Towards ready-to-use 3-D scaffolds for regenerative medicine: adhesion-based cryopreservation of human mesenchymal stem cells attached and spread within alginate-gelatin cryogel scaffolds.

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-to-use transplantation units for application in bone, cartilage or skin regenerative therapy.

Generation of bone grafts using cryopreserved mesenchymal stromal cells and macroporous collagen-nanohydroxyapatite cryogels.

Bone tissue engineering strategy involves the 3D scaffolds and appropriate cell types promoting the replacement of the damaged area. In this work, we aimed to develop a fast and reliable clinically relevant protocol for engineering viable bone grafts, using cryopreserved adipose tissue-derived mesenchymal stromal cells (MSCs) and composite 3D collagen-nano-hydroxyapatite (nanoHA) scaffolds. Xeno- and DMSO-free cryopreserved MSCs were perfusion-seeded into the biomimetic collagen/nanoHA scaffolds manufactured by cryotropic gelation and their osteoregenerative potential was assessed in vitro and in vivo. Cryopreserved MSCs retained the ability to homogenously repopulate the whole volume of the scaffolds during 7 days of post-thaw culture. Moreover, the scaffold provided a suitable microenvironment for induced osteogenic differentiation of cells, confirmed by alkaline phosphatase activity and mineralization. Implantation of collagen-nanoHA cryogels with cryopreserved MSCs accelerated woven bone tissue formation, maturation of bone trabeculae, and vascularization of femur defects in immunosuppressed rats compared to cell-free collagen-nanoHA scaffolds. The established combination of xeno-free cell culture and cryopreservation techniques together with an appropriate scaffold design and cell repopulation approach accelerated the generation of viable bone grafts.