Hello! The freezing protocol for MSCs I have been performing says to freeze 1 ml of cell suspension. I was told that in order to keep confluence, I must add only 1 ml to the pellet of cells and freeze that 1 ml in a criovial. However, my doubt is if instead of freezing in a criovial the 1 ml, I could divide by 3 criovial (around 330µl), increasing the stock of frozen cells.
Hello Denisa Amado
Your way of freezing is not the proper way of freezing cells. If you freeze cell suspension at a very low concentration, it could lead to low cell viability after thawing. When you divide 1ml cell suspension into 3 vials, the cell concentration is bound to decrease, and you will get a low cell viability on thawing. Even freezing cell suspension at a very high cell concentration is not recommended as it could lead to undesirable cell clumping.
Typically, the concentration of cells in the cryogenic vial should be within a general range of 1x10^5 - 1x10^6 cells/ml/cryovial.
If you want to increase the stock of frozen cells, you may expand the culture in bigger flasks (T-75cm^2), and on reaching 80% confluency, you may freeze the cells. In this way, you will get a much higher cell concentration. Then you may divide the cell suspension in a number of cryogenic vials with 1 x 10^6 cells/ml/vial.
Best.
Generally, people freeze 1 million cells/1ml in freezing media but this is a general guideline. We have frozen between 0.5-5 million cells per ml or per 0.5ml with good recovery. Avoid low and extremely high cell densities as this leads to increased cell loss after thawing.
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