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

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

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Alkaline Phosphatase / analysis
  • Animals
  • Cell Adhesion
  • Cell Differentiation
  • Cell Proliferation
  • Cell Survival
  • Cells, Cultured
  • Cryopreservation / methods*
  • Cryoprotective Agents / pharmacology*
  • Embryonic Stem Cells / cytology*
  • Ethylene Glycol / pharmacology
  • Lewis X Antigen / analysis
  • Mice
  • Models, Theoretical
  • Octamer Transcription Factor-3 / analysis
  • Plastics / chemistry
  • Propylene Glycol / pharmacology
  • Quartz / chemistry*
  • Thermal Conductivity
  • Trehalose / pharmacology

Substances

  • Cryoprotective Agents
  • Lewis X Antigen
  • Octamer Transcription Factor-3
  • Plastics
  • Quartz
  • Propylene Glycol
  • Trehalose
  • Alkaline Phosphatase
  • Ethylene Glycol