title: Vitrification by ultra-fast cooling at a low concentration of cryoprotectants in a quartz micro-capillary: a study using murine embryonic stem cells.
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.
authors.affiliation: 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
authors.forename: Xiaoming
authors.initials: X
authors.lastname: He
authors.forename: Eric Y H
authors.initials: EY
authors.lastname: Park
authors.forename: Alex
authors.initials: A
authors.lastname: Fowler
authors.forename: Martin L
authors.initials: ML
authors.lastname: Yarmush
authors.forename: Mehmet
authors.initials: M
authors.lastname: Toner
mesh_terms: D000469:Alkaline Phosphatase / Q000032:analysis; D000818:Animals; D002448:Cell Adhesion; D002454:Cell Differentiation; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D015925:Cryopreservation / Q000379:methods*; D003451:Cryoprotective Agents / Q000494:pharmacology*; D053595:Embryonic Stem Cells / Q000166:cytology*; D019855:Ethylene Glycol / Q000494:pharmacology; D016256:Lewis X Antigen / Q000032:analysis; D051379:Mice; D008962:Models, Theoretical; D050814:Octamer Transcription Factor-3 / Q000032:analysis; D010969:Plastics / Q000737:chemistry; D019946:Propylene Glycol / Q000494:pharmacology; D011791:Quartz / Q000737:chemistry*; D013814:Thermal Conductivity; D014199:Trehalose / Q000494:pharmacology
chemical_list: D003451:Cryoprotective Agents; D016256:Lewis X Antigen; D050814:Octamer Transcription Factor-3; D010969:Plastics; D011791:Quartz; D019946:Propylene Glycol; D014199:Trehalose; D000469:Alkaline Phosphatase; D019855:Ethylene Glycol