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Cryobiology. 2008 Jun;56(3):223-32. doi: 10.1016/j.cryobiol.2008.03.005. Epub 2008 Mar 30.

Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: a study using murine embryonic stem cells.

Author information

1
Center for Engineering in Medicine and Department of Surgical Service, Massachusetts General Hospital, Harvard Medical School, and Shriners Hospital for Children, 51 Blossom Street, Boston, MA 02114, USA. xmhe@sc.edu

Abstract

Conventional cryopreservation protocols for slow-freezing or vitrification involve cell injury due to ice formation/cell dehydration or toxicity of high cryoprotectant (CPA) concentrations, respectively. In this study, we developed a novel cryopreservation technique to achieve ultra-fast cooling rates using a quartz micro-capillary (QMC). The QMC enabled vitrification of murine embryonic stem (ES) cells using an intracellular cryoprotectant concentration in the range used for slowing freezing (1-2M). The cryoprotectants used included 2M 1,2-propanediol (PROH, cell membrane permeable) and 0.5M extracellular trehalose (cell membrane impermeable). More than 70% of the murine ES cells post-vitrification attached with respect to non-frozen control cells, and the proliferation rates of the two groups were similar. Preservation of undifferentiated properties of the pluripotent murine ES cells post-vitrification cryopreservation was verified using three different types of assays: the expression of transcription factor Oct-4, the presentation of the membrane surface glycoprotein SSEA-1, and the elevated expression of the intracellular enzyme alkaline phosphatase. These results indicate that vitrification at a low concentration (2M) of intracellular cryoprotectants is a viable and effective approach for the cryopreservation of murine embryonic stem cells.

PMID:
18462712
PMCID:
PMC2728604
DOI:
10.1016/j.cryobiol.2008.03.005
[Indexed for MEDLINE]
Free PMC Article

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