified that the antimicrobial agent was not a cellular product but was retained and released by the tissue culture plastic surface itself. This finding was reinforced by experiments showing that the antimicrobial activity of collected CM was inversely related to cellular confluency at the time of collection; lower confluency (more exposed plastic) resulted in higher apparent antimicrobial activity [3]. The activity was effectively eliminated by a simple pre-wash of the cell monolayer, with the wash solution itself containing the inhibitory activity [3].

Quantitative Data on Antimicrobial Activity

The table below summarizes the quantitative effects of conditioned medium (CM) with antibiotic carry-over on bacterial growth, as revealed by the aforementioned study [3].

Table 1: Quantitative Effects of Conditioned Medium (CM) with Antibiotic Carry-over on Bacterial Growth

Cell Line Tested	CM Concentration (v/v)	Growth Inhibition of S. aureus NCTC 6571 (Pen-S)	Growth Inhibition of S. aureus 1061 A (Pen-R)	Key Experimental Condition
All 9 lines tested (e.g., NHh, NIh, HaCaT, 10PCAh)	50% down to 6.25%	Significant inhibition (p ≤ 0.05)	No significant effect (p ≥ 0.05)	Routine CM collection
10PCAh Cell Line	12.5% and higher	Highest inhibition among all lines	No significant effect	Routine CM collection
All 9 lines tested	12.5% and higher	Significant inhibition	Not applicable	Varying cellular confluency
10PCAh Cell Line	12.5% and higher	Effectively removed (p < 0.001)	Not applicable	After a single pre-wash of cells

Consequences for Gene Expression and Cellular Physiology

Beyond microbiological assays, antibiotic carry-over can interfere with a wide range of biological studies. Genome-wide analyses have demonstrated that culturing human hepatoma cells (HepG2) with standard concentrations of penicillin-streptomycin (PenStrep) alters the expression of 209 genes and changes the enrichment of the histone mark H3K27ac at 9,514 genomic regions [54]. These changes significantly impact pathways involved in drug metabolism (PXR/RXR activation), apoptosis, and response to unfolded proteins, thereby threatening the validity of transcriptomic, epigenetic, and pharmacological studies [54].

Table 2: Impact of Penicillin-Streptomycin (PenStrep) on HepG2 Cell Genomics

Analytical Method	Total Features Altered	Key Dysregulated Pathways and Functions	Implications for Research
RNA-seq	209 genes (157 up, 52 down)	Xenobiotic metabolism, PXR/RXR activation, Apoptosis, Unfolded protein response, Insulin response [54]	Skews results in metabolism, toxicology, and stress response studies.
H3K27ac ChIP-seq	9,514 genomic regions	tRNA modification, Regulation of nuclease activity, Cell differentiation, Response to reactive oxygen species [54]	Alters the epigenetic landscape and confounds studies of gene regulation.

Experimental Protocols for Mitigation

Protocol 1: Pre-washing of Cell Monolayers to Remove Carry-Over

This protocol is designed to eliminate residual antibiotics adsorbed to the tissue culture plastic surface prior to collecting conditioned medium for downstream antimicrobial assays [3].

• Step 1: Culture Cells. Grow the donor cells to 70-80% confluency in their standard growth medium, which may contain antibiotics.
• Step 2: Aspirate Medium. Completely remove the antibiotic-containing growth medium.
• Step 3: Wash Monolayer. Gently add a sufficient volume of sterile, pre-warmed phosphate-buffered saline (PBS) or antibiotic-free basal medium to cover the monolayer.
• Step 4: Incubate and Remove. Incubate the culture vessel for 2-5 minutes at 37°C to dissociate loosely bound antibiotics.
• Step 5: Repeat. Aspirate the wash solution and repeat Steps 3 and 4 for a total of two to three washes for maximum efficacy [3].
• Step 6: Collect Conditioned Medium. After the final wash, add antibiotic-free basal medium for the desired conditioning period (e.g., 72 hours) before collection for downstream assays.

Protocol 2: Centrifugation and Resuspension for Microbial Cultures

This method is adapted from classical microbiology for determining Minimum Bactericidal Concentrations (MBCs) and is highly effective for removing antibiotics from bacterial samples before viability plating [55].

• Step 1: Aliquot Sample. Transfer the antibiotic-exposed bacterial suspension to a microcentrifuge tube.
• Step 2: Pellet Cells. Centrifuge at >10,000 × g for 2 minutes to form a firm pellet.
• Step 3: Aspirate Supernatant. Carefully aspirate and discard the supernatant, which contains the dissolved antibiotic.
• Step 4: Resuspend Pellet. Resuspend the bacterial pellet in an equal volume of fresh, antibiotic-free broth or buffer (e.g., PBS).
• Step 5: Repeat Washes. Repeat Steps 2 through 4 for a total of two washes to ensure adequate dilution of the antibiotic.
• Step 6: Plate for Viability. Proceed with serially diluting and plating the resuspended bacteria on antibiotic-free agar plates for colony counting.

Protocol 3: Strategic Streaking on Agar Plates

A simple yet effective method to physically dilute the carried-over antibiotic upon subculturing, suitable for MBC assays and colony isolation [55].

• Step 1: Inoculate Plate. Dip a sterile loop into the bacterial sample potentially containing antibiotic carry-over.
• Step 2: Primary Streak. Streak the loop in a tight, primary pattern over approximately one-quarter of the surface of an antibiotic-free agar plate.
• Step 3: Secondary Streak. Using a new sterile loop, cross-streak from the primary area into an adjacent, untouched quadrant of the plate.
• Step 4: Tertiary Streak. Using another new sterile loop, cross-streak from the secondary area into the final quadrant, ensuring that the inoculum is diluted over a large surface area (at least half the plate).
• Step 5: Incubate and Analyze. Incubate the plate and observe for colonial growth in the areas of highest dilution (tertiary streak), which indicate viable bacteria free from antibiotic inhibition.

Visualizing the Problem and Solutions

The following diagram illustrates the mechanism of antibiotic carry-over from cell culture and the two primary methods for mitigating its effects in downstream assays.

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for Mitigating Antibiotic Carry-Over

Reagent / Material	Function in Protocol	Key Considerations
Antibiotic-Free Basal Medium	Used for the final conditioning phase and for preparing wash solutions.	Essential for ensuring no new antibiotic is introduced during the critical sample preparation phase.
Sterile Phosphate-Buffered Saline (PBS)	A gentle, isotonic solution for washing cell monolayers to elute adsorbed antibiotics without stressing the cells.	Must be pre-warmed to 37°C to maintain cell viability during washing steps.
Microcentrifuge Tubes & Centrifuge	For pelleting and washing bacterial cells to remove soluble antibiotics from the suspension.	High g-forces (≥10,000 × g) ensure a firm pellet is formed quickly.
Antibiotic-Free Agar Plates	Used for viability plating after mitigation procedures.	Confirms the success of antibiotic removal and allows for accurate quantification of viable cells.
Cell Dissociation Reagent (Enzyme-Free)	For detaching adherent cells after washing, if needed for analysis.	Prevents enzymatic degradation of surface proteins or bioactive molecules of interest [39].

The antibiotic carry-over effect is a significant and demonstrable threat to experimental integrity in cell culture-based research. It can falsely suggest antimicrobial activity in biological samples and alter cellular genomics. Adherence to the detailed protocols provided—specifically the pre-washing of cell cultures and the physical removal of antibiotics via centrifugation or strategic streaking—is critical for generating valid and reproducible data. For research requiring the highest level of rigor, particularly in studies of antimicrobial activity, gene expression, or epigenetics, maintaining antibiotic-free cultures remains the most robust strategy.

Systematic Workflow for Transitioning to Antibiotic-Free Media

The use of antibiotics in primary cell culture has been a standard practice for decades, primarily to prevent microbial contamination. However, a growing body of evidence reveals that residual antibiotics in conditioned medium (CM) can act as a significant confounding variable in antimicrobial research, leading to misleading conclusions about the antimicrobial potential of cell-secreted factors or extracellular vesicles (EVs) [3]. This application note establishes a systematic, evidence-based workflow for transitioning to antibiotic-free media conditions, specifically framed within primary cell culture research. By implementing this protocol, researchers can eliminate the confounding effects of antibiotic carry-over, thereby enhancing the validity and reproducibility of research outcomes, particularly in studies investigating innate cellular antimicrobial properties.

The Case for Antibiotic-Free Culture: Quantitative Evidence

Recent investigations demonstrate that the antimicrobial activity observed in conditioned medium from various cell lines is frequently attributable not to cell-secreted factors, but to residual antibiotics, specifically the retention and release of compounds like penicillin from tissue culture plastic surfaces [3]. The tables below summarize key quantitative findings that underscore the necessity of transitioning to antibiotic-free workflows.

Table 1: Impact of Antibiotic Carry-Over on Antimicrobial Activity Assays

Experimental Factor	Finding	Quantitative Impact	Significance
Conditioned Medium (CM) from Routine Culture	Bacteriostatic effect on penicillin-sensitive S. aureus NCTC 6571	Significant growth inhibition at concentrations from 50% down to 6.25% v/v (P ≤ 0.05) [3]	Confounds assessment of true cell-secreted antimicrobial factors
Specificity of Effect	No growth inhibition on penicillin-resistant S. aureus 1061 A	No significant effect (P ≥ 0.05) across all CM concentrations [3]	Confirms antibiotic mode of action, not host-derived factor
Cellular Confluency	Antimicrobial activity of collected CM inversely correlates with cell confluency	Significant decrease (P < 0.001) as confluency increased from 70-80% to >100% [3]	Suggests antimicrobial factor is retained on uncovered plastic

Table 2: Efficacy of Mitigation Strategies in Removing Antimicrobial Activity

Mitigation Strategy	Protocol Detail	Effect on Antimicrobial Activity	Reference
Pre-washing of Cell Monolayer	Washing with sterile PBS prior to CM collection	Effective removal of antimicrobial activity after a single pre-wash (P < 0.001) [3]	[3]
Analysis of Wash Solutions	Collection and testing of PBS wash solutions	Antimicrobial activity was present in the sterile PBS wash solutions [3]	[3]
Minimizing Antibiotic Concentration	Using the lowest effective antibiotic concentration in basal medium	Reduced carry-over effect [3]	[3]

Systematic Workflow for Transitioning to Antibiotic-Free Media

Eliminating antibiotics from cell culture requires a methodical approach to maintain cell line integrity and prevent contamination. The following workflow diagram and detailed protocols guide researchers through this transition.

Systematic Workflow for Antibiotic-Free Transition

Preparatory Phase: Foundation for Success

• Bank High-Quality Stock: Before initiating the transition, create a large, cryopreserved stock of the primary cell line cultured with antibiotics. This stock serves as a backup in case of contamination events and ensures the experiment is not lost. All testing and quality control should be performed on this master stock [56].
• Implement Aseptic Technique Training: Ensure all personnel involved in the culture are trained and adhere to strict aseptic technique. This is the single most critical factor for successful antibiotic-free culture. Key practices include:
  • Regular disinfection of work surfaces and equipment.
  • Proper use of biological safety cabinets.
  • Minimizing exposure of media and cells to non-sterile environments.
  • Use of sterile pipettes and consumables to prevent cross-contamination [56].

