High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells.
OBJECTIVE
To develop an efficient freezing method suitable for large-scale cryopreservation of human embryonic stem cells (hESCs).


METHODS
Experimental study.


METHODS
Research institute.


METHODS
None.


METHODS
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.


METHODS
The hESC survival was assessed by alkaline phosphatase staining and differentiation status was determined by flow cytometry using an SSEA-4 antibody.


RESULTS
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.


CONCLUSIONS
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.
Lee
Ji Yeon
JY
Fertility Center, CHA General Hospital, CHA Research Institute, Pochon CHA University, Seoul 135-081, South Korea.
Lee
Jeoung Eun
JE
Kim
Dong Ku
DK
Yoon
Tae Ki
TK
Chung
Hyung Min
HM
Lee
Dong Ryul
DR
D000469:Alkaline Phosphatase / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001798:Blood Proteins / Q000494:pharmacology*; D022762:CCAAT-Enhancer-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D002470:Cell Survival; D015925:Cryopreservation / Q000379:methods*; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology*; D005260:Female; D005434:Flow Cytometry; D049452:Green Fluorescent Proteins / Q000235:genetics; D006801:Humans; D007621:Karyotyping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D055746:Stage-Specific Embryonic Antigens / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D014015:Tissue Banks; D020858:Tissue and Organ Harvesting
D015415:Biomarkers; D001798:Blood Proteins; D022762:CCAAT-Enhancer-Binding Proteins; D055746:Stage-Specific Embryonic Antigens; C066584:stage-specific embryonic antigen-4; D049452:Green Fluorescent Proteins; D000469:Alkaline Phosphatase