Liang Zhang1,2, Yanqing Xu1, Jiandong Xu1, Yuping Wei1, Xia Xu3. 1. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China. 2. University of Chinese Academy of Sciences, Beijing, 100049, China. 3. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, China. xuxia@ipe.ac.cn.
Abstract
OBJECTIVES: Human embryonic stem cells (hESCs) have huge potential for establishment of disease models and for treating degenerative diseases. However, the extremely low survival level of dissociated hESCs following cryopreservation is been a tremendous problem to allow for their rapid expansion, genetic manipulation and future medical applications. In this study, we have aimed to develop an efficient strategy to improve survival of dissociated hESCs after cryopreservation. MATERIALS AND 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.
OBJECTIVES:Human embryonic stem cells (hESCs) have huge potential for establishment of disease models and for treating degenerative diseases. However, the extremely low survival level of dissociated hESCs following cryopreservation is been a tremendous problem to allow for their rapid expansion, genetic manipulation and future medical applications. In this study, we have aimed to develop an efficient strategy to improve survival of dissociated hESCs after cryopreservation. MATERIALS AND 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.
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