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Hum. Reprod. Advance Access originally published online on March 10, 2005
Human Reproduction 2005 20(7):1779-1785; doi:10.1093/humrep/deh854
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© The Author 2005. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions{at}oupjournals.org

Cryopreservation of human embryonic stem cells without the use of a programmable freezer

Sung Yun Ha1, Byung Chul Jee3, Chang Suk Suh1,2,3,4, Hee Sun Kim2, Sun Kyung Oh2, Seok Hyun Kim1,2 and Shin Yong Moon1,2

1 Department of Obstetrics and Gynecology, College of Medicine, 2 Institute of Reproductive Medicine and Population, Medical Research Center, Seoul National University, Seoul, 110–744 and 3 Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, Seongnam, 463–707, Korea

4 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Seoul National University Bundang Hospital, 300 Gumi-dong, Bundang-gu, Seongnam, Kyunggi-do, 463–707, Korea. Email: suhcs{at}snu.ac.kr

BACKGROUND: An effective freezing–thawing technique is crucial for the clinical application of human embryonic stem (ES) cells. The aim of this study was to find an optimal cryopreservation protocol for human ES cells using slow freezing–rapid thawing without a programmable freezer. METHODS: The human ES cell line, SNUhES-3, was cultured on an STO feeder layer in gelatin-coated tissue culture dishes. All cryopreservation steps were performed using a simple commercial freezing container. The survival rate of cryopreserved–thawed human ES cells was estimated by counting colony numbers under a stereomicroscope. Initially, we compared the survival rates of cryopreserved human ES cells using three cryoprotectants: dimethylsulphoxide (DMSO), ethylene glycol (EG) and glycerol. In this experiment, 5% DMSO/95% fetal bovine serum (FBS) (vol/vol) showed the highest survival rate. We next tested the impact of various concentrations of FBS (95, 50 and 5%) with 5% DMSO, and then examined the effects of adding EG or glycerol to 5% DMSO + optimal FBS. RESULTS: No significant difference in survival rate was observed between 95 and 50% FBS in the presence of 5% DMSO. A significant improvement in survival rate was obtained by adding 10% EG to 5% DMSO+50% FBS. After thawing, surviving cells were found to maintain the inherent characteristics of human ES cells. CONCLUSION: 5% DMSO+50% FBS+10% EG may be an optimal cryoprotectant for the slow freezing–rapid thawing of human ES cells.

Key words: cryopreservation/dimethylsulphoxide/ethylene glycol/human embryonic stem cells


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