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Cell Culture Basics – Avoiding contamination, cell growth and passaging, and proper storage techniques
Be sure to check in to see our daily cell culture tips and tricks.
This Ask the Expert Session is 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.
This session is sponsored by
Please take advantage of the opportunity to ask our expert a question and participate in a lively discussion of Cell Culture Basics!
Questions & Answers
Is there any advantage in terms of volumetric productivity when a Mab cloning design includes light and heavy chain sequences in a bicistronic vector instead of two different ones?You know I don’t really know and that answer would be based on so many things including vector, promotors, cell type, media. culture conditions to name a few things, a simple answer is not possible. I suspect there may not be much difference though. In looking around to help me answer this question I did […]» Read More
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. […]» Read More
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. […]» Read More
What is the maximum amount of time you can wait between thaw and putting cells in culture? We are seeing a drop in viability and I think this might be the cause.I would say for most cell lines 20 minutes would be okay. If someone is having low viability following a thaw and they froze healthy cells the problem is most likely the thaw. The correct way to thaw animal cells is to bring a cup of 37C water to the freezer. Immediately upon removing the […]» Read More
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. […]» Read More
We’ve been trying to remove antibiotics from our research culture, but we usually end up going back due to contamination. Can you suggest a way to wean our lab from using them. Where would you start?This is a great question and I do recommend taking the difficult step and eliminating antibiotics. New cell culture people should learn from their mistakes and not use antibiotics. This eliminates poor techniques and reinforcing bad habits. I suggest you begin by having multiple vials of cryopreserved cells for each cell type. I would also […]» Read More
Do you routinely equilibrate your media to 37C before using it?
Are you looking for a simple way to boost productivity in many cell culture systems including recombinant protein expression?
Since many cells get limited amounts of energy (ATP) from the metabolism of glucose, other energy sources are needed. Once such source of energy comes from the metabolism of glutamine. Many cell types get somewhere between 40% to 70% of their energy from glutamine. The problem with glutamine as an energy source is that is is unstable. Glutamine spontaneously breaks down and ihas a half-life of 4-5 days at 37C. A better alternative to glutamine is GlutaMAX I. This L-Alynl-L-Glutamine dipeptide is very stable and does not breakdown in medium as monomeric glutamine. This small change can make dramatic differences in the energy status of a cells and therefore increase productivity. I f you try GLutaMAX I there is not adaptation necessary you can just substitute directly.
You might want to gain knowledge about the latest developments in cell line authentication.
Recently NIH and others have proposed Principles and Guidelines for Reporting Preclinical Research (http://www.nih.gov/about/
Know your microscope.
To get the best out of cell culture, be sure to know how to use your microscope. Most have at least an inverted phase contrast microscope. Be sure it is maintained and that you low how to use it and get the best images possible. ATCC notes that it is important to photo-document your cells at different stages of confluencey. Do you know what phase rings go with what objectives on your microscope?
Have “conditioned media” for each cell type you grow.
Conditioned media is media that has been used to grow cells. Each conditioned media is specific for a specific cell type and should be kept separate. Conditioned media is made by growing a sub-confluent culture in fresh growth media overnight. The next morning, using sterile technique, remove the partially used media and place in a sterile tube. Then centrifuge to remove any cell debris. This conditioned media has had some nutrition used by the growth of cells overnight but importantly those cells also added some compounds to the media as they grew. These factors can be useful if your cells begin to grow poorly. A 50%/50% mixture of fresh and conditioned media is very useful when growing cells at very low density or when isolating individual cells. For freezing cells us he mixture and add DMSO to about 7.5%. Once you have made conditioned media you can freeze it in small volumes for long-term storage.
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