Sometimes it’s good to get back to basics. This approach can be especially helpful in cell culture particularly in troubleshooting any culture problems. Do you have questions about aseptic techniques, contamination or the use of anitbiotics? Are you trying to troubleshoot difficulties with your cell growth or viability in culture? Are you struggling with storage, freezing or thawing of cells? If so, now is the time to get some answers. Whether it’s troubleshooting problems in your culture or just getting clarification on various cell culture techniques – visit this week’s Ask the Expert session and submit your questions.
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.Please take advantage of the opportunity to ask our expert a question and participate in a lively discussion of Cell Culture Basics!
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.
Looking for a more comprehensive study of cell culture basics:
If you are looking for a comprehensive course on cell culture basics, see Gibco’s Cell Culture Basics Course and Certification program. Learn the basic techniques for effective, reproducible laboratory cell culture, including optimized procedures and products that will help you get the consistency your lab work demands.
Cell Culture Basics is designed to introduce you to the practice of laboratory cell culture, covering topics such as laboratory set-up, safety, and aseptic technique. You’ll also learn basic methods for passaging, freezing, and thawing cultured cells. Upon the successful completion of this course, you will have a basic understanding of the principles and practice of cell culture that you can immediately apply to your research projects. Aspiring cell culturists, experienced researchers, as well as lab managers and principal investigators may all find value in Gibco® Cell Culture Basics. Students completing this course will be eligible to take the free Gibco® Cell Culture Basics certification test and earn the corresponding certificate.
0 0 0 Total: 0 A quick way is to set up several flasks of the same cell type each with different starting cell numbers. Use 5-6 flasks of each starting concentration and harvest one flask each day from each culture condition. The multiple flasks for each condition are for cell counting each day for … Continued
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?
0 0 0 Total: 0 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 … Continued
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.
0 0 0 Total: 0 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 … Continued
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.
0 0 0 Total: 0 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 … Continued
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?
0 0 0 Total: 0 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 … Continued
0 0 0 Total: 0 I suspect you are using adherent cells. Do you see debris in your cell suspension (from trypsinization) from which you freeze the cells. If you are pelleting cells from a large volume containing debris in it you may be pelleting the debris and effectively concentrating it. Do you do a … Continued
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.
0 0 0 Total: 0 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 … Continued
0 0 0 Total: 0 First, as in the first question above I would track down the source of the contamination. Mold can come from many places, including workers and heating /cooling vents. Look for the source. Does your mold contamination carry through from the original isolation of cells from mice> If you want you … Continued
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?
0 0 0 Total: 0 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 … Continued
0 0 0 Total: 0 If your adherent cells that were adapted to grow in suspension are clumping you might increase the shaking speed a little bit. You can add media that is calcium and magnesium free. Sometimes I have had clumping and when I triturated the cells the clumps they broke up. Since I … Continued
I believe there is a contaminant in my culture, but I am not sure the best way to verify and identify which one so I can find the source.
0 0 0 Total: 0 There are several good detection systems. One easy to use kit is the Cell Culture Contamination Detection Kit from Molecular Probes (https://www.lifetechnologies.com/order/catalog/product/C7028?ICID=search-product) with this kit you can detect the type of contamination. If you are trying to identify a mycoplasma contamination try MycoSEQ Mycoplasma Detection Kit (https://www.lifetechnologies.com/order/catalog/product/4460626?ICID=search-product) is a good … Continued