Document Detail

High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells.
MedLine Citation:
PMID:  19022437     Owner:  NLM     Status:  MEDLINE    
OBJECTIVE: To develop an efficient freezing method suitable for large-scale cryopreservation of human embryonic stem cells (hESCs). DESIGN: Experimental study. SETTING: Research institute. PATIENT(S): None. INTERVENTION(S): Two genetically modified hESC lines, H9-EF1-GFP and CHA-hES3-EF1-GFP, were cryopreserved in cryovials using a combination of two equilibration methods (one-step and stepwise) and two cooling vehicles (cryo-container and program-controlled freezer). After thawing, the survival and differentiation rate were compared among groups. MAIN OUTCOME MEASURE(S): The hESC survival was assessed by alkaline phosphatase staining and differentiation status was determined by flow cytometry using an SSEA-4 antibody. RESULT(S): In both H9-EF1-GFP and CHA-hES3-EF1-GFP cells, the survival rate was highest in the group using stepwise equilibration and program-controlled freezer, and lowest in the group using one-step equilibration and cryo-container. In the groups using cryo-container, the survival and the frequency of undifferentiated cells in both cell lines was highly improved in a stepwise equilibration compared with one-step. Thawed hESCs were positively stained with pluripotent markers SSEA-4, TRA-1-60, TRA-1-81, and alkaline phosphatase. The karyotypes and expression of three germ layer markers in both cell lines were not changed after freezing/thawing. CONCLUSION(S): The stepwise equilibration of Knockout Serum Replacement and cryoprotectant during freezing and thawing resulted in higher survival rates by reducing osmotic damage irrespective of cooling vehicles.
Ji Yeon Lee; Jeoung Eun Lee; Dong Ku Kim; Tae Ki Yoon; Hyung Min Chung; Dong Ryul Lee
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't     Date:  2008-11-19
Journal Detail:
Title:  Fertility and sterility     Volume:  93     ISSN:  1556-5653     ISO Abbreviation:  Fertil. Steril.     Publication Date:  2010 Feb 
Date Detail:
Created Date:  2010-02-15     Completed Date:  2010-03-16     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  0372772     Medline TA:  Fertil Steril     Country:  United States    
Other Details:
Languages:  eng     Pagination:  976-85     Citation Subset:  IM    
Copyright Information:
Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.
Fertility Center, CHA General Hospital, CHA Research Institute, Pochon CHA University, Seoul 135-081, South Korea.
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MeSH Terms
Alkaline Phosphatase / metabolism
Biological Markers / metabolism
Blood Proteins / pharmacology*
CCAAT-Enhancer-Binding Proteins / genetics,  metabolism
Cell Differentiation
Cell Survival
Cryopreservation / methods*
Embryonic Stem Cells / cytology*,  physiology*
Flow Cytometry
Green Fluorescent Proteins / genetics
Reverse Transcriptase Polymerase Chain Reaction
Stage-Specific Embryonic Antigens / genetics,  metabolism
Tissue Banks*
Tissue and Organ Harvesting
Reg. No./Substance:
0/Biological Markers; 0/Blood Proteins; 0/CCAAT-Enhancer-Binding Proteins; 0/Stage-Specific Embryonic Antigens; 0/stage-specific embryonic antigen-4; 147336-22-9/Green Fluorescent Proteins; EC Phosphatase

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