期刊
CRYOBIOLOGY
卷 56, 期 2, 页码 120-130出版社
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.cryobiol.2007.12.002
关键词
cryopreservation; mouse; embryonic stem cell; slow cooling; propylene glycol; fundamental; cryobiology; banking; stem cell
资金
- NCRR NIH HHS [U42 RR014821, U42 RR014821-08, U42 RR1482, T32-RR07004] Funding Source: Medline
Embryonic stem (ES) cell lines including the C57BL/6 genetic background are central to projects such as the Knock-Out Mouse Project, North American Conditional Mouse Mutagenesis Program, and European Conditional Mouse Mutagenesis Program, which seek to create thousands of mutant mouse strains using ES cells for the production of human disease models in biomedical research. Crucial to the success of these programs is the ability to efficiently cryopreserve these mutant cell lines for storage and transport. Although the ability to successfully cryopreserve mouse ES cells is often assumed to be adequate, the percent post-thaw recovery of viable cells varies greatly among genetic backgrounds and individual cell lines within a genetic background. Therefore, there is a need to improve the efficiency and reduce the variability of current mouse ES cell cryopreservation methods. To address this need, we employed the principles of fundamental cryobiology to improve the cryopreservation protocol of a C57BL/6 mouse ES cell line by characterizing the membrane permeability characteristics and osmotic tolerance limits. These values were used to predict optimal cooling rates, warming rates, and type of cryoprotectant, which were then verified experimentally. The resulting protocol, generated through this hypothesis-driven approach, resulted in a 2-fold increase in percent post-thaw recovery of membrane-intact ES cells as compared to the standard freezing protocol, as measured by propidium iodide exclusion. Additionally, our fundamental cryobiological approach to improving cryopreservation protocols provides a model system by which additional cryopreservation protocols may be improved in future research for both mouse and human ES cell lines. (C) 2008 Elsevier Inc. All rights reserved.
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