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Method Article
A simple improvement of the conventional cryopreservation for human ES and iPS cells
Miho K Furue
Miho K Furue Lab (Laboratory of Stem Cell Cultures,NIBIO OSAKA JAPAN)
Corresponding Author
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
In this study, a simple method for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is proposed. It is based on the conventional slow-freezing method with 10% DMSO and modified mainly in a thawing protocol without specific equipment or reagents. Recovery rate of the cells cryopreserved by this method was equally high, which is comparable to that of the cells frozen by the vitrification method. In the case of vitrification method, it requires practiced hand because cells can be terribly damaged upon failure of rapid-warming process and strict maintenance of low temperature is required throughout the cryopreservation. On the contrary, our method is available for novices and cryopreserved cells can maintain cell recovery rate for one week after transfer from -150 °C to -80 °C condition. This simple modified method would gain widespread acceptance.
Keywords
Human pluripotent stem cells, Cryopreservation, Slow-freezing method
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Supplementary Figure 2. Effect of dissociation material before cryopreservation Cells were exposed to 1 ml CTK or Dispase until the edge of colonies began to curling up from the plate in a 37 °C incubator and then harvested. The cells were frozen by modified cryopreservation method. The colonies with ALP positive were measured at 6 days (H9) or 7 days (JCRB1331) after thawing. Bars indicate the means ± S.D. (n=3).
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Supplementary Figure 3. Suspension time after dissociation The cells were detached with dispase and kept each period in suspension and then slowly frozen. The colonies with ALP positive were measured at 6 days (H9) or 7 days (JCRB1331) after thawing. Bars indicate the means ± S.D. (n=4).
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Supplementary Figure 1. Keeping time on ice before freezing The cells were harvested and kept on ice for each period and then slowly frozen. The colonies with ALP positive were measured at 6 days (H9) or 7 days (JCRB1331) after thawing. Bars indicate the means ± S.D. (n=4).
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Supplementary Figure 5. Preservation period in a -80°C deep freezer before transferring into a liquid nitrogen tank Cells were harvested and then suspended with PS medium containing 10% DMSO. The aliquot was left in a -80 °C deep freezer for each time and then transferred into a liquid nitrogen tank. The colonies with ALP positive were measured at 6 days (H9) or 7 days (JCRB1331) after thawing. Bars indicate the means ± S.D. (n=3).
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Supplementary Figure 6. Keeping period in a -80 °C deep freezer after transferring from a liquid nitrogen tank Cells were harvested and then suspended with PS medium containing 10% DMSO. The cells were frozen by modified cryopreservation method. After transferred from a liquid nitrogen tank to a -80 °C deep freezer, the vials were kept each time at -80 °C deep freezer. The colonies with ALP positive were measured at 5 days (H9) or 6 days (JCRB1331) after thawing. Bars indicate the means ± S.D. (n=3).
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Supplementary Figure 4. The volume of fluid during cryopreservation Cells were harvested and then suspended with each volume of PS medium containing 10% DMSO. The cells were frozen by modified cryopreservation method. The colonies with ALP positive were measured at 6 days (H9) or 7 days (JCRB1331) after thawing. Bars indicate the means ± S.D. (n=3).
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Method Article
A simple improvement of the conventional cryopreservation for human ES and iPS cells
Miho K Furue
Miho K Furue Lab (Laboratory of Stem Cell Cultures,NIBIO OSAKA JAPAN)
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
History
CURRENT STATUS: Posted
Version 1
Posted 27 Mar, 2014
No community comments so far
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY-NC 3.0 License. Read Full License
In this study, a simple method for the cryopreservation of human embryonic stem (ES) and induced pluripotent stem (iPS) cells is proposed. It is based on the conventional slow-freezing method with 10% DMSO and modified mainly in a thawing protocol without specific equipment or reagents. Recovery rate of the cells cryopreserved by this method was equally high, which is comparable to that of the cells frozen by the vitrification method. In the case of vitrification method, it requires practiced hand because cells can be terribly damaged upon failure of rapid-warming process and strict maintenance of low temperature is required throughout the cryopreservation. On the contrary, our method is available for novices and cryopreserved cells can maintain cell recovery rate for one week after transfer from -150 °C to -80 °C condition. This simple modified method would gain widespread acceptance.
Figure 1
Figure 2
Figure 3
Figure 4