3-dimensional Culture Of Human Adipose Tissue-derived Stromal Cells In Hydrogel

Tissue engineering aims at theories and methods of life sciences and engineering science, to develop new clinic substitute for human tissue and apparatus. Its typical method is seeding cells in the artificial extracellular matrix (ECM) to form cells/artificial ECM constructs. The methods of 3-dimensional seeding and culture of cells in scaffolds play an important role during the whole tissue engineering strategy. In this study, human adipose tissue-derived stromal cells were harvested and seeded into chitosan/β-sodium glycerophosphate/collagen (C/GP/CO) hydrogel and cultured in vitro and in vivo. The biocompatibility of the hydrogel and the cell morphology, viability the expansion fold of hADSCs were investigated.The hADSCs were harvested and seeded into above hydrogel by mixing at low temperature, gelled at 37℃, then were cultured in vitro. The expansion fold of hADSCs was assayed using CCK-8 Kit. The cell morphology and viability were also detected by fluorescence staining and scanning electron microscope (SEM). For the animal experiment, biocompatibility of the C/GP/CO hydrogel was investigated using subcutaneous injection into SD rats, using C/GP hydrogel as the negative control. For in vivo study, hADSCs which had been adipose-induced in vitro for 1 week were harvested and mixed with C/GP/CO hydrogel and subcutaneously injected into nude mice. The adipogenic differentiation of hADSCs in C/GP/CO hydrogel in vivo was further observed by immunohistochemistry staining.The results of fluorescence staining and SEM showed that within 5 days of in vitro culture, the hADSCs were well adhering to the hydrogel with good morphology and the cell viability was high. Moreover, cells number increased by 30% after 7 days of culture. Immunostaining analysis showed that no acute inflammation, infection and necrosis were found after 4 weeks'implantation, indicating that the C/GP/Co hydrogel had good biocompatibility within 4 weeks. Immunohistochemistry staining results of cell-hydrogel complex implants after 4 weeks of implantation in vivo indicated that a great number of adipocytes generated with lipid droplets and spontaneous vascularization forming while the results of complex implants with non-induced hADSCs were negative.