AIMTo observe the basical properties of adherent stromal cells in culture derived from cryopreserved bone marrow cells (BMCs), and to provide laboratory evidences for clinical application of cryopreserved stromal cells.

METHODSFresh BMCs and adherent stromal cells cultured for 14 days in Dexter long-term culture system (fresh stromal cells) plus 5% DMSO-6% HES cryopreservatives were frozen in -80 degrees C refrigerator, and cryopreserved in - 196 degrees C liquid nitrogen for 2 weeks (the former is called cryopreserved BMCs, the latter called cryopreserved stromal cells). These cells were cultured in Dexter long-term culture system after they were thawed. We have examined the growth features, constituents and stimulating functions of the culture of these cells.

RESULTSGrowth features: Cryopreserved BMCs produced adherent stromal cells, cell clusters and cell layer 1-2 days later than fresh BMCs. Cryopreserved stromal cells formed cell layer in 2nd day of culture, and were 12-18 h later than fresh stromal cells. Cryopreserved BMCs and stromal cells proliferated significantly less than fresh BMCs and stromal cells. Constituents: The ratio of fibrocytes and endothelial cells were lower, and the ratio of macrophages and fat cells were higher in culture of cryopreserved BMCs than that in fresh BMCs. The measurements in culture of cryopreserved stromal cells were much more significant compared with that in fresh stromal cells. Numbers of cells containing apoptic bodies in culture of cryopreserved BMCs and stromal cells were more than that in fresh BMCs and stromal cells. TBRR of cryopreserved BMCs and stromal cells were 92.5% and 89.5% respectively. The expression rates of CD14 and HLA-DR in culture of cryopreserved BMCs and stromal cells were higher than that in fresh BMCs and stromal cells, and just in contrast with the expression rates of CD45 and CD33. Stimulating functions: CAFA and LTC-IC on the stromal cell layer derived from cryopreserved BMCs, cryopreserved stromal cells, fresh BMCs and fresh stromal cells were all growing well, and there were no significant differences among these groups.

CONCLUSIONBiological properties of adherent stromal cells derived from BMCs and stromal cells were injured slightly and still maintained completely after cryopreservation with 5% DMSO-6% HES cryopreservatives.