A 3D Human Liver Model of Nonalcoholic Steatohepatitis

Abstract

Background and Aims: To better understand nonalcoholic steatohepatitis (NASH) disease progression and to evaluate drug targets and compound activity, we undertook the development of an in vitro 3D model to mimic liver architecture and the NASH environment. Methods: We have developed an in vitro preclinical 3D NASH model by coculturing primary human hepatocytes, human stellate cells, liver endothelial cells and Kupffer cells embedded in a hydrogel of rat collagen on a 96-well plate. A NASH-like environment was induced by addition of medium containing free fatty acids and tumor necrosis factor-a. This model was then characterized by biochemical, imaging and transcriptomics analyses. Results: We succeeded in defining suitable culture conditions to maintain the 3D coculture for up to 10 days in vitro, with the lowest level of steatosis and reproducible low level of inflammation and fibrosis. NASH disease was induced with a custom medium mimicking NASH features. The cell model exhibited the key NASH disease phenotypes of hepatocyte injury, steatosis, inflammation, and fibrosis. Hepatocyte injury was highlighted by a decrease of CYP3A4 expression and activity, without loss of viability up to day 10. Moreover, the model was able to stimulate a stable inflammatory and early fibrotic environment, with expression and secretion of several cytokines. A global gene expression analysis confirmed the NASH induction. Conclusions: This is a new in vitro model of NASH disease consisting of four human primary cell-types that exhibits most features of the disease. The 10-day cell viability and cost effectiveness of the model make it suitable for medium throughput drug screening and provide attractive avenues to better understand disease physiology and to identify and characterize new drug targets.

Bioengineered Cystinotic Kidney Tubules Recapitulate a Nephropathic Phenotype

“…For instance, reproducing the cell-to-extracellular matrix interaction, recapitulating the expression of apoptosis-related genes, the increase of drug sensitivity, and mimicking tumor invasion processes are some of the traits that have shown to be better represented in 3D cancer models [ 55 , 56 , 57 ]. 3D liver disease models have also been reported advantageous over 2D cultures since they allow for the study of biliary excretion of metabolites, the functional establishment of cell polarity, and crucial processes in liver disease such as inflammation and fibrosis [ 58 , 59 , 60 ]. Additionally, having 3D cultures made it possible to study the metabolic interaction between diseased lung cells and cancer-associated fibroblasts [ 61 ], but many more examples have been published.…”

Advancements in MAFLD Modeling with Human Cell and Organoid Models

“…Despite these advantages, the direct co-culture of the different liver cell types in suspension or matrices did not result in the formation of structures resembling the liver tissue. In most reported co-culture approaches [ 47 , 50 , 126 , 154 , 155 , 156 , 157 ] ( Table 2 ), cells were homogenously mixed and randomly distributed across the 3D liver model formed. In contrast, the PSC differentiation process to generate organoids recapitulates morphogenesis events during embryonic development.…”

“…Co-culture of different hepatic and non-hepatic cells to form 3D spheroid cultures in suspension [ 154 , 155 , 156 ] or matrices [ 157 ]. The majority of these co-cultured spheroids do not recapitulate liver tissue structure [ 154 , 155 , 156 , 157 ].…”

“…The inclusion of fibroblast and stellate cell lines enable the modeling of fibrogenesis event, and the inclusion of Kupffer cells allow the modeling of inflammatory events [ 154 , 155 , 156 , 157 ].…”

Cell Models and Omics Techniques for the Study of Nonalcoholic Fatty Liver Disease: Focusing on Stem Cell-Derived Cell Models

“…Duriez et al developed a 3D NASH model by combining the culture of four cell types embedded in collagen hydrogel with exposure to glucose, NEFA, and TNF-α for 15 days. This model showed the accumulation of lipid droplets in the cytoplasm, the generation of a proinflammatory environment determined by increased IL6 and CCL2 expressions, and the induction of early fibrosis with the expressions of MMP2 and MMP9 [ 71 ].…”