Cell Culture Basics – Avoiding contamination, cell growth and passaging, and proper storage techniques


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!

Daily Tips

Monday’s Tip:

Do you routinely equilibrate your media to 37C before using it?

Monday’s tip is that if you do, you may be doing more harm than good. Instead place you media in a dark drawer or cabinet and just take the chill off. Generally speaking, unless you are doing a special procedures or using large volumes of media, there is no advantage to pre-warming media.  Given the large surface area of a dish or flask and the relatively thin layer or media, media will cool down significantly when you take you vessel out of the incubator.  Probably after that you put your dish or flask on a room temperature microscope stage or biological safety cabinet which further speeds cooling from the incubator temperature.  The  “more harm than good” part of this tip is that there is a natural decay of critical media components such as glutamine, and others, that is sped up by 37C incubations and if you use a waterbath without a cover light can damage the media further.

Tuesday’s Tip:

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.

Wednesday’s Tip:

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/reporting-preclinical -research.htm).  Most journals will require some diligence on the part of the cell culture researcher to qualify and identify the cell type they are using.  Find out which journals at http://www.nih.gov/about/endorsing-journals.htm.  A very good LinkedIn group you may want to join is Cell Line Authentication then have a link on their site the links provides a list to over 400 cell lines that are currently known to be cross-contaminated of misidentified.  If you can not find the link the reference is as follows.  Capes-Davis A, Theodosopoulos G, Atkin I, Drexler HG, Kohara A, Macleod RA, Masters JR, Nakamura Y, Reid YA, Reddel RR, Freshney RI (2010) Check Your Cultures! A list of cross-contaminated or misidentified cell lines.  Int J Cancer 127(1) 1-8. PMID 20143388.

Thursday’s Tip:

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?

Friday’s Tip:

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.

Question 1

What is the best way to know when to feed or split my cells?

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 the 5-6 days of the test. and the subsequent development of a growth curve for each conditions. Try two or 4 different starting cell concentrations. Grow the cells without refeeding, If the cells reach confluency before the cell start to plateau then refeeding may not be necessary and splitting is in order. If the cell growth plateaus before the cells reach confluence then it is time to refeed and grow another couple of days.

Question 2

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 suggest if everyone is eliminating antibiotics at the same time I would stop culturing cells for a period and do a thorough cleaning of the cell culture labs incubators, biological safety cabinets, water baths, floors etc. Then I would begin thawing each cell type and plate them in media without antibiotics. As the cells grow I would look for low level contaminants popping up. If you find some of your cells are contaminated I would dispose of them and purchase new non-contaminated ones from a vendor. If your cells are a “one of a kind” , then a curing routine is necessary. Of course the goal is good technique and you really learn when using cells grown in antibiotic-free medium.

Question 3

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.

Question 4

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 vial from the freezer place the vial in the 37C water than proceed to the water bath to complete the thaw. Some thaw until the last small bit of ice remains, some thaw until the ice is completely gone. Do not immerse the cap of the vial under water. If you do this, I bet your recovery will be better. The damage upon thawing occurs at about -50C so the goal is to transition quickly.

Question 5

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).

Question 6

I am finding a lot of cell debris in our thawed cells. How would you address?

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 viability stain before and after freezing. If you see debris but very high viability then that is probably the origin of the debris. Also I have noticed that growing fibroblasts tend to produce more debris or bits of membranes than other cell types. If these are reasons for the debris then don’’t worry about it. One way to remove some of the debris is to allow your cells to attach then wash them with a balanced salt solution or media to wash away some debris if it bothers you.

Question 7

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?

Question 8

How to avoid mold contamination during hybridoma culture? Thanks in advance

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 can use an antimycotic/antibiotic, like Gibco Anti/Anti.

Question 9

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 find one patent for dual expression in e. coli.

Question 10

We are having a problem with our newly suspended cells clumping together. Any thoughts?

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 don’t know your culture conditions it is difficult to help more. One trick is to put the cells in a tall sterile tube like a sterile graduated cylinder and let the clumps settle for a few minutes then use the clump-free upper layer to passage the cells.

Question 11

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.

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 choice.

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