Biological and physicochemical characterization of a serum- and xeno-free chemically defined cryopreservation procedure for adult human progenitor cells

Cell Transplant. 2011;20(8):1241-57. doi: 10.3727/096368910X547426. Epub 2010 Dec 22.


While therapeutic cell transplantations using progenitor cells are increasingly evolving towards phase I and II clinical trials and chemically defined cell culture is established, standardization in biobanking is still in the stage of infancy. In this study, the EU FP6-funded CRYSTAL (CRYo-banking of Stem cells for human Therapeutic AppLication) consortium aimed to validate novel Standard Operating Procedures (SOPs) to perform and validate xeno-free and chemically defined cryopreservation of human progenitor cells and to reduce the amount of the potentially toxic cryoprotectant additive (CPA) dimethyl sulfoxide (DMSO). To achieve this goal, three human adult progenitor and stem cell populations-umbilical cord blood (UCB)-derived erythroid cells (UCB-ECs), UCB-derived endothelial colony forming cells (UCB-ECFCs), and adipose tissue (AT)-derived mesenchymal stromal cells (AT-MSCs)-were cryopreserved in chemically defined medium supplemented with 10% or 5% DMSO. Cell recovery, cell repopulation, and functionality were evaluated postthaw in comparison to cryopreservation in standard fetal bovine serum (FBS)-containing freezing medium. Even with a reduction of the DMSO CPA to 5%, postthaw cell count and viability assays indicated no overall significant difference versus standard cryomedium. Additionally, to compare cellular morphology/membrane integrity and ice crystal formation during cryopreservation, multiphoton laser-scanning cryomicroscopy (cryo-MPLSM) and scanning electron microscopy (SEM) were used. Neither cryo-MPLSM nor SEM indicated differences in membrane integrity for the tested cell populations under various conditions. Moreover, no influence was observed on functional properties of the cells following cryopreservation in chemically defined freezing medium, except for UCB-ECs, which showed a significantly reduced differentiation capacity after cryopreservation in chemically defined medium supplemented with 5% DMSO. In summary, these results demonstrate the feasibility and robustness of standardized xeno-free cryopreservation of different human progenitor cells and encourage their use even more in the field of tissue-engineering and regenerative medicine.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adipose Tissue / cytology
  • Adult
  • Adult Stem Cells / cytology*
  • Adult Stem Cells / drug effects*
  • Animals
  • Cattle
  • Cell Separation
  • Cell Shape / drug effects
  • Cells, Cultured
  • Colony-Forming Units Assay
  • Cryoelectron Microscopy
  • Cryopreservation / methods*
  • Cryoprotective Agents / pharmacology*
  • Culture Media, Serum-Free
  • Endothelial Cells / cytology
  • Endothelial Cells / drug effects
  • Endothelial Cells / ultrastructure
  • Erythroid Cells / cytology
  • Erythroid Cells / drug effects
  • Erythroid Cells / ultrastructure
  • Fetal Blood / cytology
  • Humans
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / drug effects
  • Mesenchymal Stem Cells / ultrastructure
  • Microscopy, Fluorescence, Multiphoton


  • Cryoprotective Agents
  • Culture Media, Serum-Free