Asian-Australasian Journal of Animal Sciences

Asian Australasian Association of Animal Production Societies (아세아태평양축산학회)
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Simplified Slow Freezing Program Established for Effective Banking of Embryonic Stem Cells

  • Kim, Gil Ah (Gamete and Stem Cell Biotechnology, Seoul National University) ;
  • Lee, Seung Tae (Institute for Bioengineering, Swiss Federal Institute of Technology) ;
  • Lee, Eun Ju (Clinical Research Institute, Seoul National University Hospital) ;
  • Choi, Jung Kyu (Gamete and Stem Cell Biotechnology, Seoul National University) ;
  • Lim, Jeong Mook (Gamete and Stem Cell Biotechnology, Seoul National University)
  • Received : 2008.03.24
  • Accepted : 2008.10.14
  • Published : 2009.03.01
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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 E14 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 $-80^{\circ}C$ deep freezer and subsequently into -$196^{\circ}C$ 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.

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References

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