Ultra-low content physio-chemically crosslinked gelatin hydrogel improves encapsulated 3D cell culture

Gelatin-based hydrogels are extensively used for 3D cell culture, bioprinting, and tissue engineering due to their cell-adhesive nature and tunable physio-chemical properties. Gelatin hydrogels for 3D cell culture are often developed using high-gelatin content (frequently 10-15 % w/v) to ensure fast gelation and improved stability. While highly stable, such matrices restrict the growth of encapsulated cells due to creating a dense, restrictive environment around the encapsulated cells. Hydrogels with lower polymer content are known to improve 3D cell growth, yet fabrication of ultra-low concentration gelatin hydrogels is challenging while ensuring fast gelation and stability. Here, we demonstrate that physical gelation and photo-crosslinking in gelatin results in a fast-gelling hydrogel at a remarkably low gelatin concentration of 1 % w/v (Gel^Phy/Photo). The Gel^Phy/Photo hydrogel was highly stable, allowed uniform 3D distribution of cells, and significantly improved the spreading of encapsulated 3T3 fibroblast cells. Moreover, human cholangiocarcinoma (HuCCT-1) cells encapsulated in 1 % Gel^Phy/Photo matrix grew and self-assembled into epithelial cysts with lumen, which could not be achieved in a traditional high-concentration gelatin hydrogel. These findings pave the way to significantly improve existing gelatin hydrogels for 3D cell culture applications.