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

Novel cryopreservation method for dissociated human embryonic stem cells in the presence of a... BACKGROUNDHuman 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-freezingrapid 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 RESULTSHere, 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 frozenthawed 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.CONCLUSIONThis 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. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Human Reproduction Oxford University Press

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

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References (31)

Publisher
Oxford University Press
Copyright
© Published by Oxford University Press.
Subject
ORIGINAL ARTICLES
ISSN
0268-1161
eISSN
1460-2350
DOI
10.1093/humrep/den316
pmid
18716037
Publisher site
See Article on Publisher Site

Abstract

BACKGROUNDHuman 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-freezingrapid 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 RESULTSHere, 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 frozenthawed 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.CONCLUSIONThis 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.

Journal

Human Reproduction – Oxford University Press

Published: Dec 20, 2008

Keywords: Keywords human embryonic stem cells Y-27632 cryopreservation dissociation survival

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