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An effective serum- and xeno-free chemically defined freezing procedure for human embryonic and induced pluripotent stem cells

Holm, Frida ; Ström, Susanne ; Inzunza, José ; Baker, Duncan ; Strömberg, Anne-Marie ; Rozell, Björn ; Feki, Anis ; Bergström, Rosita ; Hovatta, Outi

In: Human Reproduction, 2010, vol. 25, no. 5, p. 1271-1279

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    Titre
    • An effective serum- and xeno-free chemically defined freezing procedure for human embryonic and induced pluripotent stem cells
    Auteur
    • Holm, Frida. Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, K57, Karolinska University Hospital, Huddinge, Stockholm 141 86, Sweden
    • Ström, Susanne. Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, K57, Karolinska University Hospital, Huddinge, Stockholm 141 86, Sweden
    • Inzunza, José. Department of Biosciences and Nutrition Karolinska Institutet, Novum, Karolinska University Hospital, Huddinge, Sweden
    • Baker, Duncan. Sheffield Diagnostic Genetic Services, Sheffield Children's Foundation Trust, Western Bank, Sheffield, UK
    • Strömberg, Anne-Marie. Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, K57, Karolinska University Hospital, Huddinge, Stockholm 141 86, Sweden
    • Rozell, Björn. Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden
    • Feki, Anis. Stem Cell Research Laboratory, Geneva University Hospitals, Geneva, Switzerland
    • Bergström, Rosita. Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, K57, Karolinska University Hospital, Huddinge, Stockholm 141 86, Sweden
    • Hovatta, Outi. Division of Obstetrics and Gynecology, Department of Clinical Science, Intervention and Technology, Karolinska Institutet, K57, Karolinska University Hospital, Huddinge, Stockholm 141 86, Sweden
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • Human Reproduction, 2010, vol. 25, no. 5, p. 1271-1279. Oxford University Press
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:302578
    Summary
    BACKGROUND Both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) bear a great potential in regenerative medicine. In addition to optimized clinical grade culture conditions, efficient clinical grade cryopreservation methods for these cells are needed. Obtaining good survival after thawing has been problematic. METHODS We used a novel, chemically defined effective xeno-free cryopreservation system for cryostorage and banking of hESCs and iPSCs. The earlier established slow freezing protocols have, even after recent improvements, resulted in low viability and thawed cells had a high tendency to differentiate. The medium is a completely serum and animal substance free product containing dimethylsulfoxide, anhydrous dextrose and a polymer as cryoprotectants. The cells were directly frozen at −70°C, without a programmed freezer. RESULTS The number of frozen colonies versus the number of surviving colonies differed significantly for both HS293 (χ2 = 9.616 with one degree of freedom and two-tailed P = 0.0019) and HS306 (χ2 = 8.801 with one degree of freedom and two-tailed P = 0.0030). After thawing, the cells had a high viability (90-96%) without any impact on proliferation and differentiation, compared with the standard freezing procedure where viability was much lower (49%). The frozen-thawed hESCs and iPSCs had normal karyotype and maintained properties of pluripotent cells with corresponding morphological characteristics, and expressed pluripotency markers after 10 passages in culture. They formed teratomas containing tissue components of the three germ layers. CONCLUSION The defined freezing-thawing system described here offers an excellent simple option for banking of hESCs and iPSCs