Cryopreservation of adherent human embryonic stem cells

Authors

  • Lin Ji,

    1. Department of Chemical and Biological Engineering, University of Wisconsin–Madison, 1415 Engineering Drive, Madison, Wisconsin 53706; telephone: 608-262-8931; fax: 608-262-5434
    Search for more papers by this author
  • Juan J. de Pablo,

    1. Department of Chemical and Biological Engineering, University of Wisconsin–Madison, 1415 Engineering Drive, Madison, Wisconsin 53706; telephone: 608-262-8931; fax: 608-262-5434
    Search for more papers by this author
  • Sean P. Palecek

    Corresponding author
    1. Department of Chemical and Biological Engineering, University of Wisconsin–Madison, 1415 Engineering Drive, Madison, Wisconsin 53706; telephone: 608-262-8931; fax: 608-262-5434
    • Department of Chemical and Biological Engineering, University of Wisconsin–Madison, 1415 Engineering Drive, Madison, Wisconsin 53706; telephone: 608-262-8931; fax: 608-262-5434
    Search for more papers by this author

Abstract

Standard human embryonic stem (HES) cell cryopreservation methodologies, including slow freezing and vitrification of colonies in suspension, are plagued by poor viability and high differentiation rates upon recovery. To facilitate research studies and clinical applications of HES cells, we have developed a cryopreservation technique based on stabilizing HES colonies adherent to or embedded in a Matrigel matrix. This method increases cell viability by over an order of magnitude compared with cryopreservation in suspension and reduces differentiation. Loading adherent HES cells with the disaccharide trehalose prior to cryopreserving in a dimethylsulfoxide-containing cryoprotectant solution further improves cell viability under certain conditions. Our proposed approach has the potential to reduce the time required to amplify frozen stocks of HES cells, minimize risk of clonal selection during freeze–thaw cycles, and facilitate storage of HES cell clone libraries. © 2004 Wiley Periodicals, Inc.

Ancillary