This question is part of the following Ask The Expert session:
There are a variety of factors that are critical for successful cryopreservation such as cell harvesting, cryoprotective agents (CPAs), storage vessels, cooling rate, cryogenic storage and thawing. Unfortunately, we usually notice cell viability problems which are associated with cryopreservation after the thawing and plating steps. I would suggest four major check points that you need to consider for improving cell viability. First, cell health and cell density are critical when the cells are frozen. The healthier your cells are, the higher post thawing viability you may obtain. We recommend 2 x 106 cells for a typical Corning® cryogenic vial (Corning Cat. No. 430661 or 430489). If cell density is too high, the nutrients or CPAs might not be sufficient to maintain the health of cells. In addition, before freezing, the cells need to avoid excessive exposure to cell dissociation reagents or CPAs and keeping too long at room temperature during cell harvesting. Second, when you transfer the cryogenic vials containing resuspended cells with freezing media into a freezing container such as Corning CoolCell™ Container at -80°C, cooling rate (-1°C per minute) is an important factor for cell freezing. You need to use a suitable freezing container, type and concentration of CPAs during cell freezing. Third, you should avoid exposing the cells to warm temperatures during transfer to cryogenic storage (-196°C). Finally, thawing must be done quickly and CPAs removed properly in order to avoid toxicity or osmotic shock. I recently presented a webinar regarding a general guide to cryogenically storing animal cell cultures. Please refer to the webinar which is available on the Corning website, with particular attention to time mark 25:27 when I go over a troubleshooting methodology which may improve your viability.