Common Side Effects of Antibiotics in Mammalian Cell Culture
by Simon Currie
Antibiotics and cell selection agents are used to select for specific cell populations to, for instance, generate a stable cell line or conduct a genetic screen. The most overt phenotype of antibiotics, killing cells or stalling their growth, is their intended use. However, antibiotics can also have more subtle impacts on cells that can complicate your experiment’s results.
Antibiotics can have subtle impacts on the resistant surviving cells in mammalian cell culture such as changing gene expression, metabolism, and more. It’s important to include negative controls to disentangle confounding biological signals.
Article Table of Contents
Side effects of antibiotics in mammalian culture
Controlling for antibiotic side effects
Side effects of antibiotics in mammalian culture
The main purpose for antibiotics and cell selection agents in mammalian culture is to kill (or stall) bacterial cells and untransformed mammalian cells. So, from a big-picture perspective we often think of the outcome as binary; contaminating and untransformed cells are killed while transformed cells live and grow unperturbed.
However, the picture is a little more nuanced than this. The cells that live on can be impacted in ways that are less obvious than blatant cell death. Antibiotics can impact the surviving cells by changing their:
· Gene expression
· Metabolism
· Differentiation
Changes in gene expression
One way antibiotics can impact mammalian cells is by changing their gene expression. A notable example of this is a study that looked at the impact of penicillin and streptomycin (PenStrep) on the gene expression of HepG2 cells, a frequently used human liver cancer cell line. They identified over 200 genes whose expression was changed by PenStrep treatment (Ryu et al, 2017).
Why would this matter? Well, let’s say you were using PenStrep to overexpress your gene of interest. If you don’t have an antibiotic-only control, then you would incorrectly conclude that the 200 genes with expression changes were due to the change in your gene of interest, and not simply due to the antibiotic treatment. In this example, a negative control would be an “empty plasmid” that does not contain your gene of interest, but does contain the selection marker to enable the negative control cells to grow in the presence of antibiotic (Figure 1).
Figure 1. In the case of overexpressing Gene X from a plasmid (left), the negative control would be expressing an “empty plasmid” that still possesses the selection marker (SM) but doesn’t have Gene X (right). This will allow the cells to grow in the presence of the antibiotic.
If you did include an antibiotic-only control, which in this case would be HepG2 cells treated with PenStrep while your gene of interest is unperturbed, then you would know to discard the ~ 200 genes whose change is due to antibiotic treatment and have nothing to do with your gene of interest (Figure 2).
Figure 2. The Venn diagram on the right compares how many genes were changed by overexpressing Gene X (far right bottle) with those changed by exposure to antibiotics (overlapping circle). You would want to focus your study of the impacts of Gene X on the genes changed specifically when it is overexpressed (left circle only).
Changes in metabolism
Another way that antibiotics can impact mammalian cells is by changing their metabolism.
In one study, researchers looked at how the antibiotic gentamicin impacts cell metabolism in three different breast cancer cell lines. They found that gentamicin increased a form of metabolism called aerobic glycolysis, which in turn led to mitochondrial reactive oxygen species, and ultimately DNA oxidative damage (Elliott and Jiang, 2019).
This is another example where skipping an antibiotic-only control would have been very misleading. Without that control, they would have erroneously concluded that mitochondrial metabolism or DNA damage are important in these breast cancer cell lines without realizing that it was actually the antibiotic that was causing these changes.
No matter what your readout is, you can see why it is important to have an antibiotic-only control with cell culture studies.
Impacts on differentiation
Differentiation refers to the process by which stem cells mature into different adult cells such as liver cells, breast cells, etc. This is an important process during embryonic development as well as in maintaining tissues with high turnover such as skin cells or the intestinal lining.
Scientists can recapitulate differentiation in cell culture models by culturing stem cells and then introducing specific growth factors, small molecules, or specific culturing techniques to tilt differentiation towards the desired cell type (Figure 3).
Figure 3. In cell culture, stem cells are differentiated into different adult cells such as fat, blood, muscle, and brain cells.
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 pneumocytes, which are the epithelial cells that line the air sacs in lungs (Cohen et al, 2006).
In a similar example, stem cells had increased cell death during differentiation procedures into either liver or neuronal lineages in the presence of PenStrep or gentamicin (Varghese et al, 2017).
This effect is not limited to embryonic stem cells. Scientists can generate 3D cell culture models of skin, which are better mimics of real skin than traditional 2D cell culture because it recapitulates all of the different layers of the epidermis (Figure 4). Another study looked at the impact of PenStrep or gentamicin on keratinocytes, which are the type of cells that make up about 90% of the epidermis in human skin. They found that PenStrep disrupted cell growth and prevented the upper layers of skin from forming.
Gentamicin didn’t impact cell growth, but rather scrambled the architecture of the skin cells. That is, in the presence of gentamicin the cells grew just fine, but they no longer had distinct identities in each layer of the epidermis (Nygaard et al, 2015). It’s as if a lasagna was thoroughly mashed up so instead of distinct layers of cheese, sauce, and noodles you just have goulash instead. Tasty, just not lasagna.
Figure 4. 3D cultures of skin cells recapitulate the different layers (SB, SS, SG, and SC) of the epidermis (left). PenStrep disrupts cell growth and prevents some of the epidermal layers from forming (middle), whereas gentamicin doesn’t impact cell growth but does interfere with the identity and layering of the different cell types (right).
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.
Controlling for antibiotic side effects
How can you tease apart the biological effect that you’re investigating from any potential cellular response to the antibiotic? There are a few different options, and which one you use may depend on which type of experiment you’re doing.
Some cell cultures and experiments absolutely require antibiotics. In these situations, you will need to include a “negative control” cell culture where the cells are exposed to the same antibiotic, but don’t have the biological perturbation that you’re trying to study.
For example, let’s say you’re investigating the role of “Gene X” which is thought to drive liver cancer. So, you overexpress Gene X using a plasmid with PenStrep resistance and look at the gene expression changes caused by Gene X.
Just like the above example, there should be ~ 200 genes whose expression changes due to the exposure to PenStrep (Ryu et al, 2017). If you have a negative control of HepG2 cells that are exposed to PenStrep but don’t overexpress Gene X, then you’ll know that you aren’t interested in these genes and can manually subtract these genes that are responding to antibiotic exposure (Figure 1).
If you don’t have this negative control, however, then you will mistakenly think that these 200 genes are part of how Gene X is driving liver cancer, and perhaps you would try to use one of them as a drug target or a biomarker.
With some cell cultures and experiments you can potentially skip using antibiotics altogether. If you can wash out the antibiotics for a day or two before performing your experiment, this will likely reduce the side effects of the antibiotics on the experiment. Since this is a relatively short wash out, I would still include a negative control sample in this situation.
If you can culture your cells longer-term without antibiotic, then you may not even need this negative control. After a few passages, the cells will have divided many times without exposure to antibiotics, and in most cases that should be sufficient for erasing any cellular responses to antibiotics. However, if you’re seeing some results that you’re skeptical of, it never hurts to have a negative control that has the same exposure to antibiotics then antibiotic-free media as your sample of interest.
Antibiotics warrant careful use in mammalian cell culture to make sure that you’re not just characterizing a cellular response to the antibiotics. Yet, it’s worth keeping in mind that antibiotics and cell selection agents are still powerful tools that are often highly desirable, and in some cases irreplaceable, for cell culture and related experiments. GoldBio is a great source for a wide variety of affordable and high-quality antibiotics. The links below and throughout this article highlight just a few out of GoldBio’s vast catalog of antibiotics, so check out our website’s catalog to see even more options.
References
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(1), 28310. 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
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
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