A Discussion about Antibiotics in Cell Culture

We recently finished our Ask the Expert discussion on Antibiotics in Cell Culture – What Types Are Available and when and how to use them. The result was an interesting discussion about the issues surrounding antibiotic use including categories of antibiotics and suggested use. Topics included the use of antibiotics in hybridoma, vero and primary cell cultures and how proper cell culture techniques can reduce or eliminate the need.

Antibiotics are often chemicals and sometimes polypeptides that affect the ability of bacteria to grow. Antibiotics come in two categories, bacteriostatic and bactericidal. Bacteriostatic antibiotics don’t kill but inhibit some vital cellular process such as cell-wall synthesis or protein expression. If the baceriostatic antibiotic is removed bacterial growth can resume. Bactericidal antibiotics kill bacterial cells and therefore they are useful at preventing contaminations and eliminating them. There are many antibiotics available and it is important to know when and how to use them. These are just some of the issues discussed during our Ask the Expert discussion.

This Ask the Expert Session was Sponsored by Life Technologies and hosted by Timothy Fawcett, Ph.D. Dr. Fawcett has been in the biotechnology business for over 30 years. Trained as a biochemist he has held senior positions in both academics and industry and has been a mentor to many young scientists throughout his career. For the last 12 years Dr. Fawcett has been the Director of the BioTechnical Institute of Maryland (BTI) a non-profit institute located in Baltimore, Maryland. He is also the Founder and Director of BioSciConcepts, a social venture of BTI that provides hands-on training for professional scientists in cell culture, baculovirus based expression, as well as topics such as molecular biology, PCR and real-time PCR. BioSciConcepts is an internationally recognized provider of expertise in the biological sciences and has provided consultation services to several small and large biotechnology companies.

Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert Antibiotics in Cell Culture.

If you want to include a low level of antibiotic in your hybridoma cell culture to reduce the risk of contamination, which type would you recommend and how much?

The Answer:
My first thought would be penicillin and streptomycin at 50 ug each/ml. The two can be purchased supplied together and is called pen-strep. Penicillin kills gram positive bacteria by inhibiting cell wall synthesis and streptomycin kills by inhibiting protein synthesis in gram positive and gram negative bacteria. If you are using a serum-free medium use 25 ug/ml of each.

Which are all the antibiotics can be used in combination in large scale Vero cell culture and at what concentration?

The Answer:
This question can be answered many ways depending on the goals. Maybe you would like to be proactive and you want to use antibiotics to prevent an infection or maybe you want to cure a current infection. Of course my recommendation is not to use antibiotics unless absolutely necessary. I prefer having good frozen stocks of cells that I can thaw if needed. Having said that though, I suspect the best general antimycotic is Fungizone (Amphptericin B). At normal working concentrations (0.5-2.5 ug/ml) it works to prevent fungal contaminations. Interestingly in Vero cells, at low concentrations (250 ng/ml) amphotericin B has been show to increase viral uptake (http:/www.ncbi.nih.gov/pmc/articles/PMC3194987/). I have provided a listing below of other antibiotics and antimycotics and their working concentrations. I will mention that the range of antibiotics and antimycotics used does vary depending of the media type used. Media containing serum can contain more antibiotic/antimycotic since the high protein, mostly albumin binds much of the reagent. If you are using a low protein medium or a serum-free medium you will need to use much less for equivalent results, perhaps 10% to 50% depending on specifics of your

Antibiotics and Antimycotics and Their Working Concentrations
Anti-PPLO Agent 10-100 ug/ul Mycoplasma and gram positive bacteria 3 days
Fungizone 0.25-2.5 ug/ul Fungi and yeast 3 days
Gentamicin Sulfate 5-50 ug/ml Gram positive and gram negative bacteria and mycoplasma 3 days
Kanamcin Sulfate 100 ug/ml Gram positive and gram negative bacteria and mycoplasma 5 days
Neomycin Sulfate 50 ug/ml Gram positive and gram negative bacteria 5 days
Nystatin 100 U/ml Fungi and yeast 3 days
Penicillin G 50-100 U/ml Gram positive bacteria 3 days
Polymixin B Sulfate 100 U/ml Gram negative bacteria 5 days
Streptomycin Sulfate 50-100 ug/ml, Gram positive and gram negative bacteria 3 days
If you are using a low protein medium or a serum-free medium you will need to use much less for equivalent results,

Why would you want to use antibiotics at all? Can’t good cell culture techniques eliminate their use.

The Answer:
You are right in a perfect world and the best thing would be to not use antibiotics at all. Some might need antibiotics if their equipment or air-handling systems are not perfect. Some might use certain antibiotics for selection. Others might use antibiotics during primary culture. Sometimes it might be necessary, but if possible I try to do without it. If you are learning cell culture I strongly recommend not using antibiotics so that you can learn good technique quickly and not develop bad habits that may be masked by antibiotic use.

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