Phase 1: Antibiotic Reduction Protocol

This phase focuses on gradually reducing reliance on antibiotics.

• Objective: Acclimate cells to lower antibiotic levels while monitoring for contamination and morphological changes.
• Procedure:
  • Sub-culture cells from the high-quality stock into a new flask.
  • Reduce antibiotic concentration by 50% in the new culture medium.
  • Monitor cells daily for signs of contamination (e.g., rapid pH change, turbidity in media, unusual granularity under the microscope) and for any changes in growth rate or morphology.
  • Maintain cells for at least three passages at this reduced concentration. If no contamination occurs and cell health is stable, proceed to the next step. If contamination occurs, discard the culture and restart from the backup stock, reinforcing aseptic techniques.
  • Eliminate antibiotics entirely from the growth medium for the next passage.

Phase 2: Implementation of a Pre-washing Protocol

To remove residual antibiotics adsorbed to the cells and the tissue culture plastic, a pre-washing step is essential before any experiment, such as the collection of conditioned medium [3].

• Objective: Remove carry-over antibiotics without harming the cell monolayer.
• Procedure:
  • Aspirate the culture medium from the cells.
  • Gently wash the monolayer with a pre-warmed, sterile PBS or antibiotic-free basal medium (BM-). Use a volume equivalent to the original culture medium.
  • Rock the vessel gently to ensure the washing solution covers the entire surface.
  • Completely aspirate the wash solution. Research shows that this first wash contains the majority of antimicrobial activity [3].
  • Repeat the washing step a second time for maximum efficacy.
  • Proceed immediately with adding the fresh, antibiotic-free medium for the experimental conditioning phase.

Phase 3: Full Antibiotic-Free Culture and Experimental Validation

• Objective: Establish and validate a stable, antibiotic-free culture system.
• Procedure:
  • Culture cells exclusively in antibiotic-free medium for a minimum of five passages to ensure complete clearance of residual antibiotics.
  • Validate the absence of microbial contamination through regular tests, including mycoplasma screening.
  • Functionally validate the system by repeating the initial experiments that showed antimicrobial activity. For instance, collect Conditioned Medium (CM) from the antibiotic-free culture and re-test it against penicillin-sensitive and penicillin-resistant bacterial strains. The successful elimination of antibiotic carry-over is confirmed when:
    • The CM no longer inhibits the growth of penicillin-sensitive S. aureus [3].
    • Any remaining antimicrobial activity can be confidently attributed to genuine cell-secreted factors.

Routine Monitoring and Quality Control

• Maintain rigorous, scheduled testing for bacterial, fungal, and mycoplasma contamination.
• Keep a dedicated logbook for the antibiotic-free culture line, documenting passage number, cell morphology, confluency, and all QC results.
• Use the pre-washing protocol as a standard practice before all critical experiments to prevent any potential carry-over from reagents or previous handling [3].

The Scientist's Toolkit: Essential Reagents and Materials

Table 3: Key Research Reagent Solutions for Antibiotic-Free Culture

Reagent/Material	Function in Workflow	Application Notes
Antibiotic-Free Basal Medium	Supports cell growth during experimental phases and washing steps.	Formulate without penicillin, streptomycin, or amphotericin B. Must be validated for cell growth support.
Sterile Phosphate-Buffered Saline (PBS)	Used for the critical pre-washing step to remove residual antibiotics.	Must be pre-warmed to 37°C to avoid thermal shock to cells [3].
Cell Culture-Tested Antibiotics	Used only for maintaining the original backup stock.	Penicillin-Streptomycin (PenStrep) and solutions containing Amphotericin B (AA) are common [3] [56].
Mycoplasma Detection Kit	Essential for routine quality control.	Mycoplasma contamination is cryptic and can significantly alter cell physiology [56].
Cryopreservation Medium	For creating a large backup stock of the original cell line.	Ensures a fallback option is available if contamination occurs during transition.

Antibiotic supplementation is a standard practice in primary cell culture to prevent bacterial contamination. However, emerging evidence demonstrates that routine antibiotic use, particularly penicillin-streptomycin (PenStrep), significantly alters cellular physiology and confounds experimental outcomes [3] [7]. Dose-response testing is therefore essential to determine the optimal balance between contamination control and preservation of normal cellular function for your specific cell type.

Recent studies have revealed that antibiotic carry-over from tissue culture practices can produce misleading results in downstream applications, including false positives in antimicrobial assays [3]. Furthermore, genome-wide analyses identify hundreds of differentially expressed genes in response to PenStrep supplementation, including transcription factors like ATF3 that regulate broad cellular response networks [7]. These findings underscore the necessity of empirical dose-response testing tailored to your specific research context and primary cell type.

Quantitative Effects of Antibiotics on Cell Physiology

Gene Expression and Epigenetic Changes

Table 1: Genome-Wide Changes in HepG2 Cells Cultured with PenStrep

Parameter	Control	+ PenStrep	Change	Functional Implications
Differentially Expressed Genes	-	209	157 upregulated, 52 downregulated	Altered drug metabolism pathways
H3K27ac Peaks (Regulatory Regions)	-	9,514	5,087 enriched, 4,427 depleted	Rewiring of gene regulatory networks
Key Pathway Alterations	Baseline	Induced	PXR/RXR activation (p = 9.43E-05)	Enhanced drug metabolism capacity
Transcription Factor Changes	Normal expression	ATF3, SOX4, FOXO4 differentially expressed	Disrupted regulatory programs	Potential impact on cellular differentiation

RNA-seq and H3K27ac ChIP-seq analyses reveal that PenStrep treatment significantly alters the transcriptomic and epigenomic landscape of human cell lines. These changes are not limited to stress response pathways but extend to fundamental processes including apoptosis, unfolded protein response, and insulin signaling [7]. The activation of PXR/RXR pathways indicates initiation of xenobiotic metabolism systems normally quiescent in untreated cells.

Functional Consequences in Research Applications

Table 2: Antibiotic-Induced Artifacts in Experimental Systems

Experimental Context	Antibiotic Effect	Impact on Data Interpretation	Recommended Mitigation
Extracellular Vesicle Research	Carry-over antibiotic in conditioned medium	False antimicrobial activity against sensitive bacteria [3]	Pre-washing cells before CM collection
Antimicrobial Assays	Residual penicillin on tissue culture plastic	Bacteriostatic effects misinterpreted as bioactive secretion [3]	Multiple PBS washes, minimize uncovered plastic
Bacterial Co-culture Studies	Leached antibiotics in test systems	Inhibition of bacterial growth independent of tested variables [3]	Antibiotic-free validation controls
Transcriptomic Studies	Differential expression of 209 genes	Confounded pathway analysis results [7]	Minimum 48-hour antibiotic washout

The presence of antibiotics can fundamentally alter experimental outcomes, particularly in studies investigating host-pathogen interactions, antimicrobial mechanisms, or cellular signaling. Antibiotic carry-over effects have been shown to persist despite media changes, requiring deliberate washing procedures to eliminate [3].

Experimental Protocols for Dose-Response Testing

Determining Maximum Safe Antibiotic Concentrations

Protocol: Dose-Range Finding for Primary Cells

Objective: Establish the highest antibiotic concentration that maintains >90% viability and normal physiological function in your specific primary cell type.

Materials:

• Primary cells of interest (passage 2-4)
• Complete medium without antibiotics
• 100X penicillin-streptomycin solution (10,000 U/mL penicillin, 10,000 μg/mL streptomycin)
• 96-well tissue culture plates
• Cell viability assay kit (MTT, CCK-8, or PrestoBlue)
• RNA isolation kit for gene expression validation

Procedure:

• Preparation of antibiotic dilutions: Create a 2X dilution series of PenStrep in complete medium across these concentrations: 0.5X, 1X (standard), 2X, 5X, and 10X of the manufacturer's recommended working concentration.
• Cell seeding: Plate primary cells at optimal density (typically 5,000-20,000 cells/well depending on cell type) in 96-well plates using antibiotic-free medium. Incubate for 24 hours to allow attachment.
• Antibiotic exposure: Replace medium with the antibiotic dilution series (8 replicates per concentration). Include antibiotic-free controls.
• Viability assessment: After 72 hours of exposure, measure cell viability using your preferred assay according to manufacturer instructions.
• Morphological documentation: Capture phase-contrast images of each condition to assess morphological changes.
• Gene expression validation: For concentrations maintaining >90% viability, analyze expression of PenStrep-responsive marker genes (ATF3, SOX4) via RT-qPCR to confirm absence of stress responses [7].

Validation Criteria:

• Select the highest concentration maintaining >90% viability relative to controls
• Confirm absence of significant morphological changes
• Verify no induction of stress response markers beyond 2-fold increase

Antibiotic Wash-Out and Carry-Over Testing

Protocol: Assessing and Eliminating Antibiotic Carry-Over Effects

Objective: Determine the minimal wash procedure required to eliminate antibiotic residues that could interfere with downstream applications.

Background: Studies show that antibiotics can persist on tissue culture plastic surfaces and within cells, confounding subsequent experiments [3].

Materials:

• Sterile phosphate-buffered saline (PBS)
• Trypsin-EDTA or appropriate detachment solution
• Conditioned medium for downstream applications

Procedure:

• Culture cells to 70-80% confluence in medium containing your standard antibiotic concentration.
• Aspirate antibiotic-containing medium completely.
• Wash procedure: Add sufficient PBS to cover the monolayer (typically 2 mL for a 35-mm dish), gently swirl, and aspirate completely.
• Repeat washing for 1, 2, and 3 cycles across replicate cultures.
• After the final wash, add antibiotic-free conditioned medium collection medium.
• Incubate for the desired conditioning period (e.g., 24-72 hours).
• Collect conditioned medium and assess for residual antibiotic activity using a bacterial growth inhibition assay with penicillin-sensitive S. aureus NCTC 6571 [3].

Interpretation:

• The minimal wash cycles that eliminate antimicrobial activity against sensitive bacteria should be adopted for all pre-experimental culture processing.
• Increased cellular confluency reduces antibiotic retention, with >90% confluent cultures showing significantly less carry-over [3].

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagents for Antibiotic Dose-Response Studies

Reagent/Category	Specific Examples	Function in Dose-Response Testing	Considerations for Primary Cells
Viability Assays	MTT, CCK-8, PrestoBlue, Calcein-AM	Quantify metabolic activity and cell survival	Primary cells may have different metabolic rates; validate assay linearity
Antibiotic Solutions	Penicillin-Streptomycin, Gentamicin, Amphotericin B	Tested agents for contamination control	Gentamicin may be less disruptive than PenStrep for sensitive primary cells [7]
Bacterial Reporter Strains	S. aureus NCTC 6571 (penicillin-sensitive), S. aureus 1061 A (resistant)	Detect antibiotic carry-over in conditioned media [3]	Maintain both sensitive and resistant strains for specificity controls
Molecular Assays	RT-qPCR primers for ATF3, SOX4, FOXO4	Validate stress response activation at transcript level	Normalize to stable reference genes validated in your cell type
Cell Dissociation Reagents	Enzyme-free dissociation buffers, Mild proteases	Preserve surface markers for functional assays	Avoid trypsin for surface protein analysis; use accutase or EDTA-based solutions [39]

Signaling Pathways and Experimental Workflows

Cellular Response to Antibiotic Exposure: This diagram illustrates the cascade of molecular and functional changes triggered by antibiotic exposure in cell culture, and how dose-response testing can identify conditions that minimize these confounding effects.

