Kinetics of cell death of frozen-thawed human embryonic stem cell colonies is reversibly slowed down by exposure to low temperature

• Published in: Zygote (Cambridge) Vol. 14; no. 4; pp. 341 - 348
• Main Authors: Heng, B.C., Ye, C.P., Liu, H., Toh, W.S., Rufaihah, A.J., Cao, T.
• Format: Journal Article
• Language: English
• Published: Cambridge, UK Cambridge University Press 01.11.2006
• Subjects: Apoptosis; Cell Adhesion; Cell Death; Cell Survival; Cells, Cultured; Cold Temperature; Cryopreservation; Cryopreservation - methods; Embryonic; Embryonic Stem Cells - cytology; Human; Humans; In Vitro Techniques; Kinetics; Stem cells; Embryonic; Human; Cell death; Cryopreservation; Stem cells
• ISSN: 0967-1994; 1469-8730
• DOI: 10.1017/S0967199406003893

A major challenge in the widespread application of hES (human embryonic stem) 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 less than 5% as reported by previous studies. This study characterized cell death in 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 placed in a 37 °C incubator, there was 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 more than 90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperatures. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Hence, our observations would strongly suggest involvement of a self-induced apoptotic mechanism, as opposed to cellular necrosis arising from cryoinjury.