The ability to cryopreserve and store for long term the structure and function of therapeutic cells and tissues plays a pivotal role in clinical medicine. In fact, it is an essential pre-requisite for the commercial and clinical application of stem cells since preserves cells at low temperature and creates a reserve for future uses. This requisite may also affect the encapsulated stem cells. Several parameters should be considered on encapsulated cell cryopreservation such as the time and temperature during the cryopreservation process, or the cryoprotectant solutions used. In this study, we have compared the influence of penetrating and nonpenetrating cryoprotectants on the viability and functionality of encapsulated mesenchymal stem cells genetically modified to secrete erythropoeitin. Several cryoprotectant solutions combining DMSO, glycerol and trehalose at different concentrations were studied. Although almost no differences among the studied cryoprotectant solutions were observed on the differentiation potential of encapsulated mesenchymal stem cells, the penetrating cryoprotectant DMSO at a concentration of 10% displayed the best viability and erythropoietin secretion profile compared to the other cryoprotectant solutions. These results were confirmed after subcutaneous implantation of thawed encapsulated mesenchymal stem cells secreting erythropoeitin on Balb/c mice. The hematocrit levels of these animals increased to similar levels of those detected on animals transplanted with noncryopreserved encapsulated cells. Therefore, DMSO 10% represents the most suitable cryoprotectant solution among the solutions here studied, for encapsulated mesenchymal stem cells cryopreservation and its translation into the clinic. Similar studies should be performed for the encapsulation of other cell types before they can be translated into the clinic.