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Human Reproduction, Vol. 16, No. 10, 2187-2194, October 2001
© 2001 European Society of Human Reproduction and Embryology

Effective cryopreservation of human embryonic stem cells by the open pulled straw vitrification method

B.E. Reubinoff1,3, M.F. Pera2, G. Vajta2 and A.O. Trounson2

1 Department of Obstetrics and Gynecology and Goldyn Savad Institute of Gyne Therapy, Hadassah University Hospital, Jerusalem, Israel and 2 Department of Early Human Development, Monash Institute of Reproduction and Development, Monash University, Melbourne, Australia

BACKGROUND: Human embryonic stem (ES) cells originate from the inner cell mass of the blastocyst, and retain in culture the properties of pluripotent cells of the early embryo. The study aim was to determine whether the open pulled straw (OPS) vitrification method, which is highly effective for the cryopreservation of embryos, might be also efficient for human ES cells. METHODS AND RESULTS: All human ES cell clumps that were vitrified by the OPS method could be recovered upon thawing, and gave rise to ES cell colonies after plating. Vitrified colonies were significantly smaller and showed an increased level of background differentiation compared with control colonies. However, these unwanted effects could be overcome by additional cultivation of the colonies for 1 and 2 days respectively. The vitrified human ES cells were cultivated for prolonged periods and retained the properties of pluripotent cells, including a normal karyotype, expression of the transcription factor Oct-4 and surface markers that are characteristic to human ES cells. When grafted into SCID mice, the vitrified cells gave rise to teratomas containing derivatives of all three embryonic germ layers. CONCLUSIONS: Vitrification by the OPS method is reliable and effective for the cryopreservation of human pluripotent embryonic stem cells.

Key words: cryopreservation/human embryonic stem cells/vitrification

3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Hadassah University Hospital, Ein-Kerem, P.O.B. 12,000, Jerusalem 91120, Israel. E-mail: reubinof{at}md2.huji.ac.il


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