Bioinspired tuning of glycol chitosan for 3D cell culture

Three-dimensional (3D) cell culture systems have promising applications compared with conventional two-dimensional cell culture systems. Herein, we report a facile method for the formation of 3D spheroids using novel thermo-reversible polysaccharide-based hydrogels. A series of thermo-reversible hydrogels consisting of N -acyl glycol chitosans (NAGCs) are synthesized through a simple N -acylation reaction, and the degree of acylation is finely tuned to obtain adequate thermo-reversible properties and gel stability. Among the NAGCs, N -hexanoyl glycol chitosan is the most thermo-sensitive and is highly effective for forming multi-cellular spheroids when used to coat the surfaces of cell culture dishes. Cell spheroids are effectively formed at various cell concentrations, and their spheroid shape and cellular functions are well maintained for longer times. The hydrogel culture system is also useful for co-cultures that mimic a biological microenvironment. Our thermo-reversible hydrogels may offer a convenient method for the development of in vitro 3D cell culture systems to provide enhanced performance in tissue regeneration, organ-on-chips, drug screening research and other biomedical applications. 3D cell cultures: from shrimp shells to hydrogels Modifying the crustacean-derived molecule chitosan produces thermosensitive hydrogels for realistic, spherical cell growth studies. Three-dimensional (3D) cell environments are better platforms for exploring cell structure and function than traditional Petri dishes, but typically require complex ‘hanging drop’ substrates or micromoulds. Researchers from the Korea Institute of Toxicology and Chungnam National University have developed a simpler 3D cell culture using a water-filled polysaccharide that can transform into a gel on demand thanks to the thermo-responsive property. In experiments, cells such as cardiomyocytes spontaneously aggregated to form spheroids after only 24 hours immersion in the chitosan-based hydrogel. Harvesting was performed using temperature-reduction techniques suitable for large-scale processes. Cytotoxicity tests revealed that these cultures were safe and could be used to co-culture different cells to realize better biomimicking. We developed glycol chitosan-based thermo-reversible hydrogels that can provide a facile, convenient and reproducible method for the formation of 3D cell spheroids. The spontaneous formation of spheroids on HGC-coated plates was completed within 1 day. Furthermor