Cell Culture Freezing Media

Following on from the last post in regards to freezing cell culture stocks, I thought it would interest you in knowing the type of freezing media I generally use for each stock type. For my Master and Submaster cell stocks, I generally use a freezing media composition of 10% DMSO + 90% FBS. In contrast, for the Working cell stocks, I would use 10% DMSO + cell culture media containing 10% FBS.

High Percentage Serum

As you may know, DMSO is typically used because it has low toxicity to mammalian cells and importantly, prevents the formation of disruptive ice crystals during the freezing process. I opt for the higher serum percentage for the Master and Submaster cell stocks because it contains less water compared to cell culture media + serum. As such, the high percentage serum in combination with DMSO makes for a “gentle” freezing media capable of preserving cell viability or enhancing recovery after thawing. This is particularly important if the cell stocks are going to stay stored in liquid nitrogen for very long periods (i.e. years).

Lower Percentage Serum

Regarding the Working cells, I generally opt for a lower percentage serum for freezing because the Working cell stocks are not meant for long-term storage as compared to the Master cells and because they have a higher turnover in terms of usage. There is nothing wrong with using 10% serum; cell viability can be just as good as if you had frozen your cells using 90% serum. The main difference I have noticed comes from long-term storage (e.g. 2+ years).

Another reason to use a lower percentage serum for the Working cells is cost. A high number of Working cell stocks are generally frozen down so if you are using 90% serum, it will get expensive.

But despite what I have said above, if you are using a cell type that is just bad to freeze-thaw (i.e. not very good at recovering quickly), use the higher percentage serum to freeze down your Working cell stocks.