Dose-Response Testing Workflow: This workflow outlines the sequential steps for establishing an evidence-based antibiotic supplementation protocol for primary cell culture, incorporating viability testing, molecular validation, and carry-over assessment.

Dose-response testing for antibiotic supplementation is no longer optional but essential for rigorous cell culture research. The documented effects of antibiotics on gene expression, epigenetic regulation, and cellular function necessitate empirical determination of safe exposure levels for each primary cell type and research application.

Based on current evidence, we recommend: (1) implementing a complete antibiotic washout period (minimum 48 hours) before critical experiments, (2) using the lowest effective antibiotic concentration that maintains contamination control, (3) validating the absence of carry-over effects in conditioned media and other biological samples, and (4) establishing cell-type-specific reference ranges for antibiotic tolerance. These practices will significantly improve the reliability and reproducibility of primary cell culture research while maintaining adequate protection against microbial contamination.

Within the context of primary cell culture research, the routine use of antibiotic supplementation is a common, yet double-edged, practice. While it can suppress overt microbial growth, it inadvertently creates a significant quality control challenge: the emergence of cryptic contaminations. These are low-level, persistent infections masked by antibiotics, which can alter cellular physiology, compromise data integrity, and lead to irreproducible results without any visible signs of culture distress [57] [58] [5]. This application note details a proactive quality control framework for the routine monitoring of such cryptic contaminants, specifically mycoplasma, viruses, and cross-contamination, which are notoriously difficult to detect in antibiotic-supplemented media.

The most perilous of these is mycoplasma contamination. Due to their small size (approximately 0.3 µm) and lack of a cell wall, mycoplasma are resistant to common antibiotics like penicillin and do not cause the turbidity or pH shifts typical of bacterial contamination [58] [5]. They can persistently alter host cell metabolism, gene expression, and viability, ultimately rendering experimental data meaningless [57] [58]. Similarly, viral contamination and cross-contamination by other cell lines can proceed covertly, posing a silent threat to the validity of research findings and the safety of biopharmaceutical products [57] [5].

The Cryptic Contamination Threat in Antibiotic-Supplemented Cultures

The decision to use antibiotics in primary cell culture requires a clear understanding of the associated risks. The table below summarizes the key cryptic contaminants and their impacts.

Table 1: Characteristics of Common Cryptic Contaminants in Cell Culture

Contaminant Type	Primary Detection Challenge	Potential Impact on Primary Cells	Influence of Antibiotics
Mycoplasma	No visible turbidity; requires specialized tests (e.g., PCR, fluorescence) [58].	Alters cell growth, metabolism, and gene expression; induces chromosomal aberrations [57] [5].	Masks low-level contamination, allowing for persistent colonization and spread [58] [5].
Virus	Often latent; no cytopathic effect; requires PCR, ELISA, or electron microscopy [57] [5].	Can alter host cell genome and function; poses a safety risk to personnel [57].	No effect on viral replication; contamination remains undetected without specific screening.
Cross-Contamination	No visual indicators; misidentification requires authentication (e.g., STR profiling) [58].	Overgrowth by a fast-growing cell line completely invalidates the experimental model [57] [58].	Prevents microbial cues that might otherwise alert the researcher to a handling error.

Relying on antibiotics provides a false sense of security. Their continuous use encourages the development of antibiotic-resistant strains and, most critically for quality control, allows low-level contaminants to persist cryptically [58] [5]. This practice directly undermines the core principles of research reproducibility and data integrity. Therefore, the guidelines presented herein are designed for laboratories aiming to minimize or phase out antibiotic use, establishing a robust system to ensure culture purity.

Essential Reagents and Research Solutions

Implementing an effective monitoring program requires specific reagents and tools. The following table lists key materials essential for the protocols described in this note.

Table 2: Key Research Reagent Solutions for Contamination Monitoring

Reagent / Solution	Primary Function & Application	Specific Example
Mycoplasma Detection Kit	Specific detection of mycoplasma contamination via PCR or DNA staining.	Commercial kits based on PCR, fluorescence staining (e.g., Hoechst), or ELISA [58].
Virus-Screened FBS	Source of culture supplement that has been tested for and is guaranteed free of common viral contaminants.	Virus-screened and certified FBS to reduce risk of viral introduction [58] [5].
Cell Line Authentication Kit	Confirmation of cell line identity and detection of cross-contamination via STR profiling or isoenzyme analysis.	STR profiling service or kit for routine cell line authentication [58].
Defined, Serum-Free Medium	Eliminates the risk of contaminants introduced through serum; supports consistent cell growth.	Chemically defined, serum-free media to avoid contaminants from biological sera [58] [47].
Validated Sterilization Filters	Removal of microbial contaminants from heat-sensitive solutions (e.g., media, additives).	0.1–0.2 µm filters for sterilizing media and buffers [57].

Recommended Monitoring Protocols

Workflow for Routine Quality Control

The following workflow provides a strategic overview of the integrated monitoring process for managing cryptic contaminations in primary cell culture, from initial risk assessment to final data interpretation.

Protocol 1: Monitoring for Mycoplasma Contamination

Mycoplasma contamination is a major threat due to its subtle yet devastating effects on cell function [58]. This protocol utilizes a PCR-based method for its high sensitivity and specificity.

4.2.1 Materials

• Test cell culture (supernatant or cell lysate)
• Mycoplasma PCR detection kit (including primers, master mix, controls)
• DNA extraction kit
• PCR tubes and thermal cycler
• Gel electrophoresis equipment (if using end-point PCR)

4.2.2 Methodology

• Sample Collection: Collect 200 µL of cell culture supernatant or prepare a cell lysate from the test culture.
• DNA Extraction: Extract total DNA from the sample according to the manufacturer's instructions. Include both a known positive and a negative (sterile medium) control.
• PCR Setup: Prepare the PCR reaction mix on ice. A typical 25 µL reaction includes:
  • 12.5 µL of PCR master mix
  • 1 µL of forward primer (10 µM)
  • 1 µL of reverse primer (10 µM)
  • 5.5 µL of nuclease-free water
  • 5 µL of template DNA
• Amplification: Run the PCR in a thermal cycler using the cycling conditions specified by the detection kit. A common profile is:
  • Initial Denaturation: 95°C for 2 minutes
  • 35-40 Cycles of:
    • Denaturation: 95°C for 30 seconds
    • Annealing: 55-60°C for 30 seconds
    • Extension: 72°C for 1 minute
  • Final Extension: 72°C for 5 minutes
• Analysis: Analyze the PCR products using gel electrophoresis. A positive result is indicated by a band of the expected size when compared to the positive control.

4.2.3 Quality Control & Interpretation

• The test is invalid if the positive control shows no band or the negative control shows a band.
• A clear band in the test sample confirms mycoplasma contamination. The affected culture and all its reagents should be discarded immediately, and the incubator/hood decontaminated [58].

Protocol 2: Cell Line Authentication via STR Profiling

Cross-contamination misidentifies cell lines and invalidates research. Short Tandem Repeat (STR) profiling is the international gold standard for authentication [58].

4.3.1 Materials

• Test cell pellet (≥ 10^6 cells)
• DNA extraction kit
• STR profiling kit or service
• Capillary electrophoresis instrument (if performed in-house)

4.3.2 Methodology

• DNA Extraction: Extract high-quality genomic DNA from the cell pellet. Ensure the DNA concentration and purity (A260/A280 ratio) meet the requirements of the STR profiling service or kit.
• STR Amplification: Submit the DNA sample to a reputable cell bank or core facility for analysis. Alternatively, perform multiplex PCR with fluorescently-labeled STR primers in-house.
• Fragment Analysis: The facility or instrument will separate the amplified PCR fragments by size using capillary electrophoresis.
• Profile Generation: The output is an electrophoretogram, which is translated into an allelic profile—a unique genetic fingerprint for the cell line.

4.3.3 Quality Control & Interpretation

• Compare the obtained STR profile to reference profiles from established cell banks (e.g., ATCC).
• A match of ≥ 80% is typically required to confirm authenticity. A lower percentage indicates potential cross-contamination, and the cell line should not be used for experiments [58].

Protocol 3: Screening for Viral Contaminants

Viral screening is crucial for safety and ensuring consistent cell behavior, especially in bioproduction [57].

4.4.1 Materials

• Test sample (cell lysate or culture supernatant)
• Viral DNA/RNA extraction kit
• qPCR or RT-qPCR kit
• Specific primers and probes for target viruses (e.g., MMV, HLV)
• Real-time PCR instrument

4.4.2 Methodology

• Nucleic Acid Extraction: Extract total nucleic acid from the sample. For RNA viruses, include a reverse transcription step to generate cDNA.
• qPCR Setup: Prepare reactions in duplicate or triplicate. A typical reaction includes:
  • 10 µL of qPCR master mix
  • 1 µL of forward primer (10 µM)
  • 1 µL of reverse primer (10 µM)
  • 0.5 µL of probe (10 µM)
  • 2.5 µL of nuclease-free water
  • 5 µL of template cDNA/DNA
• Amplification and Detection: Run the plate in a real-time PCR instrument. The standard cycling conditions are:
  • Enzyme Activation: 95°C for 2 minutes
  • 40 Cycles of:
    • Denaturation: 95°C for 15 seconds
    • Annealing/Extension: 60°C for 1 minute (with data acquisition)
• Analysis: Determine the Cq (Quantification Cycle) value for each reaction.

4.4.3 Quality Control & Interpretation

• Include no-template controls and positive controls for each target virus.
• A sample is considered positive if it produces a Cq value below a predetermined threshold (e.g., Cq < 35-40) and shows a characteristic amplification curve.

Data Analysis and Quality Control Implementation

Routine monitoring generates critical data that must be integrated into the laboratory's quality management system. The frequency of monitoring should be risk-based, as outlined in the table below.

Table 3: Recommended Monitoring Schedule and Response Actions

Test	Recommended Frequency	Key Analytical Parameter	Corrective & Preventive Action (CAPA) upon Failure
Mycoplasma Screening	Every 1-2 months; with every new cell line introduction [58].	Presence/absence of specific PCR band or positive fluorescence staining.	Quarantine and discard contaminated culture. Decontaminate incubators and hoods. Retrain staff on aseptic technique [57] [58].
Cell Authentication	Every 6-12 months; upon reviving a frozen stock; before starting a major project [58].	≥80% match with reference STR profile.	Discard misidentified cell line. Audit source and handling procedures. Implement a single-cell-line-per-session rule to prevent future cross-contamination [57] [58].
Viral Screening	For critical applications (e.g., bioproduction); when introducing new cell lines, especially of human or primate origin [5].	Cq value below the validated threshold in qPCR.	Quarantine the source material. Evaluate impact on product safety. Use virus-screened or serum-free media replacements [58].

