Adoption of long-term cultures to evaluate the cryoprotective potential of trehalose for freezing hematopoietic stem cells

Dimethylsulfoxide (DMSO), which is widely used as a cryoprotectant for hematopoietic stem cells (HSC), has considerable toxicity for both the thawed cells and the patient. The aim of this study was to evaluate the cryoprotective potential of trehalose in comparison to DMSO for human HSC. Human bone marrow (BM) and peripheral blood stem cells (PBSC) of volunteer donors were cryopreserved in the presence of different concentrations of trehalose with and without insulin, as well as with DMSO 10%. After thawing to 37 degrees C colony-forming unit (CFU) assays were performed. Long-term marrow-cultures (LTMC) were established and used for the detection of long-term culture-initiating cells (LTCIC). The total amount of CFUs detected was 104+/-134 (mean+/-s.d.) in DMSO-preserved cells and 179+/-34 in trehalose-protected cells. For LTMC the best feeder layer proved to be fresh human BM and the most useful concentration of trehalose was 0.5 M. Using these culture conditions we could detect after 5 weeks LTMC a total of 172+/-28 CFUs for trehalose-protected cells and 170+/-52 for DMSO-preserved cells. In conclusion, trehalose exerts a similar cryoprotective potential for hematopoietic progenitor and stem cells like DMSO and could possibly replace DMSO at least in part as cryoprotectant in the setting of hematopoietic cell transplantation.