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Hum. Reprod. Advance Access published online on March 10, 2005
Human Reproduction, doi:10.1093/humrep/deh854
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Human Reproduction © European Society of Human Reproduction and Embryology 2005; all rights reserved
Received January 15, 2005
Revised February 11, 2005
Accepted February 18, 2005

Article

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

Sung Yun Ha 1, Byung Chul Jee 2, Chang Suk Suh 3*, Hee Sun Kim 4, Sun Kyung Oh 4, Seok Hyun Kim 5, and Shin Yong Moon 5

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

* To whom correspondence should be addressed.
Chang Suk Suh, E-mail: suhcs{at}snu.ac.kr


  Abstract

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.

Keywords: cryopreservation; dimethylsulphoxide; ethylene glycol; human embryonic stem cells.
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