Effects of cryopreservation on the adherent layer of human long-term bone marrow cultures: study of cell phenotypes

This report describes the effects of cryopreservation on the adherent layer of human long-term bone marrow cultures (HLTBMC). Stromal cells are believed to be the most important cells of medullar microenvironment to regulate hematopoiesis. To study effects of cryopreservation, we compared the cell phenotypes of adherent layers of fresh and frozen-thawed bone marrows. To characterize stromal cells we used monoclonal antibodies reacting with components of these cells (CGA-7 alpha SM and gamma SM actin isoforms; HHF-35, all muscle actin isoforms; BMS-1, stromal cell lysosomes). The other components studied were: fibronectin (BMS-2 monoclonal antibody) and hematopoietic cells (monoclonal antibodies against CD45, CD33, and CD14). Results show a decrease of cells positive for CGA-7, HHF-35, and BMS-1, in adherent layer of HLTBMC of frozen-thawed bone marrows. Expression of BMS-2 is unchanged, and CD45 and CD14-positive cells proportionately increased. These results are consistent with an impairment of stromal cell proliferation in frozen-thawed marrows, without impairment of most stromal cell functions. The difference between stromal cell and hematopoietic cell kinetics seems to be an additional fact suggesting a different origin for both cell populations.