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Hum. Reprod. Advance Access originally published online on August 20, 2008
Human Reproduction 2008 23(12):2744-2754; doi:10.1093/humrep/den316
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© The Author 2008. 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

Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a ROCK inhibitor

R. Martin-Ibañez1,2,5, C. Unger3, A. Strömberg2, D. Baker4, J.M. Canals1 and O. Hovatta2

1 Departament de Biologia Cel·lular, Immunologia i Neurociències, Facultat de Medicina, IDIBAPS, Universitat de Barcelona and Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas (CIBERNED) E-08036, Barcelona, Spain 2 Department of Clinical Science, Intervention and Technology, Karolinska Institutet, Karolinska University Hospital Huddinge SE 141 86, Stockholm, Sweden 3 Department of Medicine, Karolinska Institutet, Karolinska University Hospital Huddinge SE 141 86, Stockholm, Sweden 4 Sheffield Regional Cytogenetics Service, Sheffield Children's Trust, Western Bank S10 2TH, Sheffield, UK

5 Correspondence address. E-mail: r.martin{at}ub.edu

BACKGROUND: Human embryonic stem cells (hESCs) have potential use in clinical therapy and regenerative medicine. One of the major challenges regarding the application of these cells is the development of an efficient cryopreservation protocol, since current methods, which include slow-freezing–rapid thawing and vitrification of colonies in suspension, present poor viability and high differentiation rates. Dissociated hESC suspensions do not survive cryopreservation because they are susceptible to apoptosis upon cell detachment and dissociation. A selective Rho-associated kinase (ROCK) inhibitor has been reported to increase the survival of dissociated hESCs and their cloning efficiency.

METHODS AND RESULTS: Here, we describe a novel method for dissociated hESCs cryopreservation in the presence of the ROCK inhibitor Y-27632. The addition of this inhibitor to the freezing and post-thawing medium significantly increased the survival rate and efficiency of colony formation. Moreover, the hESC colonies obtained after the cryopreservation in the presence of the ROCK inhibitor showed a very low rate of differentiation and a reduced time of recovery. After prolonged culture of frozen–thawed dissociated hESCs, the characteristic properties of pluripotent cells were observed, including normal karyotype, morphological features, marker expression (SSEA-4, TRA-1-60, TRA-1-81 and Oct-4) and the potential to differentiate into derivatives of all three germ layers after embryoid bodies formation.

CONCLUSION: This novel method for the cryopreservation of dissociated hESCs may reduce the time required to amplify frozen stocks, and facilitate not only the storage of large numbers of hESCs but also the widespread use of these cells in regenerative medicine.

Key words: human embryonic stem cells/Y-27632/cryopreservation/dissociation/survival

Submitted on May 5, 2008; resubmitted on July 17, 2008; accepted on July 24, 2008.


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