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

Published online by Cambridge University Press:  01 November 2006

B.C. Heng ,
C.P. Ye ,
H. Liu ,
W.S. Toh ,
A.J. Rufaihah  and
T. Cao
B.C. Heng
Affiliation:
Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, Singapore.
C.P. Ye
Affiliation:
Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, Singapore.
H. Liu
Affiliation:
Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, Singapore.
W.S. Toh
Affiliation:
Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, Singapore.
A.J. Rufaihah
Affiliation:
Department of Surgery, Faculty of Medicine, National University of Singapore, Singapore.
T. Cao*
Affiliation:
Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, Singapore.
*
All correspondence to: T. Cao. Stem Cell Laboratory, Faculty of Dentistry, National University of Singapore, 5 Lower Kent Ridge Road, Singapore119074. Tel: +65 6874 4630. Fax: +65 6774 5701. e-mail: dencaot@nus.edu.sg
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Summary

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.

Keywords

Cell deathCryopreservationEmbryonicHumanStem cells
Type
Research Article
Information
Zygote , Volume 14 , Issue 4 , November 2006 , pp. 341 - 348
Copyright
Copyright © Cambridge University Press 2006

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