A proactive and systematic quality control program is non-negotiable for ensuring the integrity of primary cell culture research, particularly in the context of antibiotic supplementation. The protocols outlined here for mycoplasma, cross-contamination, and viral detection provide a defensible strategy to identify cryptic contaminations before they compromise scientific validity. By integrating these routine monitoring practices, researchers can transition away from a reliance on antibiotics with confidence, fostering a culture of rigor and reproducibility essential for meaningful scientific discovery and safe drug development.

Ensuring Data Integrity: Comparative Analyses and Validation of Experimental Outcomes

{@| Antimicrobial resistance (AMR) is a major global health threat, predicted to cause significant mortality, particularly in older populations and specific regions. The transcription of satellite DNA is highly sensitive to environmental factors and represents a source of genomic instability. Therefore, tight regulation of (peri)centromeric transcription is essential for genome maintenance. Antibiotics are routinely used for in vitro studies and for medical treatment, however, their effect on pericentromeric satellite DNA transcription was not investigated. Here we show that antibiotics geneticin and hygromycin B, conveniently used in cell culture, as well as rifampicin (along with five other antibiotics), used to treat bacterial infections, increase transcription of a major human pericentromeric alpha satellite DNA in cell lines at standard concentrations. However, response differs among cell lines - maximal increase in A-1235 cells is obtained by rifampicin while in HeLa cells and fibroblasts by geneticin. There is also a positive correlation between antibiotic concentration and the level of alpha satellite transcription. The increase of transcription is accompanied with either H3K9me3 decrease or H3K18ac increase at tandemly arranged alpha satellite arrays while H3K4me2 remains unchanged. Our results suggest that induced alpha satellite DNA transcription upon antibiotic stress could be linked to epigenetic changes - histone modifications H3K9me3 and H3K18ac, which are associated with transcription of heterochromatin.@>

Genome-Wide Changes: How Antibiotics Alter Gene Expression and Chromatin Landscapes

The routine use of antibiotic supplementation in primary cell culture is a widespread practice aimed at preventing bacterial contamination. However, a growing body of evidence demonstrates that antibiotics induce significant genome-wide alterations in eukaryotic cells, affecting both gene expression profiles and chromatin architecture. These changes present a substantial confounding variable in biological research, potentially compromising experimental reproducibility and data interpretation. Within the context of primary cell culture, where maintaining in vivo-like physiological states is paramount, understanding these antibiotic-induced effects is crucial. This Application Note synthesizes recent findings on the molecular impacts of common antibiotics, provides validated protocols for their detection, and offers practical guidance for minimizing artifacts in sensitive genomic and epigenomic studies.

The tables below summarize key quantitative findings from genome-wide studies on antibiotic effects in cultured cells.

Table 1: Genome-Wide Transcriptional and Epigenetic Changes Induced by Penicillin-Streptomycin (PenStrep) in HepG2 Cells [54]

Analysis Type	Number of Significant Changes	Key Affected Pathways/Features	Q-value Cutoff
Differentially Expressed Genes (RNA-seq)	209 genes (157 upregulated, 52 downregulated)	Xenobiotic metabolism signaling, PXR/RXR activation, Apoptosis, Unfolded protein response	≤ 0.1
Differential H3K27ac Peaks (ChIP-seq)	9,514 peaks (5,087 enriched with PenStrep, 4,427 depleted)	tRNA modification, Regulation of nuclease activity, Cellular response to misfolded protein	≤ 0.1

Table 2: Antibiotic-Induced Overexpression of Alpha Satellite DNA Across Cell Lines [59]

Cell Line	Antibiotic Treatment	Fold Increase in Alpha Satellite Transcription	P-value
A-1235 (Glioblastoma)	Rifampicin (82 µg/ml)	3.0x	P = 0.02
	Geneticin (400 µg/ml)	1.7x	P = 0.008
	Hygromycin B (50 µg/ml)	1.6x	P = 0.01
HeLa (Cervix Carcinoma)	Geneticin (400 µg/ml)	4.9x	P = 0.01
	Hygromycin B (50 µg/ml)	3.1x	P = 0.02
	Rifampicin (82 µg/ml)	1.5x	P = 0.01
MJ90hTERT (Fibroblasts)	Geneticin (600 µg/ml)	1.9x	P = 0.008
	Hygromycin B (100 µg/ml)	1.5x	P = 0.01

Experimental Protocols for Assessing Antibiotic Impact

Protocol 1: Transcriptomic Analysis via RNA-seq

This protocol identifies differentially expressed genes in cells cultured with versus without antibiotics [54].

Key Reagents:

• Cell line of interest (e.g., HepG2, primary cells)
• Standard culture media with and without 1% Penicillin-Streptomycin (PenStrep)
• TRIzol reagent or equivalent for RNA isolation
• RNA-seq library preparation kit (e.g., Illumina)

Procedure:

• Cell Culture & Treatment: Culture cells in parallel using two media conditions: (a) standard medium supplemented with 1% PenStrep, and (b) identical medium without any antibiotics. Maintain cells for at least two passages prior to harvesting to ensure acclimation.
• RNA Isolation: Harvest cells at 70-80% confluency. Extract total RNA using TRIzol according to the manufacturer's instructions. Assess RNA integrity and purity (recommended RIN > 9.0).
• Library Preparation & Sequencing: Prepare RNA-seq libraries from high-quality RNA samples. Use a standardized kit and follow the protocol for poly-A selection of mRNA and cDNA synthesis. Sequence the libraries on an appropriate platform (e.g., Illumina NovaSeq) to a minimum depth of 30 million paired-end reads per sample.
• Bioinformatic Analysis: Align sequenced reads to the reference genome (e.g., GRCh38) using a splice-aware aligner like STAR. Perform differential expression analysis using software such as DESeq2, with a significance cutoff of adjusted p-value (q-value) ≤ 0.1.

Protocol 2: Epigenetic Landscape Assessment via H3K27ac ChIP-seq

This protocol maps active enhancers and promoters by profiling histone mark H3K27ac [54].

Key Reagents:

• Antibody against H3K27ac (validated for ChIP)
• Protein A/G magnetic beads
• Cell culture samples from Protocol 1 (with and without antibiotics)
• DNA library preparation kit for ChIP-seq

Procedure:

• Crosslinking & Cell Lysis: Crosslink proteins to DNA by adding 1% formaldehyde directly to the culture medium for 10 minutes at room temperature. Quench the reaction with 125 mM glycine. Wash cells and lyse using a cell lysis buffer.
• Chromatin Shearing: Isolate nuclei and resuspend in shearing buffer. Shear chromatin to an average fragment size of 200-500 bp using a sonicator (e.g., Covaris). Confirm fragment size by agarose gel electrophoresis.
• Immunoprecipitation: Incubate the sheared chromatin with the H3K27ac antibody overnight at 4°C. Add Protein A/G magnetic beads to capture the antibody-chromatin complex. Wash beads extensively with low-salt, high-salt, and LiCl wash buffers.
• DNA Elution & Purification: Reverse crosslinks by incubating the beads with elution buffer at 65°C overnight. Treat with RNase A and Proteinase K, then purify the DNA using a PCR purification kit.
• Library Prep & Sequencing: Construct sequencing libraries from the purified ChIP DNA. Sequence and perform differential peak calling using tools like DESeq2 for ChIP-seq data, applying a q-value cutoff of ≤ 0.1.

Protocol 3: Mitigating Antibiotic Carryover in Conditioned Media

This protocol addresses the confounding effects of residual antibiotics in conditioned media (CM) collected for downstream assays [3].

Key Reagents:

• Sterile, antibiotic-free basal medium (BM-)
• Phosphate-buffered saline (PBS), sterile

Procedure:

• Cell Washing: After removing the antibiotic-containing growth medium, wash the cell monolayer thoroughly with a sufficient volume of sterile PBS. Research shows that even a single pre-wash effectively removes antimicrobial activity derived from the tissue culture plastic [3].
• Conditioned Media Collection: Replace the wash solution with antibiotic-free basal medium (BM-) for the conditioning phase. The duration of this phase can be adjusted based on experimental needs (e.g., 24-72 hours).
• Harvesting: Collect the conditioned medium and centrifuge to remove any cellular debris. Filter-sterilize (0.22 µm) the supernatant before use in downstream applications (e.g., antimicrobial assays, extracellular vesicle isolation).

Signaling Pathways and Conceptual Workflows

Diagram 1: Antibiotic-induced molecular changes in eukaryotic cells. Antibiotic exposure triggers cellular stress, leading to parallel alterations in gene expression and chromatin remodeling, which converge to alter the cellular phenotype.

Diagram 2: Experimental workflow for profiling antibiotic-induced changes. The protocol involves parallel culturing of cells, followed by transcriptomic (RNA-seq) and epigenomic (ChIP-seq) analyses to identify and validate key changes.

The Scientist's Toolkit: Essential Research Reagents

Table 3: Key Reagent Solutions for Profiling Antibiotic-Induced Changes

Reagent / Solution	Function / Application	Example Product / Specification
Penicillin-Streptomycin (PenStrep)	Standard antibiotic supplement for preventing bacterial contamination in cell culture.	Ready-to-use solution, typically at 100X concentration (e.g., 10,000 U/mL penicillin, 10,000 µg/mL streptomycin). Used at 1% v/v [54] [60].
Antibiotic-Free Basal Medium	For conditioning phases and critical experiments to avoid confounding effects of antibiotics.	DMEM, RPMI, or MEM formulations without any antibiotic additions. Essential for collecting clean conditioned media [3].
H3K27ac Antibody	Validated antibody for Chromatin Immunoprecipitation to map active enhancers and promoters.	High-quality, ChIP-validified polyclonal or monoclonal antibody. Critical for detecting epigenetic changes via ChIP-seq [54].
DNase/RNase-Free Water	Preparation of all molecular biology reagents to maintain sample integrity.	Ultra-pure, nuclease-free water for molecular applications like PCR, and library preparation.
RNA Isolation Kit	High-quality total RNA extraction for downstream transcriptomic analysis.	Kit based on spin-column technology, guaranteeing high RIN numbers suitable for RNA-seq.
Chromatin Shearing Reagents	Fragmentation of crosslinked chromatin to optimal size for ChIP-seq.	Covaris microTUBES and appropriate buffers for consistent sonication-based shearing.
Single-Cell ATAC-seq Kit	Profiling chromatin accessibility at single-cell resolution.	Commercial kit (e.g., from 10x Genomics) for analyzing heterogeneous cell populations [61].
Sterile PBS	Washing cell monolayers to remove residual antibiotics and serum.	Calcium- and magnesium-free phosphate-buffered saline, sterile-filtered. Crucial for mitigating antibiotic carryover [3].

