When to Use Antibiotics in Mammalian Cell Culture

by Simon Currie

Antibiotics are powerful tools that protect cell culture from contamination and help select for transfected cells. However, antibiotics can also have subtle impacts on resistant cells, affecting their gene expression, metabolism, and differentiation. So, while antibiotic use is widespread and important in many contexts, there are a few scenarios where you may want to avoid them.

Antibiotics and cell selection agents are used in cell culture to kill, or stall, bacterial cells and sensitive mammalian cells. However, there are times when it is better not to used them because they can have impacts on even the resistant cells that they don’t kill.

In this article, we’ll discuss when and why to use antibiotics and cell selection agents in mammalian cell culture, and when you might consider skipping them.

Antibiotics in mammalian cell culture – main effect and side effects

Antibiotics are used to prevent bacteria from growing in mammalian cell culture. Sometimes, they are also used as selection agents, to select for cells that have been transformed during stable cell line generation or a genetic screen. In all of these situations, the intended main effect of antibiotics is killing the undesired cells that are not the focus of your study.

The microscopic world is like our macroscopic lives in that there are often unintended side effects. In the case of antibiotics, this side effect is that they can impact and change the resistant cells that they don’t kill. For example, antibiotics can change the gene expression, metabolism, cell state, or even function of cells (Figure 1) (Cohen et al, 2006; Elfar et al, 2025; Elliot and Jiang, 2019; Nygaard et al, 2015; Ryu et al, 2017; Varghese et al, 2017).

Resistant vs. sensitive cells in antibiotics illustration

Figure 1. While resistant cells live and grow in antibiotics, they may have more subtle side effects such as changes in gene expression, metabolism, growth rates, differentiation, etc.

So, in most cases there is a clear benefit to using antibiotics, which is preventing infection, but they may also change the cells that you’re trying to study, which is a drawback. So how do you decide when to use antibiotics and when to avoid them?

When to use antibiotics in mammalian cell culture

Typically, antibiotics are considered a non-negotiable when cell lines are very valuable (you can’t afford to lose them) or when cell lines are particularly vulnerable. These situations include:

· Primary cell culture

· Thawing frozen cells

· Suspected contamination

· Cell selection

Primary cell culture

Primary cell cultures are cells that were recently isolated from the biological source. For example, they could be breast cancer cells that were recently biopsied or surgically excised from a patient for culture. These samples are rare and important because they are often better mimics of cancer than established breast cancer cell lines, many of which have been passaged for decades.

That’s why antibiotics are typically included in early passages until enough cells have been grown up to bank aliquots of the primary cells. In this context, “bank” is the informal term used to describe freezing aliquots of the primary cell culture for later use (Figure 2). Passage is the process of transferring a portion of cells into a new culture dish to grow more of the cells. Typically, primary cells are passaged one to five times before banking. There is a balance here, as fewer passages better retain fundamental characteristics of the primary cells, but more passages generate more cells enabling more downstream experiments.

Figure 2. New cell cultures are usually banked at an early passage to provide fresh, uncontaminated aliquots for future studies.

When thawing individual aliquots, you would be more willing to wean the cells off of antibiotics because if that single culture gets contaminated you could always go back and get another fresh aliquot out and start over. You can see why the initial primary culture had antibiotics though, because if that culture got contaminated then all of your banked aliquots would be compromised.

Thawing frozen cells

Antibiotics are usually still included when initially thawing an aliquot of cells, regardless of whether they are primary cells or regular cell lines.

Cell cultures are particularly vulnerable to contamination during the thaw process.

During the thawing process, there are additional touch points that can serve as sources of contamination such as the freezer that cell aliquots are stored in, the water bath they’re thawed in, etc. (Fountain et al, 1997).

You know how some people just take a little bit longer to wake up in the morning? Thawed cells are kind of like that. It takes them a bit to readjust to their new temperature, container, and culture media. Usually after a lag period of approximately 24 to 72 hours, cells are back to growing at their normal pace. Actively growing cells take up more nutrients that stalled cells, so this early lag phase presents an opportunity for contaminating cells to outcompete your intended cells for resources in the media.

Some cells in the culture will lyse during the freeze-thaw process, particularly if a proper cryoprotectant isn’t used (Baust et al, 2022). Those lysed cells are part of what contributes to the lag phase after thawing, and their lysed contents are thought to provide even more nutrients for contaminating cells to thrive on.

After the initial lag phase, once your cells are up and growing and are past these risks, then you could try removing antibiotics from the culture.

Suspected contamination

If you suspect contamination in your cell culture, you can always try adding antibiotics back in to get it under control. In reality, this may be more effort than it is worth depending on how precious the cells are.

If you have additional aliquots of the same cells banked, it is probably less work to trash the current, contaminated cells and start with a fresh aliquot.

If the sample is particularly valuable, or you’re down to your last few aliquots you can try to tamp down the contamination with antibiotics and continue with your experiment. Which antibiotic you would use in this scenario would depend on what you suspect that contaminating cells are (Table 1).

Table 1. Matching antibiotics to suspected contamination source.

