Designing an optimal cryopreservation protocol for human embryonic stem cells: A systematic approach

Human embryonic stem cells (hESCs) are derived from the inner cell mass of the human blastocyst. They are pluripotent cells which can differentiate into a wide range of cell types, making them potentially useful for the treatment of diseases such as diabetes, Alzheimer’s disease and heart disease. The growing numbers of hESC lines being derived demands that suitable storage conditions are identified in order to maximise their potential for future therapies. Current methods employed for cryopreserving hESCs were adopted from embryo storage (vitrification) and conventionally-frozen mouse embryonic stem cells (mESCs) with post-thaw cell survival ranging from 1-90%. Thus, generating large numbers of cells is often time consuming and potentially prone to clonal selection from the limited post-thaw population. In this work, the approach used was to determine the fundamental physical properties important for cryopreservation including the hydraulic conductivity (Lp), solute permeability (Ps) and the non-osmotic volume (Vb). The hESC lines, RH1 and SHEF3, were compared with the embryonal carcinoma (EC) cell line 2102Ep, which was used as a reference cell line. RH1 and SHEF3 had values for Vb of 0.22 and 0.19, respectively, which was comparable to that of human oocytes and human haematopoietic progenitor cells from bone marrow. The Lp and Ps values for RH1 and SHEF3 were determined in the presence of each of two cryoprotectants, dimethyl sulphoxide (Me2SO) and propylene glycol (PG), at RT and at +2°C. Cell growth and membrane integrity assays indicated that the RH1 and SHEF3 cells tolerated volume excursions between 40-130% and 40-170% of isotonic volume, respectively. These data were used to model protocols for the addition and elution of cryoprotectant that minimise osmotic stress during the storage of hESCs. The final protocols were tested and shown to support the hESC morphology and expression of TRA-1-60/81 and SSEA4.