Integrating the data and protocols presented herein leads to an unambiguous conclusion: the standard practice of antibiotic supplementation in primary cell culture induces significant, measurable, and biologically relevant changes to the eukaryotic genome. These alterations span transcriptional programs, epigenetic landscapes, and genomic stability markers. For research demanding high fidelity to in vivo states—such as drug metabolism studies, epigenetic profiling, or investigations into non-coding DNA function—the omission of antibiotics, coupled with rigorous aseptic technique, is strongly recommended. Mitigation strategies, including thorough washing steps and the use of antibiotic-free conditioned media, are essential for validating that observed biological effects are genuinely attributable to the experimental intervention and not to the unintended consequences of antimicrobial agents. Acknowledging and controlling for this variable is paramount for improving the reproducibility and translational relevance of primary cell culture research.

{Application Note & Protocol | FRAMED WITHIN A THESIS ON ANTIBIOTIC SUPPLEMENTATION IN PRIMARY CELL CULTURE}

The investigation of innate antimicrobial properties of primary cells or their secreted products (e.g., extracellular vesicles, conditioned medium) is a burgeoning field, particularly in the context of developing novel therapeutic strategies against antimicrobial resistance (AMR). A critical, yet often overlooked, confounder in this research is the routine use of antibiotic supplements in cell culture media. This application note, framed within a broader thesis on antibiotic supplementation in primary cell culture, outlines the significant risk of misleading results due to antibiotic carry-over and provides detailed protocols to distinguish genuine cellular effects from residual antibiotic activity. A foundational study demonstrated that conditioned medium (CM) from various cell lines showed potent bacteriostatic effects against penicillin-sensitive Staphylococcus aureus, but not against penicillin-resistant strains, tracing the activity back to residual antibiotics retained on tissue culture plastic rather than cell-secreted factors [21] [3]. This underscores the paramount importance of rigorous validation methods to ensure the integrity of your research in antimicrobial drug development.

Quantitative Evidence of Antibiotic Interference

The following tables summarize key quantitative findings from the literature that highlight the pervasive impact of antibiotic carry-over and its potential to alter fundamental cellular processes.

Table 1: Documented Effects of Common Cell Culture Antibiotics on Mammalian Cell Systems

Antibiotic	Reported Effect on Mammalian Cells	Experimental Context	Citation
Penicillin-Streptomycin (PenStrep)	Differential expression of 209 genes (157 upregulated, 52 downregulated) in HepG2 liver cells.	RNA-seq analysis of cells cultured with standard 1% PenStrep vs. antibiotic-free media.	[54]
Penicillin-Streptomycin (PenStrep)	Alteration of 9,514 genomic regions marked by H3K27ac (an active enhancer/promoter mark).	ChIP-seq analysis of HepG2 cells cultured with vs. without PenStrep.	[54]
Gentamicin	Increased production of reactive oxygen species (ROS) and subsequent DNA damage.	Treatment of several breast cancer cell lines.	[21]

Table 2: Key Findings on Antibiotic Carry-Over in Antimicrobial Assays

Observation	Experimental Result	Implication for Assay Validation	Citation
Carry-Over from Culture Plastic	Antimicrobial activity of CM decreased significantly as cellular confluency increased (from 70% to >100%).	The antimicrobial agent was adsorbed to the plastic surface, not secreted by cells.	[3]
Elimination via Pre-Washing	A single pre-wash with PBS was sufficient to effectively remove the antimicrobial activity from subsequent CM.	Simple washing steps can mitigate false-positive results.	[3]
Strain-Specific Activity	CM was active against penicillin-sensitive S. aureus but inactive against penicillin-resistant S. aureus.	Using isogenic resistant strains is a critical control.	[21]

Core Validation Protocol: A Step-by-Step Guide

This integrated protocol provides a definitive path to validate that observed antimicrobial activity stems from cellular factors and not residual antibiotics.

Protocol 1: Primary Validation via Resistant Strain Control

Principle: Genuine cellular antimicrobial mechanisms (e.g., antimicrobial peptides) often have a different mode of action than common culture antibiotics. Therefore, their activity should persist against bacteria resistant to those antibiotics.

• Step 1: Cell Culture and Conditioned Medium (CM) Collection

  • Culture primary cells of interest to the desired confluency.
  • CRITICAL: For the final 24-72 hours before CM collection, replace the growth medium with antibiotic-free and serum-free basal medium.
  • Collect the CM and clarify by centrifugation (e.g., 2,000 × g for 10 min) to remove cells and debris. Store at 4°C for immediate use or at -80°C.

• Step 2: Bacterial Strain Selection and Preparation

  • Select a target bacterium relevant to your research (e.g., Staphylococcus aureus).
  • KEY CONTROL: Use a pair of well-characterized bacterial isolates: one that is sensitive to the antibiotic used in your culture (e.g., Penicillin) and an isogenic or closely related strain that is resistant to it [21] [3].
  • Grow bacterial cultures to mid-log phase in an appropriate broth (e.g., Tryptic Soy Broth).

• Step 3: Antimicrobial Susceptibility Testing

  • Dilute the CM in fresh broth. Standard final test concentrations are 50%, 25%, and 12.5% (v/v) CM.
  • Inoculate the diluted CM with a standardized inoculum (~1 × 10^5 CFU/mL) of either the sensitive or resistant bacterial strain.
  • Incubate under appropriate conditions for 4-24 hours.
  • Measure bacterial growth by optical density (OD600) or by plating for colony-forming unit (CFU) counts.

• Step 4: Interpretation of Results

  • Positive for Antibiotic Carry-Over: Significant inhibition of the sensitive strain but no inhibition of the resistant strain.
  • Positive for Genuine Cellular Effect: Significant inhibition of both the sensitive and resistant strains.

Protocol 2: Confirmatory Elimination of Carry-Over

Principle: Actively remove potential residual antibiotics adsorbed to cells and cultureware.

• Step 1: Intensive Pre-Washing of Cell Monolayer

  • After aspirating the antibiotic-containing growth medium, gently wash the cell monolayer two to three times with a generous volume of pre-warmed, sterile PBS or plain basal medium [3].
  • OPTIONAL: Collect these wash solutions and test them for antimicrobial activity against the sensitive bacterial strain as a positive control for the removal of residual antibiotics.

• Step 2: Collection of Validated CM

  • After the final wash, add the antibiotic-free basal medium for the conditioning period.
  • Proceed with CM collection as in Protocol 1, Step 1.

The Scientist's Toolkit: Essential Research Reagent Solutions

Table 3: Key Reagents for Validating Antimicrobial Properties in Cell Culture

Reagent / Solution	Critical Function in Validation	Application Notes
Antibiotic-Free Basal Medium	Used during the conditioning phase to collect CM devoid of new antibiotic contamination.	Essential for all validation steps. Ensure it is matched to your cell type's nutritional requirements.
Penicillin/Streptomycin Solution (100X)	The most common source of carry-over; used to create positive controls for interference.	A stock solution (e.g., 10,000 U/mL penicillin, 10 mg/mL streptomycin) is typically used at 1X final concentration in routine culture [33].
Defined Bacterial Strains (Sensitive & Resistant)	The core biological tools for distinguishing antibiotic activity from cellular effects.	Source from reputable culture collections (e.g., NCTC). Must be well-characterized for their resistance profile.
Phosphate Buffered Saline (PBS), Sterile	For washing cell monolayers to remove adsorbed antibiotics from culture plastic and cell surfaces.	Pre-warm to 37°C to avoid thermal shock to cells.
Mycoplasma Removal Reagent	For targeted elimination of mycoplasma, a common contamination unaffected by standard antibiotics.	Used when decontaminating cell stocks, as mycoplasma is not controlled by PenStrep and can confound host response studies [33].

Advanced Techniques: Cytological Profiling & Detection Assays

For deeper mechanistic insights, consider integrating these advanced methodologies.

• Bacterial Cytological Profiling (BCP): This high-throughput microscopy technique uses fluorescent dyes to stain bacterial DNA and membrane, revealing distinct, antibiotic-induced changes in cellular morphology. The cytological profile caused by a novel cellular antimicrobial will likely differ from that of traditional antibiotics, helping to confirm a unique mechanism of action [62].
• Microbial Inhibition Assays: Simple, well-based diffusion assays can be used as a qualitative screen for the presence of antimicrobial residues in your CM or wash buffers, providing a quick yes/no answer before more quantitative tests [63].

Experimental Design & Workflow Visualization

The following diagrams outline the logical and experimental workflows for a robust validation study.

Diagram 1: Decision workflow for distinguishing antimicrobial activity sources.

Diagram 2: Core experimental protocol for validated CM collection and testing.

Antibiotic supplementation is a common practice in primary cell culture to prevent bacterial contamination. However, a growing body of evidence indicates that these antimicrobial agents are not physiologically neutral and can significantly alter fundamental cellular characteristics. This application note systematically examines how antibiotic supplementation influences core aspects of cell fitness—proliferation, metabolism, and functionality—within the context of primary cell culture research. The findings underscore the critical importance of validating culture conditions for physiologically relevant and therapeutically predictive outcomes, particularly for drug development applications.

Routine antibiotic use can mask underlying aseptic technique issues while introducing unintended experimental variables. Research demonstrates that penicillin-streptomycin (PenStrep) supplementation alters the expression of 209 genes in HepG2 liver cells, including transcription factors and genes involved in drug metabolism and stress response pathways [7]. Furthermore, antibiotics can induce persistent changes in the cellular regulatory landscape, with one study identifying 9,514 genomic regions showing differential H3K27ac enrichment (an active enhancer mark) following PenStrep treatment [7]. These molecular changes manifest in functional alterations, as antibiotic carryover from culture medium can confound subsequent antimicrobial assays, leading to misinterpretation of cellular activities [3]. This note provides structured experimental data and validated protocols to quantify these effects, supporting informed decision-making regarding antibiotic use in primary cell culture systems.

Table 1: Documented Effects of Antibiotic Supplementation on Cellular Parameters

Cell Type / System	Antibiotic Treatment	Proliferation & Viability	Metabolic & Molecular Effects	Morphological & Functional Changes
HepG2 (Liver Cell Line) [7]	Penicillin-Streptomycin (1%)	No significant change in proliferation rate reported.	Differential expression of 209 genes (157 upregulated, 52 downregulated); Altered pathways: PXR/RXR activation, apoptosis, unfolded protein response.	Altered regulatory landscape with 9,514 differential H3K27ac peaks.
Various Mammalian Cell Lines [33]	Gentamicin (10-50 µg/mL)	Can impair membrane function and slow proliferation, especially in sensitive cell types (e.g., stem cells).	Increased production of reactive oxygen species (ROS) and DNA damage in breast cancer cell lines.	Cytotoxic effects at higher doses.
Various Mammalian Cell Lines [33]	Amphotericin B (0.25-2.5 µg/mL)	Higher concentrations may impact viability.	N/D	Cytotoxic effects observed at higher doses.
Cardiomyocytes [3]	Penicillin-Streptomycin	N/D	Altered action and field potential of cardiomyocytes.	N/D
Hippocampal Pyramidal Neurons [3]	Penicillin-Streptomycin	N/D	Altered electrophysiological properties.	N/D
Bacterial Cells (E. coli) [64]	Sub-MIC Antibiotics	Concentration-dependent inhibition of growth rate.	N/D	Concentration-dependent changes in cell volume, surface-to-volume ratio, and aspect ratio.

