Non-cell adhesive hexanoyl glycol chitosan hydrogels for stable and efficient formation of 3D cell spheroids with tunable size and density

Three-dimensional (3D) culture systems that provide a more physiologically similar environment than conventional two-dimensional (2D) cultures have been extensively developed. Previously we have provided a facile method for the formation of 3D spheroids using non-adhesive N-hexanoyl glycol chitosan (HGC) hydrogel-coated dishes, but with limitations such as low gel stability and weak mechanical properties. In this study, chemically crosslinked hydrogels were prepared by photocrosslinking of methacrylated HGCs (M-HGCs), and their spheroidforming abilities were evaluated for long-term 3D cell cultures. The M-HGC hydrogels demonstrated not only enhanced gel stability, but also good spheroid-forming abilities. Furthermore, the M-HGC-coated dishes were effective in generating spheroids of larger size and higher cell density depending on the crosslinking density of the M-HGCs. These results indicate that our hydrogel-coated dish system could be widely applied as an effective technique to produce cell spheroids with customized sizes and densities that are essential for tissue engineering and drug screening.