- 作者: Boon Chin Heng; Chao Peng Ye; Hua Liu; Wei Seong Toh; Abdul Jalil Rufaihah; Zheng Yang; Boon Huat Bay; Zigang Ge; Hong Wei Ouyang; Eng Hin Lee; Tong Cao
- 作者服務機構: 1 Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, 119074, Singapore, Singapore; ; 2 Department of Surgery, Faculty of Medicine, National University of Singapore, 5 Lower Kent Ridge Road, 119074, Singapore, Singapore; ; 3 Department of Orthopedic Surgery, Faculty of Medicine, National University of Singapore, 5 Lower Kent Ridge Road, 119074, Singapore, Singapore; ; 4 Department of Anatomy, Faculty of Medicine, National University of Singapore, 5 Lower Kent Ridge Road, 119074, Singapore, Singapore;; 5 Tissue Engineering Center, School of Medicine, Zhejiang University, 353 Yan'an Road, Hangzhou, 310031, China
- 中文摘要: --
- 英文摘要: 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 (~98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 ℃ 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 ul-trastructural 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.
- 中文關鍵字: --
- 英文關鍵字: apoptosis, cryopreservation, embryonic, human, stem cells