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

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Ha, Sung Yun; Jee, Byung Chul; Suh, Chang Suk; Kim, Hee Sun; Oh, Sun Kyung; Kim, Seok Hyun; Moon, Shin Yong
Issue Date
Oxford University Press
Hum Reprod 20,1779-1785
cryopreservationdimethylsulphoxideethylene glycolhuman embryonic stem cells
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.
0268-1161 (Print)
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College of Medicine/School of Medicine (의과대학/대학원)Obstetrics & Gynecology (산부인과전공)Journal Papers (저널논문_산부인과학전공)
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