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Cell Culture Basics – A discussion of some of the most common problems in cell culture
We recently finished our Ask the Expert discussion on Cell Culture Basics. This week we had many interesting questions and helpful suggestions on topics including avoiding contamination, cell growth and passaging and proper storage techniques. Specific questions involved troubleshooting contamination, the use of antibiotics, and problems with freezing and thawing cells. We also featured a tip of the day with useful cell culture tips and tricks.
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.
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Below is a sneak peek of the discussion. For a full transcript of the discussion, please see – Ask the Expert – Cell Culture Basics.
I sometimes have a contamination in the culture bottles (usually fungus). However, when I examine all the used media I do not find any contamination in these media.
Often fungus contamination does not affect the media color ( if phenol red is present) or clarity.. If you are using original media bottles and not transferring the media I would worry about how the contamination is occurring. If you transfer your media for some reason, to a glass bottle I would heat treat them. Maybe use an antimycotic?
We are moving away from manual cell viability assays and are looking for an automated process. What is most important to consider in making our evaluation of which process to choose?
First, the gold standard for viability determination is the trypan blue exclusion assay where viable cells exclude the dye and dying and dead cells do not. This is a short-term assay and easy to do. Many automated viability assays use trypan blue as the dye but then just automatically calculate viability based on some algorithm. The advantage to the automated system is that they tend to count many more cells and counting squares than done when counting manually. This means the statistical probability is greater, meaning more accuracy in the count. Another nice feature about automation is better documentation and the ability for validation. The drawback to automation is that one might stop looking at the cells under a microscope, and I think it is always a good idea to see the cells under a microscope. Personally, if I were evaluating I would compare directly a manual count against a automated instrument. I would compare multiple cell types and media and between instrument I would compare ease of use, upkeep and cost of use. So you know, other methods are available for mammalian cell viability assays such as fluorescent assays developed by Molecular Probes (http://www.lifetechnologies.com/us/en/home/life-science/cell-analysis/cell-viability-and-regulation/cell-viability.html).
Our group is looking for a way to cryopreserve our iPSCs without using DMSO. Any suggestions for ways to remove or at a minimum reduce DMSO? Thx.
Although I do not know of specific recipes for iPSCs freezing medium that don’t use DMSO here is a web link to a site with a good protocol that I know works with DMSO as the freezing adjuvent (http://www.lifetechnologies.com/us/en/home/references/protocols/cell-culture/stem-cell-protocols/ipsc-protocols/reprogramming-fibroblasts-with-cytotune-ips-reprogramming-it.html). An alternative to DMSO is glycerol and can be substituted at the same concentration as DMSO. Recently people have been having success at reducing the level of DMSO from the standard 10% to 7.5% or even 5% without problems. This may work for you. Also, if you are finding toxicity with DMSO or glycerol, make sure you are using cell culture grade reagents from small volumes that are purged with nitrogen gas. Old and oxidized DMSO or glycerol can be toxic to animal cells.