A 3D primary human cell-based in vitro model of non-alcoholic steatohepatitis for efficacy testing of clinical drug candidates

Abstract

Non-alcoholic steatohepatitis (NASH) is a progressive and severe liver disease, characterized by lipid accumulation, inflammation, and downstream fibrosis. Despite its increasing prevalence, there is no approved treatment yet available for patients. This has been at least partially due to the lack of predictive preclinical models for studying this complex disease. Here, we present a 3D in vitro microtissue model that uses spheroidal, scaffold free co-culture of primary human hepatocytes, Kupffer cells, liver endothelial cells and hepatic stellate cells. Upon exposure to defined and clinically relevant lipotoxic and inflammatory stimuli, these microtissues develop key pathophysiological features of NASH within 10 days, including an increase of intracellular triglyceride content and lipids, and release of pro-inflammatory cytokines. Furthermore, fibrosis was evident through release of procollagen type I, and increased deposition of extracellular collagen fibers. Whole transcriptome analysis revealed changes in the regulation of pathways associated with NASH, such as lipid metabolism, inflammation and collagen processing. Importantly, treatment with anti-NASH drug candidates (Selonsertib and Firsocostat) decreased the measured specific disease parameter, in accordance with clinical observations. These drug treatments also significantly changed the gene expression patterns of the microtissues, thus providing mechanisms of action and revealing therapeutic potential. In summary, this human NASH model represents a promising drug discovery tool for understanding the underlying complex mechanisms in NASH, evaluating efficacy of anti-NASH drug candidates and identifying new approaches for therapeutic interventions.

Targeted therapeutics and novel signaling pathways in non-alcohol-associated fatty liver/steatohepatitis (NAFL/NASH)

“… 318 , 319 In a 3D in vitro microtissue model, administration of Selonsertib decreased the measurements of specific disease parameters, such as the secretion of the profibrotic factor (procollagen type I), proinflammatory cytokines (TNF-α and IL-6), and chemokines (MCP-1, MIP-1α, IL-8, IP-10), in accordance with clinical observations. 320 In a short-term phase 2 trial, the administration of Selonsertib improved NASH and fibrosis (at least a one-stage improvement) in some patients (NCT02466516). 247 , 248 …”

Advancements in MAFLD Modeling with Human Cell and Organoid Models

“…This reduces the heterogeneity of organoids generated across batches, favoring drug testing and screening applications. These advantages were well demonstrated by the in vitro microtissue model reported by Ströbel et al, where the team co-cultured primary human hepatocytes, Kupffer cells, liver endothelial cells, and HSCs [ 154 ]. Human liver microtissues (hLiMTs) were cultured in suspension devoid of scaffolds, and MAFLD modeling was achieved with a 10-day treatment regime using NASH-inducing media composed of elevated glucose, fructose, and FFA.…”

“…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 ].…”