Hi. I am involved in a project in the area of gastric cancer and I should make a culture from KATO III cell line. I revived my stock. But after 48 hours I saw apoptotic cells. The culture media, serum, incubator items, azote tank, amount of DMSO and serum were standard. I don’t know what item I should check. I would appreciate it if you guide me.
Hello Azar Gharoonpour
Since you have checked the culture media, serum, etc…… I feel you should revisit your freezing protocol.
For good results, you should harvest the cells during their maximum growth phase (or log phase) and should have greater than 80% confluency before freezing. Also, it is important to consider the optimal concentration for freezing cells which may vary depending on cell type. If you freeze cells at a very low concentration it could lead to low cell viability after thawing and a high concentration could cause 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.
Additionally, when you freeze cells, you should follow the standard protocol of freezing namely, gradual freezing at -1 degree C/min using Mr. Frosty freezing container. Have you checked the cell viability after thawing?
Best.
@Malcolm Nobre. Thank you much for your complete answer. I think I should recheck the freezing protocol. I feel I have had problem with the time and the duration of freezing cells.
@Malcom Nobre.
Dear Dr Nobre I checked the process of cell freezing and figured out that the -80 cold room had been out of order for a day. I think the problem was related to cold room.
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