Bioprinting is an emergent technology that has already demonstrated the capacity to create complex and/or vascularized multicellular structures with defined and organized architectures, in a reproducible and high throughput way. Here, we present the implementation of a complex liver model by the development of a three-dimensional extrusion bioprinting process, including parameters for matrix polymerization of methacrylated gelatin, using two hepatic cell lines, Huh7 and HepaRG. The printed structures exhibited long-term viability (28 days), proliferative ability, a relevant hepatocyte phenotype and functions equivalent to or better than those of their 2D counterparts using standard DMSO treatment.This work served as a basis for the bioprinting of complex multicellular models associating the hepatic parenchymal cells, HepaRG, with stellate cells (LX-2) and endothelial cells (HUVECs), able of colonizing the surface of the structure and thus recreating a pseudo endothelial barrier. When bioprinted in 3D monocultures, LX-2 expression was modulated by TGF-β-1 toward the induction of myofibroblastic genes such as ACTA2 and COL1A1. In 3D multicellular bioprinted structures comprising HepaRG, LX-2 and endothelial cells, we evidenced fibrillar collagen deposition, which is never observed in monocultures of either HepaRG or LX-2 alone. These observations indicate that a precise control of cellular communication is required to recapitulate key steps of fibrogenesis. Bioprinted 3D co-cultures therefore open up new perspectives in studying the molecular and cellular basis of fibrosis development and provide better access to potential inducers and inhibitors of collagen expression and deposition.

Generating the proliferation of differentiated normal adult human hepatocytes is a major challenge and an expected central step in understanding the microenvironmental conditions that regulate the phenotype of human hepatocytes in vitro. In this work, we described optimized 3D culture conditions of primary human hepatocytes (PHH) to trigger two waves of proliferation and we identified matrix stiffness and cell–cell interactions as the main actors necessary for this proliferation. We demonstrated that DNA replication and overexpression of cell cycle markers are modulate by the matrix stiffness while PHH cultured in 3D without prior cellular interactions did not proliferate. Besides, we showed that PHH carry out an additional cell cycle after transient inhibition of MAPK MER1/2-ERK1/2 signaling pathway. Collagen cultured hepatocytes are organized as characteristic hollow spheroids able to maintain survival, cell polarity and hepatic differentiation for long-term culture periods of at least 28 days. Remarkably, we demonstrated by transcriptomic analysis and functional experiments that proliferating cells are mature hepatocytes with high detoxication capacities. In conclusion, the advanced 3D model described here, named Hepoid, is particularly relevant for obtaining normal human proliferating hepatocytes. By allowing concomitant proliferation and differentiation, it constitutes a promising tool for many pharmacological and biotechnological applications.