ROCK inhibitor可提高胚胎幹細胞之繁殖率與冷凍解凍存活率

添加Rho-associated kinase (ROCK) inhibitor Y-27632可提高胚胎幹細胞之繁殖率與冷凍解凍存活率

Y-27632可穩定胚胎幹細胞之染色體與複製過程相關marker

http://humrep.oxfordjournals.org/content/24/10/2468.full

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

1. Sepideh Mollamohammadi1, 
2. Adeleh Taei1, 
3. Mohammad Pakzad1,
4. Mehdi Totonchi1,2, 
5. Ali Seifinejad1, 
6. Najmehsadat Masoudi2 and
7. Hossein Baharvand1,3,4

+Author Affiliations

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

1. 4Correspondence address. Tel: +98-21-22306485; Fax: +98-21-22310406; E-mail:baharvand@royaninstitute.org

• Received April 12, 2009.
• Revision received June 9, 2009.
• Accepted June 12, 2009.

 

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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.