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

Abstract

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.

Three-Dimensional Liver Culture Systems to Maintain Primary Hepatic Properties for Toxicological Analysis In Vitro

“…Albumin synthesis was elevated at day 6 of spheroid culture, and mRNA levels of CYP1A2 , CYP2B6 and CYP3A4 first dropped, then slightly increased at day 4 and were elevated 1.2–3 -fold at day 7 when compared to day 0 [ 77 ]. Additionally, others have shown that HepaRG spheroids (self-aggregated or bioprinted) could be cultured over several weeks and that those cultures maintained several hepatic properties: HepaRG spheroids were capable of (a) albumin [ 87 , 88 , 89 , 90 , 91 ] and urea [ 87 , 90 ] production, (b) displayed expression and activity of CYP1A2 [ 88 , 89 ], CYP2B6 [ 89 ] and CYP3A4 [ 86 , 88 , 89 , 90 , 92 ], (c) showed phase II enzyme activity [ 89 , 91 ], and (d) displayed cellular polarization shown by MRP2 [ 87 , 88 , 90 ] and Pgp [ 91 ] expression, and by F-actin bands, indicative of bile canalicular structures [ 89 , 91 , 92 ]. Regarding expression and activity of phase I enzymes, however, several studies have shown that basal levels in spheroids were unaffected, in part even slightly lower than in monolayer cultures.…”

“…This might be explained by the relatively high basal expression of several CYP enzymes in HepaRG cells. However, the authors of those studies have deemed it necessary to elevate expression and activity of CYPs by induction using β-naphthoflavone (for CYP1A2), phenobarbital (for CYP2B6) or rifampicin (for CYP2C9 and CYP3A4) to reach more relevant levels [ 87 , 89 , 91 , 93 ].…”

Recent advances in the development of in vitro liver models for hepatotoxicity testing

“…Drug hepatotoxicity testing showed increased sensitivity to APAP compared to 2D adhesion controls. In addition, 3D extrusion bioprinting was used to fabricate a complex liver model [106]. The bioprinted structure showed 28-day cell viability and liver function.…”

Bioprinting Au Natural: The Biologics of Bioinks

“…GelMA maintains high cellular viability with high shape integrity post-printing making it an excellent bioink for larger tissue constructs ( Figure 2 H) [ 69 ]. It has been extensively used in the bioprinting community to create a multitude of constructs, including skin, cartilage, tumors, cornea, blood vessels, and liver and cardiac tissues, capitalizing on the strong bioink–cell interactions and resulting in a bioink that can be used for a wide number of tissues [ 43 , 76 , 106 , 107 , 108 , 109 , 110 , 111 , 112 , 113 , 114 , 115 ].…”