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Integration of stem cells to injured tissues requires an appropriate delivery device and scaffolding system. In the present study we have developed an in vitro strategy to load and release adipose-derived mesenchymal stem cells (ASC) from chitosan microspheres (CSM) into a collagen gel scaffold. Porous CSM of uniform size and composition were prepared and used as a stem cell carrier. ASC were allowed to attach to the microspheres and infiltrate through the microsphere pores. The number of viable cells was counted in vitro, using MTT and Calcein acetoxymethyl ester (AM) assays, and it showed a proportional increase with seeding density and reached a maximum cell number by 24 h. The cells inside the microspheres remained metabolically active and viable, could be retrieved from the spheres, and maintained expression of stem-cell-specific markers. Electron microscopic evaluation of the cell-microsphere complex showed that the CSM were able to support cell attachment and that the cells had infiltrated into the pores of the microspheres. The ability of the cells to proliferate and differentiate into adipogenic- and osteogenic-like precursors indicates that the cells have maintained their multipotency after migration out of the microspheres. To mimic cell delivery into a tissue, ASC-loaded CSM were embedded in type-1 collagen scaffold by mixing them with type-1 collagen solution while inducing gelation. By 14 days the cells released into the collagen gel and were able to populate the entire scaffold. When observed through transmission electron microscopy, the cells align along the collagen fibrils with a characteristic fibroblast-like morphology. This study provides a model to capture pluripotent stem cells, expand their cell number within a biomaterial scaffold in vitro, and deliver within an appropriate matrix to repair damaged tissue.

Adipose-Derived Stem Cell Delivery into Collagen Gels Using Chitosan Microspheres