Preparation of three‐dimensional macroporous chitosan–gelatin B microspheres and HepG2‐cell culture

Chitosan–gelatin B microspheres with an open, interconnected, highly macroporous (100–200 µm) structure were prepared via a three‐step protocol combining freeze‐drying with an electrostatic and ionic cross‐linking method. Saturated tripolyphosphate ethanol solution (85% ethanol) was chosen as the crosslinking agent to prevent destruction of the porous structure and to improve the biostability of the chitosan–gelatin B microspheres, with N‐(3‐dimethylaminopropyl)‐N′‐ethyl‐carbodiimide/N‐hydroxysuccinimide as a second crosslinking agent to react with gelatin A and fixed chitosan–gelatin B microspheres to attain improved biocompatibility. Water absorption of the three‐dimensional macroporous chitosan–gelatin B microspheres (3D‐P‐CGMs) was 12.84, with a porosity of 85.45%. In vitro lysozyme degradation after 1, 3, 5, 7, 10, 14, and 21 days showed improved biodegradation in the 3D‐P‐CGMs. The morphology of human hepatoma cell lines (HepG2 cells) cultured on the 3D‐P‐CGMs was spherical, unlike that of cells cultured under traditional two‐dimensional conditions. Scanning electron microscopy and paraffin sections were used to confirm the porous structure of the 3D‐P‐CGMs. HepG2 cells were able to migrate inside through the pore. Cell proliferation and levels of albumin and lactate dehydrogenase suggested that the 3D‐P‐CGMs could provide a larger specific surface area and an appropriate microenvironment for cell growth and survival. Hence, the 3D‐P‐CGMs are eminently suitable as macroporous scaffolds for cell cultures in tissue engineering and cell carrier studies. Copyright © 2014 John Wiley & Sons, Ltd.