3D bioprinting of multicellular constructs using HepG2 and HUVEC cells for in-vitro liver models

Abstract

Drug-induced liver injury is a major concern in drug development, often leading to drug failures, withdrawals, and cases of acute liver failure. Accurately predicting the liver’s response to the cytotoxic effects of drugs remains a challenge. While animal models have traditionally been used to study liver toxicity, they often fail to fully replicate human liver physiology. In-vitro models are being actively explored as more relevant alternatives. Among these, 3D bioprinting offers the potential for creating liver constructs that better mimic the complex architecture and cellular composition of the liver. However, a significant challenge with 3D bioprinting is the crosslinking of bioinks, which typically involves chemicals, high temperatures, or UV light conditions that can compromise cell viability and function. To address this issue, this study developed a chitosan–gelatin bioink that forms a gel at body temperature, eliminating the need for harsh crosslinking steps. We optimized bioprinting parameters, including deposition rate, substrate properties, and cell density, to improve cell viability and function. Results showed that slower deposition rates (30 µL/min) and soft chitosan substrates enhanced cell viability compared to rigid substrates. Multicellular bioprinting of HepG2 hepatocytes and human umbilical vein endothelial cells (HUVECs) maintained over 70 % viability by day 5, similar to controls. A 2:1 HUVEC-to-HepG2 ratio further increased urea production to physiological levels (200 mg per 10⁶ cells per day), with stability maintained through day 7. We also demonstrate that these chitosan–gelatin bioinks can be used to print co-culture models that physiologically mimic liver architecture. These models hold potential for future applications in studying drug-induced liver injury.