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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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References

Amit, M., Shariki, C., Margulets, V. & Itskovitz-Eldor, J. (2004). Feeder layer- and serum-free culture of human embryonic stem cells. Biol. Reprod. 70, 837–45.CrossRefGoogle ScholarPubMed
Chan, S.Y. & Evans, M.J. (1991). In situ freezing of embryonic stem cells in multiwell plates. Trends Genet. 7, 76.CrossRefGoogle ScholarPubMed
Chen, D., Lewis, R.L. & Kaufman, D.S. (2003). Mouse and human embryonic stem cell models of hematopoiesis: past, present and future. Biotechniques 35, 1253–61.CrossRefGoogle ScholarPubMed
Cowan, C.A., Klimanskaya, I., McMahon, J., Atienza, J., Witmyer, J., Zucker, J.P., Wang, S., Morton, C.C., McMahon, A.P., Powers, D. & Melton, D.A. (2004). Derivation of embryonic stem-cell lines from human blastocysts. N. Engl. J. Med. 350, 1353–6.CrossRefGoogle ScholarPubMed
Eisenberg, L.M., Kubalak, S.W. & Eisenberg, C.A. (2004). Stem cells and the formation of the myocardium in the vertebrate embryo. Anat. Rec. A Discov. Mol. Cell. Evol. Biol. 276, 2–12.CrossRefGoogle ScholarPubMed
Gerecht-Nir, S. & Itskovitz-Eldor, J. (2004). Cell therapy using human embryonic stem cells. Transpl. Immunol. 12, 203–9.CrossRefGoogle Scholar
Gribaldo, L., Alison, M., Andrews, P.W., Bremer, S., Donovan, P.J., Knaan-Shanzer, S., Mertelsmann, R., Spielmann, H., Testa, N.G., Triffitt, J.T., Zipori, D. & de Wynter, E. (2002). Meeting summary: European Workshop on Stem Cells, European Centre for the Validation of Biomedical Testing Methods, Institute for Health and Consumer Protection, Joint Research Centre, Ispra, Italy, November 21–23, 2001. Exp. Hematol. 30, 628–33.CrossRefGoogle ScholarPubMed
Heng, B.C., Kuleshova, L.L., Bested, S.M., Liu, H. & Cao, T. (2005). The cryopreservation of human embryonic stem cells. Biotechnol. Appl. Biochem. 41, 97–104.CrossRefGoogle ScholarPubMed
Ji, L., de Pablo, J.J. & Palecek, S.P. (2004). Cryopreservation of adherent human embryonic stem cells. Biotechnol. Bioeng. 88, 299–312.CrossRefGoogle ScholarPubMed
National Institute of Health (NIH) backgrounder on stem cells. (2005). Accessible at: http://www.nih.gov/news/pr/mar2003/stemcellbackgrounder.htm. Accessed: May 10.Google Scholar
Raff, M.C., Barres, B.A., Burne, J.F., Coles, H.S., Ishizaki, Y. & Jacobson, M.D. (1994). Programmed cell death and the control of cell survival. Philos. Trans. R. Soc. Lond. B Biol. Sci. 345, 265–8.Google ScholarPubMed
Reubinoff, B.E., Pera, M.F., Fong, C.Y., Trounson, A. & Bongso, A. (2000). Embryonic stem cell lines from human blastocysts: somatic differentiation in vitro. Nat. Biotechnol. 18, 399–404.CrossRefGoogle ScholarPubMed
Reubinoff, B.E., Pera, M.F., Vajta, G. & Trounson, A.O. (2001). Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum. Reprod. 16, 2187–94.CrossRefGoogle ScholarPubMed
Richards, M., Fong, C.Y., Tan, S., Chan, W.K. & Bongso, A. (2004). An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells. Stem Cells 22, 779–89.CrossRefGoogle ScholarPubMed
Richards, M., Tan, S.P., Tan, J.H., Chan, W.K. & Bongso, A. (2004). The transcriptome profile of human embryonic stem cells as defined by SAGE. Stem Cells 22, 51–64.CrossRefGoogle ScholarPubMed
Rohwedel, J., Guan, K., Hegert, C. & Wobus, A.M. (2001). Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects. Toxicol. In Vitro 15, 741–53.CrossRefGoogle Scholar
Sathananthan, H., Pera, M. & Trounson, A. (2002). The fine structure of human embryonic stem cells. Reprod. Biomed. Online 4, 56–61.CrossRefGoogle ScholarPubMed
Thomson, J.A., Itskovitz-Eldor, J., Shapiro, S.S., Waknitz, M.A., Swiergiel, J.J., Marshall, V.S. & Jones, J.M. (1998). Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–7.CrossRefGoogle ScholarPubMed
Udy, G.B. & Evans, M.J. (1994). Microplate DNA preparation, PCR screening and cell freezing for gene targeting in embryonic stem cells. Biotechniques 17, 887–94.Google ScholarPubMed
Ure, J.M., Fiering, S. & Smith, A.G. (1992). A rapid and efficient method for freezing and recovering clones of embryonic stem cells. Trends Genet. 8, 6.CrossRefGoogle ScholarPubMed
Wicell Research Institute Inc. (2005). Appendix – Freezing Human Embryonic Stem Cells. Accessible at: http://www.wicell.org/uploads/media/Freezing_Human_ESC_04.pdf. Accessed: May 10.Google Scholar