N/D: Not specifically documented in the provided search results.

Table 2: Antibiotic Working Concentrations and Key Considerations

Antibiotic	Common Stock Concentration	Common Working Concentration	Key Considerations & Potential Effects
Penicillin-Streptomycin (Pen-Strep) [33]	100X (e.g., 10,000 U/mL Pen, 10 mg/mL Strep)	1X (e.g., 100 U/mL Pen, 100 µg/mL Strep)	Alters gene expression [7]; masks contamination; low cytotoxicity at standard concentration.
Gentamicin Sulfate [33]	50 mg/mL	10 – 50 µg/mL	Broad-spectrum; dose-dependent cytotoxicity; can stress sensitive cell lines.
Amphotericin B [33]	250 µg/mL	0.25 – 2.5 µg/mL	Antifungal; light-sensitive; higher doses can harm mammalian cells.
Antibiotic-Antimycotic (e.g., Pen-Strep + Amphotericin B) [33]	100X	1X	Convenient for broad contamination control. See individual component considerations.

Experimental Protocols

Protocol: Assessing Proliferation and Viability in the Presence of Antibiotics

Objective: To quantitatively compare the growth kinetics and viability of primary cells cultured with versus without standard antibiotic supplementation.

Materials:

• Primary cells of interest (e.g., human dermal fibroblasts)
• Complete growth medium with and without antibiotics (e.g., 1x PenStrep)
• Sterile PBS (phosphate-buffered saline)
• Trypsin-EDTA or other dissociation reagent
• Hemocytometer or automated cell counter
• Vital dye (e.g., Trypan Blue)
• 12-well or 24-well cell culture plates
• CO₂ incubator

Method:

• Cell Seeding: Harvest and count the primary cells. Seed triplicate wells of a culture plate at a density of 5 x 10³ to 1 x 10⁴ cells/cm² in complete growth medium with antibiotics. Seed an identical set of triplicate wells in medium without antibiotics.
• Media Conditioning: Twenty-four hours post-seeding, carefully aspirate the media from all wells. Replace the media in the "with antibiotic" wells with fresh complete medium containing antibiotics. Replace the media in the "without antibiotic" wells with fresh antibiotic-free complete medium.
• Daily Sampling & Counting: Every 24 hours for 5-7 days (or until control wells reach confluence):
  • Trypsinize and resuspend cells from one triplicate set for each condition.
  • Mix a small aliquot of the cell suspension with Trypan Blue (e.g., 1:1).
  • Count total and viable cells using a hemocytometer or automated counter.
• Data Analysis: Plot the mean cell number (and viability percentage) for each condition over time to generate growth curves. Calculate population doubling times and compare final cell yields.

Protocol: Metabolic Profiling via Extracellular Flux Analysis

Objective: To evaluate the functional impact of antibiotics on cellular metabolic pathways by measuring mitochondrial respiration and glycolysis.

Materials:

• Primary cells cultured with/without antibiotics for at least 72 hours
• Extracellular Flux Analyzer (e.g., Seahorse XF Analyzer)
• XF Cell Culture Microplates
• XF Assay Medium
• Substrates and inhibitors (e.g., Glucose, Oligomycin, FCCP, Rotenone/Antimycin A)
• Sterile PBS

Method:

• Cell Preparation: Seed XF microplates with cells from both culture conditions (with/without antibiotics) at an optimal density (e.g., 2 x 10⁴ cells/well) 24 hours before the assay. Use at least 5-8 replicate wells per condition.
• Assay Day Preparation:
  • Wash cells twice with XF assay medium and incubate in a non-CO₂ incubator for 1 hour.
  • Load injector ports with compounds for a Mitochondrial Stress Test (e.g., Port A: Oligomycin; Port B: FCCP; Port C: Rotenone/Antimycin A).
• Run Assay: Execute the pre-programmed assay protocol on the analyzer. The instrument will measure the Oxygen Consumption Rate (OCR, indicator of mitochondrial respiration) and Extracellular Acidification Rate (ECAR, indicator of glycolysis) in real-time.
• Data Analysis: Normalize data to cell number/protein content. Compare key metabolic parameters (Basal Respiration, ATP-linked Respiration, Maximal Respiration, Proton Leak, Glycolysis, Glycolytic Capacity) between the two culture conditions using statistical tests.

Protocol: Evaluating Functional Competence – An Example with Antimicrobial Activity

Objective: To determine if observed antimicrobial activity in conditioned medium is a genuine cell-secreted factor or a result of antibiotic carryover.

Materials:

• Conditioned Medium (CM) from test cells cultured with/without antibiotics
• Basal medium (antibiotic-free)
• Sterile PBS
• Indicator bacterial strains (e.g., penicillin-sensitive S. aureus NCTC 6571 and a penicillin-resistant strain)
• Spectrophotometer or colony counting equipment
• Luria-Bertani (LB) agar plates

Method:

• Generate Conditioned Medium: Culture donor cells to 70-80% confluence. Wash cell monolayers thoroughly with sterile PBS (3x) to remove residual antibiotics [3]. Add fresh, antibiotic-free basal medium and incubate for the desired conditioning period (e.g., 72 hours). Collect CM and centrifuge to remove cells/debris.
• Prepare Bacterial Inoculum: Grow indicator bacteria to mid-log phase and dilute to a standard density (~10⁵ CFU/mL) in fresh broth.
• Co-culture Assay: Mix the bacterial inoculum with CM (or basal medium as a negative control) at various dilutions (e.g., 50%, 25%, 12.5%). Incubate the mixtures for 4-6 hours with shaking.
• Assess Bacterial Growth: Measure the optical density (OD₆₀₀) of the cultures. Alternatively, perform serial dilutions and plate on LB agar to enumerate viable bacteria (CFU/mL).
• Interpretation: Genuine cellular antimicrobial activity will inhibit both antibiotic-sensitive and resistant strains. Inhibition that is specific to the antibiotic-sensitive strain and is abolished by pre-washing the cells indicates antibiotic carryover as the cause [3].

Visualizations and Workflows

Diagram 1: Experimental Workflow for Comparative Cell Fitness Analysis

Workflow for Comparative Cell Fitness

Diagram 2: Antibiotic-Induced Cellular Stress and Signaling Pathways

Antibiotic-Induced Signaling Pathways

The Scientist's Toolkit: Research Reagent Solutions

Table 3: Essential Materials and Reagents for Antibiotic Impact Studies

Reagent / Solution	Function / Purpose	Example & Notes
Antibiotic/Antimycotic Solutions	Prevent bacterial and fungal contamination in cell cultures.	Penicillin-Streptomycin (Pen-Strep): Common, broad-spectrum. Gentamicin: Broad-spectrum, stronger against Gram-negatives. Amphotericin B: Antifungal. Use working concentrations to minimize cytotoxicity [33].
Antibiotic-Free Basal Medium	Serves as the base for creating experimental and control media.	Essential for preparing conditioned medium free of antibiotic carryover and for maintaining control cultures [3].
Cell Dissociation Reagents	Detach adherent cells for subculturing or endpoint analysis.	Trypsin-EDTA solution, non-enzymatic cell dissociation buffers. Critical for accurate cell counting during proliferation assays.
Cell Viability Stains	Distinguish between live and dead cells.	Trypan Blue: Used for manual counting with a hemocytometer. Propidium Iodide & Fluorescent-based dyes for flow cytometry.
Extracellular Flux Assay Kits	Measure real-time metabolic function.	Seahorse XF Cell Mito Stress Test Kit, XF Glycolysis Stress Test Kit. Provides direct measurement of OCR and ECAR [7].
Mycoplasma Detection Kits	Detect silent mycoplasma contamination.	PCR-based or luminescence-based detection kits. Cruignant for validating sterile technique in antibiotic-free cultures [33].
Mycoplasma Removal Reagents	Eliminate mycoplasma contamination.	Targeted reagents are required, as standard antibiotics are ineffective against mycoplasma due to its lack of a cell wall [33].

The data and protocols presented herein demonstrate that antibiotic supplementation is a significant experimental variable with measurable consequences for primary cell fitness. The documented alterations in gene expression, metabolic function, and secretory profile confirm that antibiotics extend beyond their intended antimicrobial role to actively influence cell physiology [7] [3]. These effects pose a substantial risk to the translational validity of research findings, particularly in drug development where predictive in vitro models are paramount.

The decision to use antibiotics should be a deliberate one, weighed against the specific research objectives. While their use may be justified for short-term applications, such as stabilizing primary cultures post-thaw or working with high-risk samples, their routine inclusion in long-term cultures or studies focused on metabolism, gene regulation, or functional secretion is not recommended [33]. Robust aseptic technique, regular mycoplasma testing, and the use of antibiotic-free media represent best practices for preserving authentic cellular phenotypes.

In conclusion, researchers must critically evaluate the necessity of antibiotics in their primary cell culture systems. The comparative analysis of cell fitness with versus without antibiotics is not merely a quality control measure but a fundamental step in ensuring the biological relevance and reproducibility of scientific data.

Within primary cell culture research, antibiotic supplementation is a common practice aimed at preventing microbial contamination. However, the physiological media in which cells are cultured can significantly alter the apparent efficacy of these antibiotics, leading to misleading predictions in downstream drug development assays. This case study explores the critical, yet often overlooked, interplay between culture media composition, antibiotic activity, and cellular function. We examine how standard in vitro susceptibility testing can fail to predict treatment outcomes by not accounting for the media's influence on both the antibiotic compound and the target cells or bacteria. Framed within a broader thesis on rational antibiotic use in cell culture, this analysis provides application notes and protocols to help researchers generate more physiologically relevant and predictive data.

Background and Significance

Antimicrobial resistance (AMR) is a global health threat, and the development of new antibiotics is paramount [65]. The initial stages of drug discovery heavily rely on in vitro models to predict antibiotic efficacy. A critical flaw in this pipeline, however, is the disparity between the controlled environment of laboratory media and the complex in vivo conditions of an infection [66].

Physiological media are not inert backgrounds; their composition—ranging from rich nutrient broths to minimal defined salts—directly influences cellular metabolism, stress responses, and gene expression. For instance, the production of hydrogen sulfide (H2S) in bacteria, a mechanism linked to antibiotic tolerance, is strongly dependent on whether cysteine is synthesized by the cell or readily imported from cystine-rich media [67]. Furthermore, the phenomenon of "phenotypic resilience"—including bacterial tolerance, persistence, and heteroresistance—is regulated by environmental signals and can be induced or suppressed by specific media conditions [66]. These factors mean that an antibiotic showing high efficacy in a standard rich medium like LB might demonstrate reduced activity in a more minimal, physiologically relevant medium, and vice-versa.