Antibiotic	Use
Penicillin + Streptomycin (PenStrep)	Gram-positive and gram-negative bacteria
Gentamicin	Gram-negative bacteria, often used when PenStrep isn’t sufficient
Amphotericin B	Fungal and yeast contamination
Ciprofloxacin	Mycoplasma
Tetracycline or Doxycycline	Mycoplasma – resistance and variable efficacy are limitations

However, if you’re comparing the results of these cells with other cells that were not contaminated then you’ll have to keep in mind that there could be some pretty substantial differences due to the contamination and subsequent antibiotic treatment. That’s why it is usually advisable to start with a fresh aliquot of cells if possible.

Cell selection

Antibiotics such as puromycin, blasticidin, hygromycin, and G418 are used as cell selection agents to retain cells that have been transformed while killing off untransformed cells. In contexts like stable cell line generation or a genetic screen, cells that have been transformed will have a resistance gene that allows them to grow in the presence of antibiotics. Untransformed cells lack the resistance gene and will die or struggle to grow (Figure 1).

Antibiotics are required for these types of experiments. If you skip the cell selection agent, you will not have any selection and will end up with a mixture of cells in your stable cell line or in your genetic screen. Basically, the whole effort is fundamentally a lost cause without selection.

When to skip antibiotics in mammalian cell culture

Given the side effects that antibiotics can impose on resistant cells, it will sometimes benefit your experiments to skip antibiotics, as long as you can keep your culture from getting contaminated. These situations include:

· Sensitive cell lines like stem cells

· Assays like gene expression or metabolism studies

· Long-term maintenance

· Potential for mycoplasma contamination

Sensitive cell lines

The primary case of antibiotics influencing sensitive cell lines is stem cells. Stem cells are frequently differentiated into different lineages of adult cell types by introducing specific growth factors, small molecules, or through specific culturing techniques.

Antibiotics can negatively impact the ability of stem cells to differentiate into different types of adult cells. For example, gentamicin or penicillin plus streptomycin (PenStrep) reduced the differentiation of stem cells into lung cells, liver cells, and neurons (Cohen et al, 2006; Varghese et al, 2017).

So, if you’re performing any cell culture experiments involving differentiation, it is probably worth testing out if antibiotics are having an impact on the cells during your procedure.

Gene expression or metabolism assays

Antibiotics can change the gene expression or metabolism of mammalian cells (Elliott and Jiang, 2019; Ryu et al, 2017). So, if you are studying gene expression or metabolism using mammalian cell culture, you have a couple of options: you could either not use antibiotics in the culture, or you need to control for the antibiotics to make sure that the effect you’re observing isn’t just a cellular response to antibiotics.

Skipping antibiotics is probably the simplest way of accomplishing this. If your cells have been cultured long-term in the absence of antibiotics, then you should be good to go.

If your cells require antibiotics, for whatever reason, then you need to make sure that you have a control condition where the cells are exposed to antibiotics, but not the experimental variable that you’re trying to investigate.

For example, if you’re overexpressing “Gene X” to understand it’s impact on metabolism, then you’ll need to have a negative control where the cells have been exposed to antibiotics but do not have Gene X overexpressed (Figure 3). Without this control you may erroneously attribute effects due to the cellular response to antibiotics to the biological variable that you’re trying to study.

Figure 3. It is important to include an antibiotic-only control to ensure that the parameter that you’re measuring isn’t just a cellular response to the antibiotics. In this case, you wouldn’t be interested in the changes in metabolites 2 and 5 since they also occur in the antibiotic-only control, and would instead focus your efforts on metabolites 1, 3, and 4.

If your cells need antibiotics, a middle ground solution is to culture your cells with antibiotics, but wash them out shortly (a day or a few days) before you run your assay. If you choose this route, again make sure to include a control that goes through the same culturing routine without the experimental perturbation.

Long-term maintenance

Long-term culturing in antibiotics poses risks for cell culture, even with cells that tolerate antibiotics well.

One main risk is that bacteria or other contaminants will develop resistance over time and then take over the culture once the antibiotic doesn’t impact them anymore.

Another risk is related to the gene expression and metabolism assays we discussed above. Cells are malleable or have a certain plasticity in that they can change their gene expression, metabolism, etc. to help them adapt to changing conditions, such as the introduction of an antibiotic. But with chronic exposure, cells may lose their plasticity and become dependent on the antibiotic in a sense. Meaning the cells may be so entrenched in that state that if the antibiotics are withdrawn, the cells may struggle to revert back to their original state.

In general, this is one advantage for primary culture over established cell lines. After cells have been living for months, years, or even decades in plastic dishes, they are in many ways quite different than when they lived in their original 3D context of a lung, brain, or wherever the cells came from.

Potential for mycoplasma contamination

At first glance this might seem a little bit backwards. If you have contamination, wouldn’t you want to use antibiotics to try to combat it?

The issue is antibiotics tend to work weakly, if at all, against mycoplasma. So, if you’re harboring mycoplasma contamination, antibiotics may keep them from exploding like a wildfire, but this would give you the false sense that your culture isn’t contaminated. Ultimately, your experiment is reading out results from both your cells of interest and the contaminating mycoplasma.

