Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

An efficient and easy-to-use cryopreservation protocol for human ES and iPS cells

Abstract

Here we describe a simple and efficient human embryonic stem (ES) and induced pluripotent stem (iPS) cells cryopreservation protocol. This protocol involves the use of Rho-associated kinase (ROCK) inhibitor, Y-27632, for the feeder-free dissociated cells. The addition of ROCK inhibitor to both pre- and post-thaw culture media enhanced the cloning efficiency. The presence of Y-27632 in Matrigel further increased the cloning efficiency. As compared with other available protocols for human ES and iPS cells cryopreservation, our protocol differs in the technical simplicity, high cloning efficiency and post-thawing passaging. We believe that this protocol could be a generally applicable and robust platform for feeder-free cryopreservation and the expansion of present and future applications of human ES and iPS cells. The treatment with ROCK inhibitor, cell harvesting and the freezing–thawing process usually takes about 2 h excluding overnight incubation at −80 °C.

This is a preview of subscription content, access via your institution

Access options

Rent or buy this article

Prices vary by article type

from$1.95

to$39.95

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Photomicrographs of human ES and iPS cells or pluripotent stem cells (hPSCs) in tissue culture plate.
Figure 2: Human ES and iPS cells or pluripotent stem cells (hPSC) harvesting, growing and passaging.

Similar content being viewed by others

References

  1. Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).

    Article  CAS  Google Scholar 

  2. Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).

    Article  CAS  Google Scholar 

  3. Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).

    Article  CAS  Google Scholar 

  4. Nishikawa, S., Goldstein, R.A. & Nierras, C.R. The promise of human induced pluripotent stem cells for research and therapy. Nat. Rev. Mol. Cell Biol. 9, 725–729 (2008).

    Article  CAS  Google Scholar 

  5. Fujioka, T., Yasuchika, K., Nakamura, Y., Nakatsuji, N. & Suemori, H. A simple and efficient cryopreservation method for primate embryonic stem cells. Int. J. Dev. Biol. 48, 1149–1154 (2004).

    Article  Google Scholar 

  6. Ji, L., de Pablo, J.J. & Palecek, S.P. Cryopreservation of adherent human embryonic stem cells. Biotechnol. Bioeng. 88, 299–312 (2004).

    Article  CAS  Google Scholar 

  7. Heng, B.C., Kuleshova, L.L., Bested, S.M., Liu, H. & Cao, T. The cryopreservation of human embryonic stem cells. Biotechnol. Appl. Biochem. 41, 97–104 (2005).

    Article  CAS  Google Scholar 

  8. Ware, C.B., Nelson, A.M. & Blau, C.A. Controlled-rate freezing of human ES cells. Biotechniques 38, 879–880, 882–883 (2005).

    Article  CAS  Google Scholar 

  9. Reubinoff, B.E., Pera, M.F., Vajta, G. & Trounson, A.O. Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method. Hum. Reprod. 16, 2187–2194 (2001).

    Article  CAS  Google Scholar 

  10. Richards, M., Fong, C.Y., Tan, S., Chan, W.K. & Bongso, A. An efficient and safe xeno-free cryopreservation method for the storage of human embryonic stem cells. Stem Cells 22, 779–789 (2004).

    Article  Google Scholar 

  11. Zhou, C.Q., Mai, Q.Y., Li, T. & Zhuang, G.L. Cryopreservation of human embryonic stem cells by vitrification. Chin. Med. J. (Engl.) 117, 1050–1055 (2004).

    Google Scholar 

  12. Li, T., Zhou, C., Liu, C., Mai, Q. & Zhuang, G. Bulk vitrification of human embryonic stem cells. Hum. Reprod. 23, 358–364 (2008).

    Article  CAS  Google Scholar 

  13. Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).

    Article  CAS  Google Scholar 

  14. Heng, B.C. et al. Loss of viability during freeze-thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to apoptosis rather than cellular necrosis. J. Biomed. Sci. 13, 433–445 (2006).

