In his essay “More is Different,” Nobel laureate Dr. Phillip Anderson cautioned against overly reductionist approaches and advised that the complexity found within each scientific field must be grappled with to unlock new scientific understanding (Anderson, 1972).

A mundane example of reductionism that Dr. Anderson almost assuredly was not thinking of is: “what difference will it make if I combine two antibiotics in my cell culture? They will probably work exactly the same.”

Combining antibiotics in mammalian cell culture can cause changes in potency and unintended side effects for the antibiotics. It is important to check for interactions between the antibiotics and ensure they are not obscuring the biological signal that you’re investigating.

We’ll discuss these considerations to keep in mind when combining antibiotics and provide an example of when using multiple antibiotics is really worth it in mammalian cell culture.

 

Article Table of Contents

Considerations when combining antibiotics in mammalian cell culture

Changes in antibiotic potency

Side effects caused by antibiotics

Using multiple antibiotics when necessary

References

 

 

Considerations when combining antibiotics in mammalian cell culture

First let’s discuss issues you should be thinking about when combining antibiotics in mammalian cell culture. Two of the main considerations are:

·         Changes in potency when combining antibiotics

·         Side effects caused by antibiotics

 

Changes in antibiotic potency

When combining antibiotics, one plus one is not always two. Often it is two, but sometimes it can be less or more (Figure 1).

Figure 1. Simple math equations representing the difference between additive, antagonistic, and synergistic antibiotic combinations.

 

Two antibiotics often do combine as expected. That is called additive and means that each antibiotic has the same potency in the presence of the other antibiotic.

An antibiotic combination that is less potent when used together is an antagonistic combination. This means you would need to use higher concentrations of each antibiotic when using them together compared to the concentration you would use as single agents.

An antibiotic combination that is more potent at killing cells when used together is called a synergistic combination. In this case, you can use less of each antibiotic when using them together compared to the concentration you would use them at individually.

Whether an antibiotic pairing is additive, synergistic, or antagonistic is cell-type specific (Brochado et al, 2018). So, it is important to test your antibiotic combination in the specific type of cells that you’re using, unless there is already an established protocol with the same cells and antibiotics.

The most common way to check how antibiotics work together is the checkerboard assay. Check out this article to learn more about the checkerboard assay.

 

Side effects caused by antibiotics

Ideally, antibiotics are meant to kill or stall the growth of bacterial cells or untransformed mammalian cells and to have no impact on the surviving cells.

However, even cells that grow in the presence of antibiotics can be impacted in other, more subtle ways such as changes in gene expression, metabolism, or differentiation (Figure 2). These are considered side effects of the antibiotics.

Figure 2. Side effects caused by antibiotics include changes in metabolism (left), gene expression (middle), and reduced differentiation (right).

 

 

Side effects are observed even with common combinations of antibiotics such as PenStrep which is the combination of penicillin and streptomycin (Nygaard et al, 2015; Ryu et al, 2017; Varghese et al, 2017).

To avoid side effects, it is best to use antibiotics only when necessary. If possible, wash the antibiotics out and grow your cells without antibiotics for a few days before starting your assay. If antibiotics are absolutely required even during your assay, then make sure you use a proper negative control to be able to differentiate between the impact of antibiotics and the impact of the actual biological perturbation that you’re studying. We discuss more about setting up antibiotic-only controls in this article. 

 

Using multiple antibiotics when necessary

Given the complexities of using antibiotics with mammalian cell culture, it’s worth asking whether you really need to use multiple antibiotics, or really any antibiotics at all, for your experiment.

There are certain instances where it really is crucial to use multiple antibiotics. One recent example is a research group that used multiple antibiotic selection to increase the efficiency of CRISPR gene editing.

CRISPR frequently edits only a single allele of the gene that it is targeting. In certain diseases, such as macular degeneration or Alzheimer’s disease, it is important to fix both copies of a gene to obtain the desired therapeutic effect.

These researchers demonstrated how selecting with two antibiotics, one for each allele, improved the efficiency of CRISPR for editing both gene copies in cell culture (Supharattanasitthi et al, 2019). They used puromycin and blasticidin as the two antibiotics. The researchers noted that it is important to select antibiotics that don’t interact to change each other’s potency, which was true for puromycin and blasticidin in the cells they tested.  

 

GoldBio is your go-to source for high-quality and affordable antibiotics and cell selection agents to power mammalian cell culture experiments.

Check out the product links below for more information about the wide-range of antibiotics that GoldBio carries. Also, if you want to learn more about antibiotics and cell culture, click the links below and throughout this article.  

 

References

Anderson P. W. (1972). More is different. Science (New York, N.Y.), 177(4047), 393–396. https://doi.org/10.1126/science.177.4047.393

Brochado, A. R., Telzerow, A., Bobonis, J., Banzhaf, M., Mateus, A., Selkrig, J., Huth, E., Bassler, S., Zamarreño Beas, J., Zietek, M., Ng, N., Foerster, S., Ezraty, B., Py, B., Barras, F., Savitski, M. M., Bork, P., Göttig, S., & Typas, A. (2018). Species-specific activity of antibacterial drug combinations. Nature, 559(7713), 259–263. https://doi.org/10.1038/s41586-018-0278-9

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

Supharattanasitthi, W., Carlsson, E., Sharif, U., & Paraoan, L. (2019). CRISPR/Cas9-mediated one step bi-allelic change of genomic DNA in iPSCs and human RPE cells in vitro with dual antibiotic selection. Scientific reports, 9(1), 174. https://doi.org/10.1038/s41598-018-36740-2

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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antibiotics cell culture Simon Currie

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