Cultivation and proliferation of stem cells in three-dimensional (3-D) scaffolds is a promising strategy for regenerative medicine. Mesenchymal stem cells with their potential to differentiate in various cell types, cryopreserved adhesion-based in fabricated scaffolds of biocompatible materials can serve as ready-to-use transplantation units for tissue repair, where pores allow a direct contact of graft cells and recipient tissue without further preparation. A successful cryopreservation of adherent cells depends on attachment and spreading processes that start directly after cell seeding. Here, we analyzed different cultivation times (0.5, 2, 24 h) prior to adhesion-based cryopreservation of human mesenchymal stem cells within alginate-gelatin cryogel scaffolds and its influence on cell viability, recovery and functionality at recovery times (0, 24, 48 h) in comparison to non-frozen control. Analysis with confocal laser scanning microscopy and scanning electron microscopy indicated that 2 h cultivation time enhanced cryopreservation success: cell number, visual cell contacts, membrane integrity, motility, as well as spreading were comparable to control. In contrast, cell number by short cultivation time (0.5 h) reduced dramatically after thawing and expanded cultivation time (24 h) decreased cell viability. Our results provide necessary information to enhance the production and to store ready-touse transplantation units for application in bone, cartilage or skin regenerative therapy.

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We have developed a novel wide-pore scaffold for cell 3D culturing, based on the technology of freeze-drying of Ca-alginate and gelatin. Two different preparation methodologies were compared: (i) freeze-drying of Na-alginate + gelatin mixed solution followed by the incubation of dried polymer in saturated ethanolic solution of CaCl₂; (ii) freeze-drying of the Na-alginate solution followed by the chemical "activation" of polysaccharide core with divinylsulfone with subsequent gelatin covalent attachment to the inner surfaces of pore walls. The scaffolds produced using the first approach did not provide adhesion and proliferation of human bone marrow mesenchymal stromal cells (MSCs). Conversely, the second approach allowed to obtain scaffolds with a high adherence ability for the cells. When cultured within the latter type of scaffold, MSCs proliferated and were able to differentiate into adipogenic, osteogenic and chondrogenic cell lineages, in response to specific induction stimuli. The results indicate that Ca-alginate wide-pore scaffolds with covalently attached gelatin could be useful for stem cell-based bone, cartilage and adipose tissue engineering.

New gelatin-based cryostructurates have been elaborated and tested as scaffolds for three-dimensional (3D) cell culturing. Scaffold preparation included dissolution of Type A gelatin in dimethylsulfoxide, freezing of such solution, cryoextraction of crystalline phase with cold ethanol, cross-linking of gelatin with carbodiimide in ethanol medium, treatment of the matrix with ethanolic solution of Tris and tanning of the matrix with formaldehyde dissolved in ethanol. The use of organic media during all the preparation stages ensured the sterility of the scaffolds. The matrices thus prepared were seeded with human adipose tissue multipotent mesenchymal stromal cells to confirm the biocompatibility of scaffolds and their possibility to provide necessary environment for the cell growth and differentiation. The cells attached onto the surface of the pore walls, proliferated and differentiated into osteogenic and adipogenic lineages. These results demonstrate that gelatin-based cryostructurates prepared in the sterility ensuring organic media can be used as scaffolds for tissue engineering purposes.

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We performed a comparative study of the localization, distribution, metabolic activity, and surface properties of human bone marrow mesenchymal stromal cells after static and perfusion seeding to macroporous alginate cryogels. A simple perfusion system for mesenchymal stromal cell seeding to macroporous alginate cryogel sponges proposed in this study resulted in rapid and uniform distribution of cells within the whole volume of the scaffold preserving functional and morphological properties of the cells.

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