Multipotent Mesenchymal Stem Cells Derived from Sheep Bone Marrow: Isolation and Cryopreservation

Sheep are often used as preclinical large animal models for testing the therapeutic potential of multipotent mesenchymal stem cells (MMSCs). A cell culture with characteristics typical for MMSCs was obtained due to optimization of the method of cell isolation from sheep bone marrow (BM) in a Ficoll density gradient. The advantage of using SepMate-15 tube compared to the isolation by the classical method has been demonstrated. The obtained cells demonstrated a strong adhesion to the culture plastic, fibroblast-like morphology, and, being induced in vitro, the ability to differentiate into cells of adipose, bone, and cartilage tissues. Adipogenic differentiation was confirmed on day 14, when adipocytes with lipid vesicles positive for Oil Red O specific staining had been formed. On day 14, specific alkaline phosphatase activity appeared in cells in osteogenic medium. Von Kossa staining revealed the presence of insoluble calcium salts in the intercellular space. Chondrogenic differentiation appeared on day 14 and was accompanied with the formation of multilayer structures with an abandon matrix, in which the isogenic groups that resemble hyaline cartilage lacunae were visualized. On day 21, the cells formed tight microgranules with glycosaminoglycan matrixes. Cells were cryopreserved in three different media at the zero passage. A comparative analysis of cell viability was then performed, which revealed a relatively good cell state (more than 70% of live cells). The MMSCs culture obtained from sheep BM was deposited at two to three passages in the specialized Collection of Somatic Cell Cultures of Agricultural and Industrial Animals at the All-Russian Research Institute of Experimental Veterinary Medicine of Russian Academy of Science and could be used in preclinical studies.