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Fertil Steril. 2010 Feb;93(3):976-85. doi: 10.1016/j.fertnstert.2008.10.017. Epub 2008 Nov 19.

High concentration of synthetic serum, stepwise equilibration and slow cooling as an efficient technique for large-scale cryopreservation of human embryonic stem cells.

Author information

1
Fertility Center, CHA General Hospital, CHA Research Institute, Pochon CHA University, Seoul 135-081, South Korea.

Abstract

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.

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