In vitrolong term differentiation and functionality of three-dimensional bioprinted primary human hepatocytes: application forin vivoengraftment.

In recent decades, 3Din vitrocultures of primary human hepatocytes (PHHs) have been increasingly developed to establish models capable of faithfully mimicking main liver functions. The use of 3D bioprinting, capable of recreating structures composed of cells embedded in matrix with controlled microarchitectures, is an emergent key feature for tissue engineering. In this work, we used an extrusion-based system to print PHH in a methacrylated gelatin (GelMa) matrix. PHH bioprinted in GelMa rapidly organized into polarized hollow spheroids and were viable for at least 28 d of culture. These PHH were highly differentiated with maintenance of liver differentiation genes over time, as demonstrated by transcriptomic analysis and functional approaches. The cells were polarized with localization of apico/canalicular regions, and displayed activities of phase I and II biotransformation enzymes that could be regulated by inducers. Furthermore, the implantation of the bioprinted structures in mice demonstrated their capability to vascularize, and their ability to maintain human hepatic specific functions for at least 28 d was illustrated by albumin secretion and debrisoquine metabolism. This model could hold great promise for human liver tissue generation and its use in future biotechnological developments.

DMSO-free highly differentiated HepaRG spheroids for chronic toxicity, liver functions and genotoxicity studies.

The liver is essential in the elimination of environmental and food contaminants. Given the interspecies differences between rodents and humans, the development of relevant in vitro human models is crucial to investigate liver functions and toxicity in cells that better reflect pathophysiological processes. Classically, the differentiation of the hepatic HepaRG cell line requires high concentration of dimethyl sulfoxide (DMSO), which restricts its usefulness for drug-metabolism studies. Herein, we describe undifferentiated HepaRG cells embedded in a collagen matrix in DMSO-free conditions that rapidly organize into polarized hollow spheroids of differentiated hepatocyte-like cells (Hepoid-HepaRG). Our conditions allow concomitant proliferation with high levels of liver-specific functions and xenobiotic metabolism enzymes expression and activities after a few days of culture and for at least 4 weeks. By studying the toxicity of well-known injury-inducing drugs by treating cells with 1- to 100-fold of their plasmatic concentrations, we showed appropriate responses and demonstrate the sensitivity to drugs known to induce various degrees of liver injury. Our results also demonstrated that the model is well suited to estimate cholestasis and steatosis effects of drugs following chronic treatment. Additionally, DNA alterations caused by four genotoxic compounds (Aflatoxin B1 (AFB1), Benzo[a]Pyrene (B[a]P), Cyclophosphamide (CPA) and Methyl methanesulfonate (MMS)) were quantified in a dose-dependent manner by the comet and micronucleus assays. Their genotoxic effects were significantly increased after either an acute 24 h treatment (AFB1: 1.5-6 µM, CPA: 2.5-10 µM, B[a]P: 12.5-50 µM, MMS: 90-450 µM) or after a 14-day treatment at much lower concentrations (AFB1: 0.05-0.2 µM, CPA: 0.125-0.5 µM, B[a]P: 0.125-0.5 µM) representative to human exposure. Altogether, the DMSO-free 3D culture of Hepoid-HepaRG provides highly differentiated and proliferating cells relevant for various toxicological in vitro assays, especially for drug-preclinical studies and environmental chemicals risk assessment.

Generation of proliferating human adult hepatocytes using optimized 3D culture conditions.

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.

3D culture of HepaRG cells in GelMa and its application to bioprinting of a multicellular hepatic model.

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 TGFß-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.