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

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.

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.

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.

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.

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).

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.

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?

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.

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.

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.

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.