This gap is exacerbated in cell culture-based research where antibiotics are used as a supplement. A stark example is the "antibiotic carry-over" effect, where residual antibiotics from tissue culture can be released into conditioned medium, creating a false impression of cell-secreted antimicrobial activity [3]. Such effects can severely confound the validation of novel antimicrobial therapies, such as extracellular vesicles (EVs) or cell-secreted products. Therefore, understanding and controlling for the impact of physiological media is not merely a technical detail but a fundamental requirement for improving the predictive power of early-stage antibiotic research.

Key Experimental Findings

Antibiotic Carry-Over as a Confounding Factor

A 2025 study directly demonstrated that the routine inclusion of antibiotics in cell culture can lead to significant confounding results. Researchers investigating the antimicrobial properties of conditioned medium (CM) from various cell lines initially found that the CM exhibited bacteriostatic effects against a penicillin-sensitive strain of Staphylococcus aureus [3].

Key Findings:

• The antimicrobial activity was traced not to cell-secreted factors, but to the retention and release of penicillin from the tissue culture plastic surfaces into the CM [3].
• This carry-over effect was so potent that the antimicrobial activity remained in CM collected after a 72-hour conditioning period in antibiotic-free medium [3].
• The effect was inversely correlated with cellular confluency, suggesting the uncovered plasticware acted as a reservoir for the antibiotic. Crucially, a simple pre-wash step before medium conditioning effectively removed this confounding activity [3].

Implication: This finding highlights a critical artifact that can lead to the false attribution of antimicrobial properties to novel biological therapeutics, potentially misdirecting research efforts.

Media-Dependent Bacterial Physiological Responses

The physiological response of bacteria to antibiotics is not absolute but is heavily modified by the growth medium. Research on Escherichia coli exposed to ciprofloxacin showed dramatically different outcomes in different media [67].

Quantitative Data:

Physiological Parameter	Minimal M9 Medium	Rich LB Medium
Optimal Bactericidal Concentration (OBC) of Ciprofloxacin	0.3 µg/mL	3 µg/mL
Bacterial Survival (CFU)	2-3 orders of magnitude higher	Lower
Metabolic Activity at High Ciprofloxacin	Not retained	Retained at higher concentrations
H2S Production	Transient, induced by antibiotics	Constitutive, due to cystine content

Implication: The same bacterial strain can appear tolerant or susceptible based solely on the culture medium used for testing. Rich media like LB may mask true bactericidal activity and alter metabolic responses to stress, leading to inaccurate predictions of drug efficacy [67].

Off-Target Effects on Eukaryotic Cells

The common practice of using antibiotic cocktails like penicillin-streptomycin (Pen-Strep) in cell culture media can directly impact the physiology of the host cells under study, thereby indirectly affecting antibiotic efficacy predictions.

Impact of Streptomycin on C2C12 Myotubes [19]:

• Protein Synthesis: ~40% reduction in global protein synthesis rates.
• Morphology: ~40% reduction in myotube diameter, with a 25% lower differentiation rate and a 60% lower fusion index.
• Mitochondrial Health: Fragmentation of the mitochondrial network, a 64% smaller mitochondrial footprint, and reduced content of mitochondrial complex I subunits.

These off-target effects demonstrate that antibiotics in culture media can alter fundamental cellular processes, including differentiation and metabolism, which are critical endpoints in many drug discovery assays [33] [19]. This can be particularly detrimental when studying primary cells, stem cells, or conducting high-content phenotypic screenings.

Application Notes

Interpreting Susceptibility Data in Context

Researchers must recognize that Minimum Inhibitory Concentration (MIC) values are not intrinsic properties of a bacterium-antibiotic pair but are context-dependent. An MIC determined in a standard rich medium may not predict efficacy in a more physiologically relevant environment or at an infection site where nutrients are limited [67] [66]. Susceptibility testing should be interpreted with the media context in mind, and where possible, assays should be designed to mimic the target physiological environment.

Recommendations for Primary Cell Culture

The following guidelines are proposed for the use of antibiotics in primary cell culture within a research setting:

• Avoid Routine Use: For long-term maintenance of established, clean cultures, avoid antibiotics to prevent masked contamination and off-target effects on cell biology [33].
• Limit to Specific Scenarios: The use of antibiotics can be justified during primary cell isolation, thawing of frozen stocks, or when working in shared incubator spaces with a high risk of contamination [33].
• Implement a "Wash-Out" Period: When conditioning medium for downstream analysis (e.g., EV isolation), include a pre-wash step and use a sufficient period of culture in antibiotic-free medium to eliminate carry-over effects [3].
• Validate Critical Assays: For experiments investigating antimicrobial activity, gene expression, cellular differentiation, or metabolism, an antibiotic-free culture condition is strongly recommended as a control to rule out confounding effects [33] [19].

Detailed Protocols

Protocol 1: Mitigating Antibiotic Carry-Over in Conditioned Medium Collection

Objective: To collect cell-conditioned medium (CM) for antimicrobial or extracellular vesicle studies without confounding effects from residual antibiotics.

Materials:

• Primary cells of interest
• Standard culture medium with antibiotics (e.g., Pen-Strep)
• Antibiotic-free basal medium (BM-)
• Sterile phosphate-buffered saline (PBS)
• Tissue culture plasticware

Method:

• Culture Cells: Grow primary cells to 70-80% confluency in standard medium containing antibiotics.
• Pre-Wash Monolayer: Aspirate the antibiotic-containing medium. Gently wash the cell monolayer with sterile PBS. Aspirate and repeat this wash step two more times for a total of three washes.
• Collect Wash Eluate (Optional Control): Retain the PBS from the final wash if a control for carry-over is needed.
• Condition Medium: Add antibiotic-free basal medium (BM-) to the washed cells.
• Incubate: Culture the cells for the desired conditioning period (e.g., 24-72 hours) in a 37°C, 5% CO₂ incubator.
• Collect CM: Harvest the conditioned medium and centrifuge to remove cells and debris (e.g., 300 × g for 10 min). Aliquot and store at -80°C.

Validation: The collected CM should be tested against antibiotic-resistant bacterial strains as a negative control. No growth inhibition should be observed, confirming the absence of functional antibiotic carry-over [3].

Protocol 2: Assessing Antibiotic Efficacy in Physiologically Relevant Media

Objective: To determine the bactericidal activity of an antibiotic compound against a target pathogen in different physiological media.

Materials:

• Bacterial pathogen (e.g., E. coli)
• Test media (e.g., Rich LB, Minimal M9, M9 with casamino acids and cystine)
• Antibiotic stock solution (e.g., Ciprofloxacin)
• Sterile flasks and culture tubes

Method:

• Prepare Inoculum: Grow bacteria overnight in a standard medium. Dilute in fresh test media to an OD₆₀₀ of ~0.05.
• Grow to Mid-Log Phase: Incubate the cultures with shaking at 37°C until they reach mid-exponential phase (OD₆₀₀ ~0.4).
• Add Antibiotic: Add the antibiotic at a range of concentrations (e.g., from 0.1× to 10× the expected MIC) to the cultures. Include a no-antibiotic control.
• Incubate and Sample: Continue incubation for a set period (e.g., 2-4 hours). Sample at time zero and various time points post-addition.
• Determine Viability: Serially dilute samples and plate on non-selective agar to determine colony-forming units (CFU/mL).
• Monitor Physiology (Optional): Use sensors to monitor dissolved oxygen, pH, or H₂S production in parallel cultures [67].

Analysis: Plot kill curves (CFU/mL vs. time) for each antibiotic concentration in the different media. Compare the optimal bactericidal concentration (OBC) and the rate of killing across media types [67].

Protocol 3: Evaluating Off-Target Effects of Antibiotics on Primary Cell Phenotype

Objective: To assess the impact of culture-grade antibiotics on the differentiation capacity and health of primary cells.

Materials:

• Primary cells (e.g., myoblasts, stem cells)
• Growth and differentiation media
• Antibiotic stocks (e.g., Penicillin, Streptomycin, Gentamicin)
• Assay kits for protein synthesis, ATP levels, etc.

Method:

• Culture Setup: Split primary cells into multiple culture vessels.
• Apply Conditions: Maintain parallel cultures in:
  • Control: Antibiotic-free medium.
  • Test Groups: Medium supplemented with standard working concentrations of antibiotics (e.g., Pen-Strep, Gentamicin alone).
• Induce Differentiation: Once cells reach confluence, switch to differentiation-specific medium, maintaining the same antibiotic conditions.
• Monitor Differentiation: Track morphological changes daily under a microscope.
• Quantify Endpoints:
  • Fusion Index: After fixation and immunostaining for cell-specific markers (e.g., Myosin Heavy Chain for myotubes), calculate the percentage of nuclei within multinucleated cells [19].
  • Protein Synthesis: Use a non-radioactive assay (e.g., SUnSET) to measure global protein synthesis rates.
  • Metabolic Function: Measure ATP levels or mitochondrial respiration using a Seahorse Analyzer.

Analysis: Compare the differentiation efficiency, protein synthesis rates, and metabolic parameters of antibiotic-treated cells to the antibiotic-free control. A significant reduction indicates a deleterious off-target effect [19].

The Scientist's Toolkit: Research Reagent Solutions

The following table details key reagents and their considerations for the featured experiments.

Research Reagent	Function	Key Considerations & Rationale
Penicillin-Streptomycin (Pen-Strep)	Broad-spectrum antibiotic combo for preventing bacterial contamination.	Can alter gene expression in host cells; penicillin component is a common source of carry-over. Use for short-term protection, not long-term culture [33].
Gentamicin Sulfate	Broad-spectrum aminoglycoside antibiotic.	Offers good Gram-negative coverage but has dose-dependent cytotoxicity on sensitive cell types [33].
Amphotericin B	Antifungal agent.	Higher doses can harm mammalian cells. It is light-sensitive and requires careful handling and storage [33].
Antibiotic-Antimycotic (100X)	Pre-mixed solution of Pen-Strep and Amphotericin B.	Convenient for short-term protection against fungi and bacteria. Does not protect against mycoplasma [33].
Mycoplasma Removal Reagent	Targeted reagent for eliminating mycoplasma.	Essential for eradicating a common, silent contaminant unaffected by standard antibiotics. Requires a dedicated treatment protocol [33].
Defined Minimal Media (e.g., M9)	For assessing antibiotic efficacy under nutrient-restricted conditions.	Promotes physiological stress responses and can reveal tolerance mechanisms not seen in rich media [67].
Rich Media (e.g., LB Broth)	For standard bacterial propagation and high-yield protein production.	May mask true antibiotic lethality and alter metabolic responses to drugs, leading to over-optimistic efficacy predictions [67].

Visualizations

Antibiotic Carry-Over Confounds Antimicrobial Assays

Media Dictates Bacterial Response to Antibiotics

This case study underscores that physiological media are active determinants of antibiotic efficacy, not passive backdrops. The documented phenomena of antibiotic carry-over, media-dependent bacterial responses, and off-target effects on eukaryotic cells collectively present a significant risk to the predictive validity of in vitro models. For researchers in drug development, acknowledging and controlling for these factors is crucial. By adopting the recommended protocols—such as implementing wash-out steps, testing efficacy in multiple media types, and validating key assays in antibiotic-free conditions—scientists can generate more robust, reliable, and physiologically relevant data. This rigorous approach will enhance the translation of promising antibiotic candidates from cell culture to the clinic, ultimately contributing to the global fight against antimicrobial resistance.

