title: High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells.
abstract: 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.
authors.affiliation: Fertility Center, CHA General Hospital, CHA Research Institute, Pochon CHA University, Seoul 135-081, South Korea.
authors.forename: Ji Yeon
authors.initials: JY
authors.lastname: Lee
authors.forename: Jeoung Eun
authors.initials: JE
authors.lastname: Lee
authors.forename: Dong Ku
authors.initials: DK
authors.lastname: Kim
authors.forename: Tae Ki
authors.initials: TK
authors.lastname: Yoon
authors.forename: Hyung Min
authors.initials: HM
authors.lastname: Chung
authors.forename: Dong Ryul
authors.initials: DR
authors.lastname: Lee
mesh_terms: 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
chemical_list: 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