Authors: Mollamohammadi, Sepideh; Taei, Adeleh; Pakzad, Mohammad; Totonchi, Mehdi; Seifinejad, Ali; Masoudi, Najmehsadat; Baharvand, Hossein

Source: Human Reproduction, Volume 24, Number 10, 24 October 2009 , pp. 2468-2476(9)

Publisher: Oxford University Press

Abstract:

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.

Keywords: human embryonic stem cells; human induced pluripotent stem cells; ROCK inhibitor Y-27632; culture; cryopreservation

Document Type: Research article

DOI: http://dx.doi.org/10.1093/humrep/dep244

Publication date: 2009-10-24

More about this publication?

• Human Reproduction features full-length, peer-reviewed papers reporting original research, clinical case histories, as well as opinions and debates on topical issues. Papers published cover the scientific and medical aspects of reproductive physiology and pathology, endocrinology, andrology, gonad function, gametogenesis, fertilization, embryo development, implantation, pregnancy, genetics, genetic diagnosis, oncology, infectious disease, surgery, contraception, infertility treatment, psychology, ethics and social issues. The highest scientific and editorial standard is maintained throughout the journal along with a rapid rate of publication.

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