    Article  CAS  Google Scholar 

  15. Shi, J. & Wei, L. Rho kinase in the regulation of cell death and survival. Arch. Immunol. Ther. Exp. (Warsz.) 55, 61–75 (2007).

    Article  Google Scholar 

  16. Koyanagi, M. et al. Inhibition of the Rho/ROCK pathway reduces apoptosis during transplantation of embryonic stem cell–derived neural precursors. J. Neurosci. Res. 86, 270–280 (2008).

    Article  CAS  Google Scholar 

  17. Li, X., Krawetz, R., Liu, S., Meng, G. & Rancourt, D.E. ROCK inhibitor improves survival of cryopreserved serum/feeder-free single human embryonic stem cells. Hum. Reprod. 24, 580–589 (2009).

    Article  CAS  Google Scholar 

  18. Li, X., Meng, G., Krawetz, R., Liu, S. & Rancourt, D.E. The ROCK inhibitor Y-27632 enhances the survival rate of human embryonic stem cells following cryopreservation. Stem Cells Dev. 17, 1079–1085 (2008).

    Article  CAS  Google Scholar 

  19. Lingor, P. et al. ROCK inhibition and CNTF interact on intrinsic signaling pathways and differentially regulate survival and regeneration in retinal ganglion cells. Brain 131, 250–263 (2008).

    Article  Google Scholar 

  20. Martin-Ibañez, R. et al. Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor. Hum. Reprod. 23, 2744–2754 (2008).

    Article  Google Scholar 

  21. Claassen, D.A., Desler, M.M. & Rizzino, A. ROCK inhibition enhances the recovery and growth of cryopreserved human embryonic stem cells and human induced pluripotent stem cells. Mol. Reprod. Dev. 76, 722–732 (2009).

    Article  CAS  Google Scholar 

  22. Mollamohammadi, S. et al. A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells. Hum. Reprod. 24, 2468–2476 (2009).

    Article  CAS  Google Scholar 

  23. Krawetz, R.J., Li, X. & Rancourt, D.E. Human embryonic stem cells: caught between a ROCK inhibitor and a hard place. Bioessays 31, 336–343 (2009).

    Article  CAS  Google Scholar 

  24. Klimanskaya, I., Chung, Y., Becker, S., Lu, S.J. & Lanza, R. Derivation of human embryonic stem cells from single blastomeres. Nat. Protoc. 2, 1963–1972 (2007).

    Article  CAS  Google Scholar 

  25. Suemori, H. & Nakatsuji, N. Growth and differentiation of cynomolgus monkey ES cells. Methods Enzymol. 365, 419–429 (2003).

    PubMed  Google Scholar 

  26. Pakzad, M. et al. Presence of a ROCK inhibitor in extracellular matrix supports more undifferentiated growth of feeder-free human embryonic and induced pluripotent stem cells upon passaging. Stem Cell Rev. published online, doi:10.1007/s12015-009-9103-z (12 December 2009).

  27. Bajpai, R., Lesperance, J., Kim, M. & Terskikh, A.V. Efficient propagation of single cells Accutase-dissociated human embryonic stem cells. Mol. Reprod. Dev. 75, 818–827 (2008).

    Article  CAS  Google Scholar 

  28. Masters, J.R. & Stacey, G.N. Changing medium and passaging cell lines. Nat. Protoc. 2, 2276–2284 (2007).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported by Royan Institute.

Author information

Authors and Affiliations

Authors

Contributions

H.B. designed experiments, analyzed data and wrote the paper; G.H.S. wrote the paper; A.T. carried out the experiments, as well as designed experiments; S.M. carried out the experiments.

Corresponding author

Correspondence to Hossein Baharvand.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Baharvand, H., Salekdeh, G., Taei, A. et al. An efficient and easy-to-use cryopreservation protocol for human ES and iPS cells. Nat Protoc 5, 588–594 (2010). https://doi.org/10.1038/nprot.2009.247

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2009.247

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing