Cost-effective robust synthesis of methacrylic cellulosic sponge for organoid culture

Abstract

We previously reported the development of a three-dimensional cellulosic sponge capable of constraining hepatocytes within macropores to support the rapid formation of organoids with preserved hepatocyte functions for hepatotoxicity testing applications. Fabrication of this macroporous sponge involves conjugating allyl groups onto hydroxypropyl cellulose (HPC) to serve as crosslinking sites during gamma irradiation following thermally-induced phase separation. However, this method requires the use of moisture-sensitive reagents and unstable organic solvents which introduces batch-to-batch variability. To address this problem, we developed a cellulosic sponge system which replaces the use of allyl groups as cross-linkers with methacrylic groups to generate methacrylic-HPC (MA-HPC) under fully aqueous conditions. The resulting MA-HPC sponge contains macropores (94 ± 8 μm, 90% porosity) to constrain cells to form organoids, and has an average elastic modulus of 8.5 kPa that is close to the modulus of native rat and human livers. We demonstrate that similar to the allyl-based sponge, the MA-HPC sponge reliably supports human hepatocyte organoid culture and maintains high level cellular functions for at least 1 week in culture, thereby providing a reliable alternative to the existing allyl-based sponge for organoid culture.