Comparison of three methods for cryopreservation of human embryonic stem cells.

OBJECTIVE: To establish reliable methods for cryopreservation of human embryonic stem cells (hESCs).
DESIGN: Prospective experimental study.
SETTING: University laboratory. PATIENT(S): One hESC line. INTERVENTION(S): The attachment rates and recovery rates of cryopreserved hESCs using three different cryopreservation methods were compared. MAIN OUTCOME MEASURE(S): The hESCs were frozen and thawed by conventional cryopreservation, programmable cryopreservation, and vitrification method. The efficiency of cryopreservation was assessed by attachment rate and recovery rate. RESULT(S): The attachment rate and recovery rate after thawing of hESCs frozen by the conventional cryopreservation method were significantly lower than those of hESCs frozen by programmed cryopreservation and vitrification methods. Vitrification resulted in the highest attachment rate and recovery rate compared with the other two methods. Human ESCs after vitrification and programmable cryopreservation still expressed pluripotent markers, maintained normal karyotype, and retained their pluripotency. CONCLUSION(S): Our data show that programmable cryopreservation and vitrification methods are appropriate for cryopreservation of hESCs, whereas the conventional slow-rate freezing method is not appropriate for cryopreservation of hESCs. Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.