Human mesenchymal stromal cells (MSCs) can differentiate into various cell types, which makes them attractive for regenerative medicine and tissue engineering. Encapsulation of MSCs in alginate microspheres (AMS) is a novel and promising approach of tissue engineering. Application and research of such cell-hydrogel systems require selection of adequate cryopreservation protocols. In this study we investigated the response of MSCs encapsulated in AMS to different cryopreservation protocols. Bone marrow MSCs either encapsulated in AMS and or as cells in suspension, were cryopreserved with 5% and 10% of dimethyl sulfoxide (ME₂SO) using conventional 2-step slow cooling (protocol 1). The viability and metabolism of MSCs in AMS following cryopreservation with 5% Me₂SO were lower than in the group cryopreserved with 10% Me₂SO. MSCs in suspension were more resistant to cryopreservation than cells in AMS when cryopreserved with 5% Me₂SO, although when using a concentration of 10% Me₂SO, no differences were detected. Comparisons of the viability and metabolic activity of MSC cryopreserved either in AMS or as cell suspensions with 10% ME₂SO using protocol 1 (2-step cooling), protocol 2 (3-step slow cooling with induced ice nucleation) or protocol 3 (rapid 1-step freezing), showed that the highest viabilities and metabolic rates were obtained following cryopreservation of MSCs in AMS by protocol 2 (with controlled ice nucleation). Cryopreservation with protocol 3 resulted in critical damage of the encapsulated MSCs. After cryopreservation by protocol 2, AMS encapsulated MSCs were capable of achieving multilineage differentiation directed towards osteogenic, adipogenic and chondrogenic lineages. The data obtained indicate that cryo-banking of AMS encapsulated MSCs is feasible for future regenerative medicine projects.