Framework for Authenticating Cell Lines and Confirming Phenotypic Stability

The integrity of biomedical research hinges on the use of authentic and stable biological tools. Within the context of primary cell culture research, where antibiotic supplementation remains a common practice, ensuring cell line identity and phenotypic stability becomes paramount. Cell line misidentification and cross-contamination pose significant threats to scientific reproducibility, with studies indicating that at least 5% of human cell lines used in manuscripts are misidentified [68]. Furthermore, the customary use of antibiotic supplements in mammalian cell cultures introduces confounding variables, including cytotoxic effects and alterations in biological patterns that can compromise phenotypic stability [4]. Recent investigations have demonstrated that residual antibiotics from culture media can be carried over into experimental systems, potentially leading to misleading conclusions about antimicrobial properties of cell-secreted factors [3]. This application note establishes a comprehensive framework for authenticating cell lines and confirming phenotypic stability, with particular emphasis on addressing challenges amplified by antibiotic supplementation regimes. By implementing standardized authentication protocols and continuous monitoring strategies, researchers can significantly enhance the reliability of cell-based data in basic research and drug development applications.

The Problem: Cell Line Misidentification and Instability

Prevalence and Impact of Misidentification

The scale of cell line misidentification in biomedical research is substantial, with far-reaching scientific and economic consequences. Analyses of manuscripts considered for peer review reveal that approximately 5% of human cell lines are misidentified, leading to approximately 4% of manuscripts being rejected due to severe cell line problems [68]. A retrospective analysis of leukemia-lymphoma cell lines received by the German Collection of Microorganisms and Cell Culture (DSMZ) demonstrated a concerning cross-contamination prevalence of 14-18% for cell lines obtained from secondary sources, indicating approximately one in six shared cell lines is misidentified [68]. The financial impact is equally staggering, with estimates suggesting roughly $990 million were spent to publish 9,894 manuscripts using just two known HeLa-contaminated cell lines (HEp-2 and Intestine 407) [68]. With the International Cell Line Authentication Committee (ICLAC) register currently listing 576 misidentified cell lines, the cumulative economic damage likely amounts to billions of research dollars [68].

Antibiotic Supplementation as a Confounding Factor

The routine use of antibiotic supplements in cell culture systems introduces critical variables that can compromise phenotypic stability and experimental outcomes:

• Cytotoxic and Cytostatic Effects: Customary antibiotic supplements exhibit cytotoxic and cytostatic activity at standard concentrations, altering biological patterns of cultured mammalian cells [4].
• Carry-Over Effects: Recent studies demonstrate that residual antibiotics persist in conditioned media and can bind to tissue culture plastic surfaces, leading to potentially misleading conclusions about antimicrobial properties of cell-secreted factors [3].
• Transcriptomic Alterations: Penicillin-streptomycin supplementation has been shown to alter the expression of 209 genes in HepG2 cells, including transcription factors, suggesting widespread effects on multiple pathways [3].
• Functional Impairment: Antibiotic supplements alter the action potential of cardiomyocytes and electrophysiological properties of hippocampal pyramidal neurons, indicating profound functional consequences [3].

Table 1: Common Antibiotic Supplements and Their Potential Impacts on Cell Cultures

Antibiotic	Standard Concentration	Class	Reported Effects on Cells
Penicillin-Streptomycin	100 U/mL-100 µg/mL	β-lactam & Aminoglycoside	Alters electrophysiological properties, transcriptome changes
Gentamicin	50 µg/mL	Aminoglycoside	Increases ROS production and DNA damage
Ampicillin	100 µg/mL	β-lactam	Cytostatic effects at high concentrations
Amphotericin B	2.5 µg/mL	Antifungal	Alters membrane properties

Authentication Framework: Principles and Methodologies

Short Tandem Repeat (STR) Profiling Protocol

STR profiling represents the international reference standard for human cell line authentication. The following protocol is adapted from the American National Standards Institute standard (ANSI/ATCC ASN-0002-2021) [68]:

Materials and Reagents:

• DNA extraction kit (commercial systems recommended)
• STR amplification kit (commercially available systems)
• Capillary electrophoresis system
• Cellosaurus database access (for reference STR profiles)
• Quality control DNA samples

Procedure:

• DNA Extraction: Harvest approximately 10⁶ cells and extract genomic DNA using a validated method. Assess DNA quality and quantity using spectrophotometry or fluorometry.
• PCR Amplification: Amplify 8-17 core STR loci using commercial STR profiling kits. Include positive and negative controls in each run.
• Capillary Electrophoresis: Separate PCR products using capillary electrophoresis according to manufacturer specifications.
• Data Analysis: Use specialized software to convert raw data into allele calls at each locus.
• Interpretation: Compare the resulting STR profile to reference databases:
  • Match: ≥80% match with reference profile indicates authentication
  • Mismatch: <80% match suggests misidentification
  • Mixed Profile: Multiple peaks at multiple loci indicate contamination

• Documentation: Record all quality metrics, including peak height ratios, signal strength, and inter-locus balance.

Frequency Guidelines:

• Upon cell line acquisition
• When expanding from frozen stocks
• Every 3 months for continuous cultures
• Before and after generating experimental data
• Prior to publication or deposition

The Cellosaurus database serves as a comprehensive knowledge resource documenting over 102,000 human cell lines and provides STR profiles for more than 8,000 distinct human cell lines [68]. The database includes critical information on misidentified, partly contaminated, and misclassified cell lines, with unique Research Resource Identifiers (RRIDs) assigned to all cell lines [68]. The Cellosaurus STR similarity search tool (CLASTR) enables researchers to compare obtained STR profiles with those available in the database, facilitating authentication [68]. The ICLAC register of misidentified cell lines provides an essential resource for checking known problematic cell lines, currently listing 576 misidentified cell lines, including 531 with no known authentic stock [68].

Assessing Phenotypic Stability in Antibiotic-Supplemented Cultures

Morphological and Growth Kinetics Assessment

Regular monitoring of phenotypic stability is essential, particularly in antibiotic-supplemented cultures where sublethal effects may influence cellular characteristics:

Morphological Documentation:

• Capture high-resolution phase-contrast images at each passage
• Document cell size, shape, granularity, and organization
• Note any deviations from established morphological characteristics

Growth Kinetics Analysis:

• Seed cells at standardized density (e.g., 10⁴ cells/cm²)
• Perform daily cell counts using automated or manual methods
• Calculate population doubling time using the formula: Doubling time = (t × ln2) / ln(N₂/N₁) Where t is time in hours, N₁ is initial cell number, N₂ is final cell number
• Compare growth parameters against baseline established without antibiotics

Functional Stability Assessment: For specialized cell types, include functional assessments relevant to the research context:

• Neuronal cultures: Patch-clamp recordings to confirm excitable properties [47]
• Epithelial cells: Transepithelial electrical resistance measurements
• Secretory cells: ELISA-based quantification of specific secreted factors

Protocol for Antibiotic Carry-Over Testing

The following protocol addresses the confounding effects of antibiotic carry-over in conditioned media collection:

Materials:

• Antibiotic-free basal medium
• Sterile PBS
• Sensitive bacterial strain (e.g., penicillin-sensitive S. aureus NCTC 6571)
• Resistant control strain (e.g., S. aureus 1061 A)
• Tissue culture vessels identical to those used for experiments

Procedure:

• Culture cells in antibiotic-containing medium following standard protocols
• Prior to conditioned media collection, wash cell monolayers thoroughly (minimum 3× with PBS)
• Collect wash solutions and retain for testing
• Add antibiotic-free medium for conditioned media collection
• Test both wash solutions and conditioned media for antimicrobial activity:
  • Prepare dilutions of test solutions (50% to 6.25%) in bacterial culture medium
  • Inoculate with standardized bacterial suspension
  • Monitor bacterial growth via optical density for 24 hours
  • Compare growth inhibition between test and control media

• Interpret results:
  • Antimicrobial activity in wash solutions indicates antibiotic carry-over
  • Activity in conditioned media from pre-washed cells suggests genuine antimicrobial properties
  • Differential effects on sensitive vs. resistant strains confirm antibiotic-specific activity

Integrated Workflow for Continuous Authentication and Stability Monitoring

The following diagram illustrates the comprehensive framework for maintaining authenticated and phenotypically stable cell cultures:

Table 2: Key Research Reagent Solutions for Cell Authentication and Phenotypic Assessment

Reagent/Resource	Function	Application Notes
STR Profiling Kit	DNA fingerprinting for authentication	Select kits with 8-17 core loci; ensure compatibility with analysis software
Cellosaurus Database	Reference STR profiles	Access online for comparison of obtained STR profiles [68]
CultureOne Supplement	Serum-free defined supplement	Controls astrocyte expansion in neuronal cultures without antibiotics [47]
Penicillin-Streptomycin	Antibiotic combination	Standard concentration: 100 U/mL penicillin, 100 µg/mL streptomycin; pH and temperature sensitive [4]
Collagenase IV / Hyaluronidase	Tissue dissociation enzymes	Enzyme combination for primary cell isolation; used at 1.6 mg/mL and 0.14 mg/mL respectively [48]
B-27 Plus Supplement	Serum-free neuronal culture	Supports neuronal differentiation and survival in antibiotic-free conditions [47]
BSA Solution	Protein carrier in enzymatic digestion	Used at 2 mg/mL in primary fibroblast isolation protocols [48]

The framework presented herein provides researchers with a comprehensive strategy for authenticating cell lines and confirming phenotypic stability within the context of antibiotic supplementation in primary cell culture research. Implementation of regular STR profiling, coupled with systematic phenotypic monitoring and antibiotic carry-over testing, addresses critical vulnerabilities in contemporary cell culture practices. As the scientific community moves toward enhanced reproducibility, such standardized approaches will prove indispensable for generating reliable, translatable data in both basic research and drug development applications. By prioritizing cell line integrity through these verification processes, researchers can substantially strengthen the foundation upon which scientific discoveries are built.

Conclusion

The integration of antibiotic supplements in primary cell culture demands a critical, evidence-based reassessment. While offering a practical solution for contamination control, their documented effects—from altering fundamental electrophysiological properties and global gene expression to confounding downstream antimicrobial assays—pose a significant threat to experimental validity and reproducibility. The key takeaway is a paradigm shift towards minimizing or eliminating routine antibiotic use, reinforced by rigorous aseptic technique. Future directions should prioritize the standardization of antibiotic-free protocols, a deeper investigation into the long-term functional consequences of antibiotic exposure on primary cell phenotypes, and the development of more physiologically relevant culture media that improve the predictive accuracy of in vitro models for clinical outcomes. Embracing this cautious approach is paramount for enhancing the reliability and translational impact of biomedical research.

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