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).

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.