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Hum. Reprod. Advance Access originally published online on July 14, 2009
Human Reproduction 2009 24(10):2468-2476; doi:10.1093/humrep/dep244
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© The Author 2009. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

A simple and efficient cryopreservation method for feeder-free dissociated human induced pluripotent stem cells and human embryonic stem cells

Sepideh Mollamohammadi1, Adeleh Taei1, Mohammad Pakzad1, Mehdi Totonchi1,2, Ali Seifinejad1, Najmehsadat Masoudi2 and Hossein Baharvand1,3,4

1 Department of Stem Cells and Developmental Biology, Royan Institute for Stem Cell Biology and Technology, ACECR, PO Box 19395-4644, Tehran, Iran 2 Department of Genetics, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran 3 Department of Developmental Biology, University of Science and Culture, ACECR, Tehran, Iran

4 Correspondence address. Tel: +98-21-22306485; Fax: +98-21-22310406; E-mail: baharvand{at}royaninstitute.org

BACKGROUND: An essential prerequisite for the future widespread application of human induced pluripotent (hiPSCs) and embryonic stem cells (hESCs) is the development of efficient cryopreservation methods to facilitate their storage and transportation.

METHODS: We developed a simple and effective freezing/thawing method of single dissociated hESCs and hiPSCs in a feeder-free culture in the presence of Rho-associated kinase (ROCK) inhibitor Y-27632.

RESULTS: Exposure to ROCK inhibitor Y-27632 in freezing solution alone does not significantly enhance the post-thaw survival rate of single dissociated hESCs and hiPSCs. However, when ROCK inhibitor was added to both pre- and post-thaw culture media, there was an enhancement in the survival rate, which further increased when ROCK inhibitor was added to Matrigel as well. Under these treatments, hESCs and hiPSCs retained typical morphology, stable karyotype, expression of pluripotency markers and the potential to differentiate into derivatives of all three germ layers after long-term culture.

CONCLUSIONS: This method is an effective cryopreservation procedure for single dissociated hESCs in feeder-free culture, which is also applicable for single dissociated hiPSCs using a ROCK inhibitor. The cloning efficiency of hiPSCs and hESCs improves when ROCK inhibitor is added both in Matrigel and in medium in comparison with conventional addition to medium. Therefore, we believe this method would be useful for current and future applications of the pluripotent stem cells.

Key words: human embryonic stem cells/human induced pluripotent stem cells/ROCK inhibitor Y-27632/culture/cryopreservation

Submitted on April 12, 2009; resubmitted on June 9, 2009; accepted on June 12, 2009.


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