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
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
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
Similar content being viewed by others
References
Thomson, J.A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998).
Takahashi, K. et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 131, 861–872 (2007).
Yu, J. et al. Induced pluripotent stem cell lines derived from human somatic cells. Science 318, 1917–1920 (2007).
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).
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).
Ji, L., de Pablo, J.J. & Palecek, S.P. Cryopreservation of adherent human embryonic stem cells. Biotechnol. Bioeng. 88, 299–312 (2004).
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).
Ware, C.B., Nelson, A.M. & Blau, C.A. Controlled-rate freezing of human ES cells. Biotechniques 38, 879–880, 882–883 (2005).
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).
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).
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).
Li, T., Zhou, C., Liu, C., Mai, Q. & Zhuang, G. Bulk vitrification of human embryonic stem cells. Hum. Reprod. 23, 358–364 (2008).
Watanabe, K. et al. A ROCK inhibitor permits survival of dissociated human embryonic stem cells. Nat. Biotechnol. 25, 681–686 (2007).
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).
Shi, J. & Wei, L. Rho kinase in the regulation of cell death and survival. Arch. Immunol. Ther. Exp. (Warsz.) 55, 61–75 (2007).
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).
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).
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).
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).
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).
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).
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).
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).
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).
Suemori, H. & Nakatsuji, N. Growth and differentiation of cynomolgus monkey ES cells. Methods Enzymol. 365, 419–429 (2003).
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).
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).
Masters, J.R. & Stacey, G.N. Changing medium and passaging cell lines. Nat. Protoc. 2, 2276–2284 (2007).
Acknowledgements
This work was supported by Royan Institute.
Author information
Authors and Affiliations
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
Rights 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
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2009.247
This article is cited by
-
Generation of iPSC-derived human forebrain organoids assembling bilateral eye primordia
Nature Protocols (2023)
-
Rho Kinases in Embryonic Development and Stem Cell Research
Archivum Immunologiae et Therapiae Experimentalis (2022)
-
Cryopreservation of NK and T Cells Without DMSO for Adoptive Cell-Based Immunotherapy
BioDrugs (2021)
-
A single-cell identification and capture chip for automatically and rapidly determining hydraulic permeability of cells
Analytical and Bioanalytical Chemistry (2020)
-
Generation of human muscle fibers and satellite-like cells from human pluripotent stem cells in vitro
Nature Protocols (2016)
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.