Abstract
This study was designed to simplify a cryopreservation program for embryonic stem cells (ESCs) by selection of cooling method and cryoprotectant. Commercially available mouse El 4 embryonic stem cells (ESCs) were cryopreserved with various protocols, and morphology and viability of the frozen-thawed ESCs and their reactive oxygen species (ROS) production were subsequently monitored. Post-thaw colony-formation of ESCs was detected only after a slow freezing using dimethyl sulfoxide (DMSO) by stepwise placement of a freezing container into a -80°C deep freezer and subsequently into -196°C liquid nitrogen, while no proliferation was detected after vitrification. When the simplified protocol was employed, the replacement of DMSO with a mixture of DMSO and ethylene glycol (EG) further improved the post-thaw survival. ROS generation in ESCs frozen-thawed with the optimized protocol was not increased compared with non-frozen ESCs. The use of fresh mouse embryonic fibroblasts as feeder cells for post-thaw subculture did not further increase post-thaw cell viability. In conclusion, a simplified slow-freezing program without employing programmable freezer but using DMSO and EG was developed which maintains cell viability and colony-forming activity of ESCs during post-thaw subculture.
Highlights
An effective cryopreservation program for embryonic stem cells (ESCs) is essential for establishing ESC bank (Gearhart, 1998), as well as for developing a strategy for resisting ESCs against extremely low temperature
The suggested program only takes the merits of slow freezing method; it does not utilize programmable freezer and freezing cells are placed into a -80°C deep freezer and liquid nitrogen
Morphology of ESCs frozen with a simplified slow freezing protocol or vitrification
Summary
An effective cryopreservation program for embryonic stem cells (ESCs) is essential for establishing ESC bank (Gearhart, 1998), as well as for developing a strategy for resisting ESCs against extremely low temperature. In the case of ESC freezing that employed only small number of colony-forming cells are provided for the cryopreservation, the simplified program ought to further be optimized for increasing post-thaw survival (Miyamoto et al, 2006).
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