We have had challenges with cryopreservation and post-thaw recovery of our PSCs from cryopreservation? The post-thaw viability is high upon directly post-thaw, but the following day very few PSCs are shown to recover. Any advice for support of cells during post-thaw recovery?

Answer

While many cryopreservation solutions are shown to have high viability direct post-thaw (i.e., 80-90% viability), during the first 24 hours post-thaw significant apoptosis and necrosis occurs, resulting in substantial loss in PSC viability and thus poor post-thaw recovery.
The following are points to consider to improve post-thaw recovery of your PSCs:

(1) Choice of cryopreservation solution-The cryopreservation solution is an essential component that can have a significant impact on the recovery of cells 24 hours post-thaw. While the viability of cells may appear artificially high direct post-thaw, the stress of thawing of cells and osmotic stress associated with the process of diluting the cryomedium solution itself is often not immediately evident.

(2) Temperature and time of exposure to cryopreservation solution-A key step in the cryopreservation process is ensuring that the cryopreservation medium is prechilled (i.e., at 2 to 8 °C) prior to addition to the PSCs. This is vital as it greatly impacts the permeation rate of the cryoprotectant into the cytosol of the cell. The quicker the influx of the solution into the cell, the greater the impact of osmotic shock on cell health. Additionally, the longer duration exposure to the cryoprotectant –commonly DMSO−at a range of temperatures, the higher the toxicity of the cryoprotectant. Therefore, the duration of exposure of cells to the prechilled cryopreservation medium, should be minimized in order to minimize the toxicity of the cryoprotectant on the cells.

(3) Quick Recovery-Just as incubation of the cells prior to cryopreservation is a vital step, minimizing exposure of the cells post-thaw to the cryoprotectant is also critical to achieving high post-thaw recovery.

(4) Additives to Support Post-Thaw Recovery-In order to support cells during post-thaw recovery, additives may be spiked into the growth medium for the first 24 hours followed by feeding of cells in growth medium alone for the remainder of culture. Traditional additives include ROCK inhibitors, antioxidants, and free radical scavengers. Through mechanisms of decreasing apoptosis and necrosis and facilitating cell attachment with the extracellular matrix, these compounds significantly improve recovery of cryopreserved pluripotent stem cells.

Thermo Fisher Scientific now offers a complete, ready-to-use kit – PSC Cryopreservation Kit (Cat #A26446-01)– for cryopreservation and recovery of clump passaged or single cell passaged PSCs. Together these solutions minimize the stress associated with cryopreservation of pluripotent stem cells, providing recovery of 50% greater cells 24 hours post-thaw than best-in-class commercial solutions.