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.
A 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 (approximately 98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 degrees 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 degrees C, with >90% of cells remaining viable after 90 min of incubation at 4 degrees 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 degrees 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 degrees 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.
Heng
Boon Chin
BC
Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, Singapore.
Ye
Chao Peng
CP
Liu
Hua
H
Toh
Wei Seong
WS
Rufaihah
Abdul Jalil
AJ
Yang
Zheng
Z
Bay
Boon Huat
BH
Ge
Zigang
Z
Ouyang
Hong Wei
HW
Lee
Eng Hin
EH
Cao
Tong
T
D017209:Apoptosis; D002454:Cell Differentiation; D002470:Cell Survival; D002843:Chromatin / Q000737:chemistry; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology; D053938:DNA Fragmentation; D004622:Embryo, Mammalian / Q000166:cytology*; D005615:Freezing; D006801:Humans; D020287:In Situ Nick-End Labeling; D007700:Kinetics; D046529:Microscopy, Electron, Transmission; D009336:Necrosis / Q000473:pathology*; D013234:Stem Cells / Q000166:cytology*; D013696:Temperature
D002843:Chromatin; D003451:Cryoprotective Agents