Biocasting bioorthogonal gelatin with expanded primary hepatocytes to engineer therapeutic liver grafts for acute liver failure

Therapeutic liver grafts (Livergraft, 10 mm × 3 mm) were developed by biocasting bioorthogonal gelatin with in vitro -expanded primary hepatocytes (3 × 10 7 cells/mL). The bioorthogonal gelatin composed of methyltetrazine-modified gelatin (Gel-T) and norbornene-modified gelatin (Gel N), which were synthesized by reacting methyltetrazine-NHS ester and cis-5-Norbornene- endo -2,3-dicarboxylic anhydride with the amino groups of gelatin, respectively. Uniform mixing of in vitro -expanded primary hepatocytes with Gel-T and Gel-N triggered an immediate bioorthogonal reaction between norbornene and methyltetrazine, resulting in solidification to form Livergraft within 10 min. The resulting Livergraft exhibited a well-defined 3D architecture with pore sizes of 100–400 μm. Remarkably, hepatocytes within Livergraft proliferated and self-organized into cell aggregates with an average diameter of 118 μm after three days of in vitro 3D culture. This strategy was faclie and efficient, requiring neither external triggers ( e.g. , UV light), Matrigel pre-incubation, nor specialized equipment. Livergraft transplantation effectively compensated liver function in mice undergoing 90 % hepatectomy, with five out of twelve mice surviving long-term. On the 5th day after hepatectomy, the caudate lobe was significantly enlarged, and the liver function indicators (ALT, AST, and ALP) were consistent with those of normal mice. Comparative characterization demonstrated that bioorthogonal gelatin outperformed GelMA in supporting hepatocyte function and tissue formation, demonstrating its potential as a promising material for functional liver tissue fabrication.