Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Abstract

SummaryA major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen-thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (%98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37°C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4°C, with >90% of cells remaining viable after 90 min of incubation at 4°C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37°C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen-thawed hES cells after incubation at 37°C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze-thawing with conventional slow-cooling protocols.

Cryopreservation of Human Pluripotent Stem Cells: Are We Going in the Right Direction?

“…Results obtained in a previous work from the same group showing that apoptosis rather than necrosis was the responsible mechanism involved in the loss of viability during hESCs cryopreservation encouraged them to test a broad-spectrum irreversible inhibitor of caspase enzymes (Heng et al, 2006). Exposure to 100 mM Z-VAD-FMK in the freezing solution alone did not significantly enhace the postthaw survival rate.…”

“…Another study optimizing the same critical factors described an improved protocol consisting in: cooling the sample from 0 ºC to -35 ºC at a cooling rate of -0.5ºC/min, seeding at -10 ºC before being plunged immediately into the liquid nitrogen and rapid thawing. Under these conditions a survival rate of 80% was obtained (Yang et al, 2006). A successful usage of programmable freezing for the cryopreservation of adherent iPSCs has also been recently described (Katkov et al, 2011).…”

“…Heng et al postulated for the first time that apoptosis instead of cellular necrosis, was the major mechanism inducing the loss of viability of cryopreserved hESCs during freezethawing with conventional slow-cooling protocols (Heng et al, 2006). They showed that most of the cells were viable (~98%) immediately after thawing (determined by the Trypan blue dye exclusion method) and that cell viability was gradually decreasing with time in culture at 37ºC.…”

“…These characteristics could explain why most of the cryopreservation protocols rely on hPSCs small clumps to improve survival rates (Heng et al, 2006;Reubinoff et al, 2001;Richards et al, 2004;Zhou et al, 2004). However, the cryopreservation of clumps presents some associated problems such as limitations on cryoprotectant exposure inside the clump.…”

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“…Cells were then tested for viability, which is reported to be maintained more effectively at 4°C than at 37°C. 17 cell sampling and propidium iodide staining Syringes of cells stored from years 2007 to 2015 were thawed, and cell viability was tested by PI staining. Propidium iodide is an intercalating membraneimpermeable dye.…”