Microspheres with porous structures have been applied as microcarriers for cell culture in past decades.However, the utilization of internal pores in microcarriers for cell attachment,growth and interactions has been rarely reported.Meanwhile,microcarriers from green and sustainable raw materials are on an increasing demand in terms of biocompatibility,safety and abundant source.In this work,naturally-derived porous chitosan microspheres(CSM) free of toxic crosslinking and porogenic agents were developed ,which allowed cell attachment,growth and cell-cell interactions in the internal pores.Firstly, CSM with a macroporous structure were prepared through a combination method of emulsification and thermally induced phase separation(Fig.1).The morphology of CSM were studied, followed by the evaluations of cytotoxicity and blood compatibility (Fig.2).Then, L-02 was used to evaluate CSM as microcarriers for 3D cell culture.SEM and LSCM combining Live/Dead assay indicated the hepatocytes could well adhere and grow into the pores besides on the outer surface of the CSM.Furthermore,the liver function studies including the expressions of related genes and proteins,albumin secretion and urea synthesis demonstrated that the metabolism of hepatocytes cultured with CSM were normal compared with the free cells.To the best of our knowledge,this work presents the first report that cell attachment and growth as well as the cell-cell interactions were observed in the internal pores of CSM,indicating a “real” 3D cell culture with the potential high cell activity and density.Together with the green and sustainable nature and superior biocompatibility and safety,these porous CSM may have great potential as cell microcarriers for 3D cell culture in biomedical applications.

Green porous Chitosan microspheres as microcarriers for “real” 3D culture of hepatocytes

255th National Meeting (2018)

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