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


CONCLUSIONS
5% DMSO+50% FBS+10% EG may be an optimal cryoprotectant for the slow freezing-rapid thawing of human ES cells.
Ha
Sung Yun
SY
Department of Obstetrics and Gynecology, College of Medicine, Seoul National University, Seoul, 110-744, Korea.
Jee
Byung Chul
BC
Suh
Chang Suk
CS
Kim
Hee Sun
HS
Oh
Sun Kyung
SK
Kim
Seok Hyun
SH
Moon
Shin Yong
SY
D001483:Base Sequence; D002454:Cell Differentiation; D002470:Cell Survival; D003114:Colony-Forming Units Assay; D015925:Cryopreservation / Q000295:instrumentation / Q000379:methods*; D003451:Cryoprotective Agents; D004268:DNA-Binding Proteins / Q000235:genetics; D004121:Dimethyl Sulfoxide; D019855:Ethylene Glycol; D015870:Gene Expression; D005990:Glycerol; D018398:Homeodomain Proteins / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; D039904:Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism; D012313:RNA / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics
D003451:Cryoprotective Agents; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C486780:NANOG protein, human; D000071317:Nanog Homeobox Protein; D050814:Octamer Transcription Factor-3; C494173:POU5F1 protein, human; D014157:Transcription Factors; D012313:RNA; D019855:Ethylene Glycol; D005990:Glycerol; D004121:Dimethyl Sulfoxide