Conclusions
This new approach for cryopreservation of single hESCs, using H89, can promote potential use of hESCs in regenerative medicine in the future.
Methods
Human embryonic stem cells (H9 line), dissociated into single cells, were cryopreserved using the slow-freezing method. Viable cells and their colony numbers in culture after cryopreservation were evaluated when treated with protein kinase A inhibitor H89. Western blotting was carried out to investigate mechanisms of low survival levels of dissociated hESCs following cryopreservation. Immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), in vitro and in vivo differentiation were performed to testify to pluripotency and differentiation ability of hte cryopreserved cells treated with H89.
Results
H89 significantly improved survival level of dissociated hESCs after cryopreservation through ROCK inhibition. H89-treated cells still maintained their pluripotency and differentiation capacity. Conclusions: This new approach for cryopreservation of single hESCs, using H89, can promote potential use of hESCs in regenerative medicine in the future.