By skipping antibiotics, you allow the mycoplasma to grow like crazy, if they’re there, at which point you realize your culture is compromised and you can switch to using uncontaminated cells before wasting your experiment on bad cells.

The importance of proper aseptic technique

Regardless of antibiotic usage, proper aseptic technique is critical when working with cell cultures. Aseptic technique refers to using sterile personal protective equipment (gloves, goggles, lab coats, and sometimes masks), establishing and maintaining sterile working areas, and using sterile lab equipment.

If your aseptic technique is lacking, then your cultures will quickly become contaminated.

Most cell culture rooms are shared spaces. So, despite good personal aseptic technique, contamination may still occur. Rather than fault-finding, if contamination is rampant in your lab, you will want to make sure that your aseptic technique is on point and that you’re still using antibiotics whenever possible.

Consider your context

The pros and cons for using antibiotics in this article are suggestions for your consideration. They are not necessarily hard and fast rules (Table 2). You will, of course, need to also consider the environment within which you work.

Table 2. When to use, or skip, antibiotics in mammalian cell culture.

Mammalian Cell Culture
Use Antibiotics	Skip Antibiotics
Primary or other valuable, irreplaceable cells	Sensitive cell lines like stem cells
Thawing cell aliquots	Long-term maintenance
Messy shared space with frequent contamination	Clean working space without contamination
Cell selection assays	Assays such as gene expression or metabolism

If you are new to cell culture or your cell culture room is a shared space with frequent and wide-spread contamination issues, then I would certainly lean towards using antibiotics for any fringe cases.

Alternatively, if you’re experienced with cell culture, it’s been years since the last contamination in your lab, and there’s no new scientists starting cell culture work recently, then you likely can be a little more aggressive in skipping antibiotics in your mammalian cultures.

When your mammalian cell culture does warrant them, GoldBio is your go-to source with reliable and affordable antibiotics and cell selection agents. You can check out the antibiotics we carry by looking at the tabs below or by searching on our website. Additionally, check out the related resources below if you’re interested in learning more about antibiotics and cell selection agents.

References

Baust, J. M., Snyder, K. K., Van Buskirk, R. G., & Baust, J. G. (2022). Assessment of the Impact of Post-Thaw Stress Pathway Modulation on Cell Recovery following Cryopreservation in a Hematopoietic Progenitor Cell Model. Cells, 11(2), 278. https://doi.org/10.3390/cells11020278

Cohen, S., Samadikuchaksaraei, A., Polak, J. M., & Bishop, A. E. (2006). Antibiotics reduce the growth rate and differentiation of embryonic stem cell cultures. Tissue engineering, 12(7), 2025–2030. https://doi.org/10.1089/ten.2006.12.2025

Elfar, M. Y., Brown, H. L., Clayton, A., & Stephens, P. (2025). Antibiotic carry over is a confounding factor for cell-based antimicrobial research applications.Scientific reports https://doi.org/10.1038/s41598-025-14186-7

Elliott, R. L., & Jiang, X. P. (2019). The adverse effect of gentamicin on cell metabolism in three cultured mammary cell lines: "Are cell culture data skewed?". PloS one, 14(4), e0214586.https://doi.org/10.1371/journal.pone.0214586

Fountain, D., Ralston, M., Higgins, N., Gorlin, J. B., Uhl, L., Wheeler, C., Antin, J. H., Churchill, W. H., & Benjamin, R. J. (1997). Liquid nitrogen freezers: a potential source of microbial contamination of hematopoietic stem cell components. Transfusion, 37(6), 585–591. https://doi.org/10.1046/j.1537-2995.1997.37697335152.x

Gautier-Bouchardon A. V. (2018). Antimicrobial Resistance in Mycoplasmaspp. Microbiology spectrum, 6(4), 10.1128/microbiolspec.arba-0030-2018.https://doi.org/10.1128/microbiolspec.ARBA-0030-2018

Nygaard, U. H., Niehues, H., Rikken, G., Rodijk-Olthuis, D., Schalkwijk, J., & van den Bogaard, E. H. (2015). Antibiotics in cell culture: friend or foe? Suppression of keratinocyte growth and differentiation in monolayer cultures and 3D skin models. Experimental dermatology, 24(12), 964–965. https://doi.org/10.1111/exd.12834

Ryu, A. H., Eckalbar, W. L., Kreimer, A., Yosef, N., & Ahituv, N. (2017). Use antibiotics in cell culture with caution: genome-wide identification of antibiotic-induced changes in gene expression and regulation. Scientific reports, 7(1), 7533. https://doi.org/10.1038/s41598-017-07757-w

Varghese, D. S., Parween, S., Ardah, M. T., Emerald, B. S., & Ansari, S. A. (2017). Effects of Aminoglycoside Antibiotics on Human Embryonic Stem Cell Viability during Differentiation In Vitro. Stem cells international, 2017, 2451927. https://doi.org/10.1155/2017/2451927

When to Use Antibiotics in Mammalian Cell Culture

In this article:

Antibiotics in mammalian cell culture – main effect and side effects