Experimental and clinical applications of mesenchymal stromal cells (MSCs) require the development of new approaches and improvements of existing methods of cell cryopreservation. Cryoprotective solutions for effective cell preservation usually contain 10% dimethyl sulfoxide (Me2SO) and fetal bovine serum (FBS) limiting clinical application of MSCs. We have developed a novel approach to the cryopreservation of human MSCs comprising inclusion of sugars into incubation medium for 24 hrs prior to cryopreservation (pretreatment) with their obligatory presence in cryoprotective solution. Such combined application of mannitol, lactose, sucrose, trehalose or raffinose on cultivation and cryopreservation stages resulted in a significant increase of MSCs viability. Optimal concentration of sugars for cell pretreatment was 200 mM, as an additive in cryoprotective solution - 300 mM. Highest cell viability and metabolic activity assessed by Alamar Blue test were achieved with sucrose, trehalose and raffinose. Using these sugars about 50% of pretreated cells after cryopreservation in the absence of Me2SO and FBS preserved their survival and metabolic activity. During following recultivation cryopreserved MSCs were able to attachment, proliferation and multilineage differentiation towards osteogenic and adipogenic lineages. The data obtained indicate that the approach included pretreatment of cells with sugars combined with their presence in cryoprotective solution is feasible for future regenerative medicine projects.