Hierarchical microspheres with macropores fabricated from chitin as 3D cell culture.

3D cell culture is an urgent requirement for large-scale cell manufacture and structure-based tissue engineering; however, their preparation is still challenging due to the lack of a highly biocompatible culture environment for various types of cells. The excellent biocompatibility of chitin derived from crabs and shrimps has been proved before. Herein, for the first time, we designed and prepared hierarchical chitin microspheres with open tunable macropores using chitosan microspheres as a sacrificial template, based on their selective solubility. In our findings, the chitosan microspheres with various sizes were embedded in the chitin microsphere fabricated from NaOH/urea aqueous solution, and then were removed using a weak acid to form macropores with mean pore sizes of 63 μm, 94 μm and 135 μm. Importantly, the chitin microspheres having an average diameter of 240 μm with the macroporous structure exhibited good strength, as a result of the existence of nanofibrous networks. The chitin nanofibers played important roles in both enhancing the mechanical properties of PCMS and improving the adhesion, growth, and proliferation of cells due to the inherent bioactivity of chitin. Moreover, the chitin macroporous microspheres could support the growth of various mammalian cells, such as monkey fibroblasts, human hepatocytes and even human stem cells. The stem cells cultured in this microsphere maintained their 3D structure and multi-lineage differentiation capacity. This study provides a new "bottom-up" approach to create macroporous microspheres from a chitin solution, which could be used for large-scale human stem cell 3D culture and also as a promising candidate for the fabrication of excellent scaffolds.