Maintenance of multipotent mesenchymal stem cells of farm animals in cryogels based on naturally-derived polymers

Multipotent mesenchymal stem cells (MMSCs) of farm animals, whose growth in culture is determined by attachment to a solid substrate, are promising cellular material for veterinary medicine and biotechnology, as well as virology. One of the methods to overcome cell adhesion in suspension bioreactors in order to obtain a large number of cells with permanent properties of acceptable quality is the use of porous carriers formed from polymers of natural origin. Thus, for the first time we obtained data that allow us to make a scientific substantiation of the parameters for the cultivation of adhesive cultures of animal MMSCs using spatial protein-based cryogel carriers for subsequent suspension cultivation of the obtained constructs. The purpose of the work is to study the possibility to culture MMSC of farm animals in three-dimensional matrix sponges, i.e. the cryogels based on gelatin, blood plasma total protein and fetal bovine serum (FBS). MMSCs isolated from bovine bone marrow (BM) and adipose tissue (АТ) and ovine BM, as well as mouse fibroblast STO cell line were used. We found that the optimal cell concentration for the settlement of the cell suspension by the method of natural absorption with swelling of squeezed sponges (0.24 cm3 in volume) is 1.0×106 cells per 100 µl of medium for 2 hrs of saturation. The loading efficiency of MMSCs in sponge scaffolds is 98 %. The analysis of histological slices (at least 10 per sample) of three cryogels demonstrated the ability of all three-dimensional porous scaffolds to maintain cell culture for 14 days. Sponges were filled with cells that preserved morphology and proliferated in places of attachment to the polymer surface. The results of experiments on the effect of the matrix material on cell migration showed that all cells migrate from the monolayer in the volume of cryogel from the bottom and are not detected on the upper sides of the cryogels under study. On day 10 of culture, fibroblast STO cell line were detected in the volume of sponge scaffolds based on gelatin, blood plasma and FBS protein at a distance of 2990, 2871 and 1930 μm, respectively. MMSCs isolated from bovine AT migrated into the porous structure of matrix sponges to a depth of 607, 1364 and 657 μm, respectively. Expansion of MMSCs isolated from bovine and ovine BM in cryogels on the basis of different materials did not differ significantly from the migration of AT-MMSCs. The ability of farm animals’ MMSCs on the early passages (2 to 3) and late passages (9 to 10) to attach to macroporous cryogels was not significantly different. Comparative analysis of the results of the experiments obtained in three replicates showed that the macroporous matrices based on gelatin, bovine blood plasma proteins and FBS support the viability of MMSCs during short-term culture, promote cellular adhesion, proliferation and migration. The obtained data allow us to predict the use of these cryogels as matrices for MMSCs of farm animals for research and practical use.