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 spheroid-forming 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. Schematic illustration of 3D cell spheroid formation using non-cell-adhesive hydrogel-coated dishes. [Display omitted] • Photocrosslinkable thermogelling M-HGCs were synthesized as new non-cell adhesive hydrogels for 3D cell culture. • The photocrosslinked M-HGC hydrogels exhibited enhanced physical stability and mechanical properties. • M-HGC-coated dishes demonstrated good spheroid-forming abilities and produced large spheroids with higher cell density. • M-HGC-coated dish system could be useful to produce diverse cell spheroids with customized sizes and densities. [ABSTRACT FROM AUTHOR]