Microengineered cultures containing human hepatic stellate cells and hepatocytes for drug development

Abstract

In non-alcoholic steatohepatitis (NASH), hepatic stellate cells (HSC) differentiate into myofibroblast-like cells that cause fibrosis, which predisposes patients to cirrhosis and hepatocellular carcinoma. Thus, modeling interactions between activated HSCs and hepatocytes in vitro can aid in the development of anti-NASH/fibrosis therapeutics and lead to a better understanding of disease progression. Species-specific differences in drug metabolism and disease pathways now necessitate the supplementation of animal studies with data acquired using human liver models; however, current models do not adequately model the negative effects of primary human activated HSCs on the phenotype of otherwise well-differentiated primary human hepatocytes (PHHs) as in vivo. Therefore, here we first determined the long-term effects of primary human activated HSCs on PHH phenotype in a micropatterned co-culture (MPCC) platform while using 3T3-J2 murine embryonic fibroblasts as the control cell type since it has been shown previously to stabilize PHH functions for 4-6 weeks. We found that HSCs were not able to stabilize the PHH phenotype to the same magnitude and longevity as the fibroblasts, which subsequently inspired the development of a micropatterned tri-culture (MPTC) platform in which (a) micropatterned PHHs were functionally stabilized using fibroblasts, and (b) the PHH phenotype was modulated by culturing HSCs within the fibroblast monolayer at physiologically-relevant ratios with PHHs. Transwell inserts containing HSCs were placed atop MPCCs containing fibroblasts to confirm the effects of paracrine signaling between PHHs and HSCs. We found that while albumin and urea secretions were relatively similar in MPTCs and MPCCs (suggesting well-differentiated PHHs), increasing HSC numbers within MPTCs downregulated hepatic cytochrome-P450 (2A6, 3A4) and transporter activities, and caused steatosis over 2 weeks. Furthermore, MPTCs secreted higher levels of pro-inflammatory interleukin-6 (IL-6) cytokine and C-reactive protein (CRP) than MPCCs. Treatment of MPCCs with HSC-conditioned culture medium confirmed that HSC secretions mediate the altered phenotype of PHHs observed in MPTCs, partly via IL-6 signaling. Lastly, we found that NADPH oxidase (NOX) inhibition and farnesoid X receptor (FXR) activation using clinically relevant drugs alleviated hepatic dysfunctions in MPTCs. In conclusion, MPTCs recapitulate symptoms of NASH- and early fibrosis-like dysfunctions in PHHs and have utility for drug discovery in this space.

3D Primary Hepatocyte Culture Systems for Analyses of Liver Diseases, Drug Metabolism, and Toxicity: Emerging Culture Paradigms and Applications

“…Under hyperglycemic conditions, hepatocytes developed insulin resistance and subsequent steatosis . Furthermore, in the same model, coculture with activated stellate cells promoted steatogenesis that could be ameliorated by treatment with obeticholic acid, a bile acid analogue in clinical trials for the treatment of NASH …”

“…[99] Furthermore, in the same model, coculture with activated stellate cells promoted steatogenesis that could be ameliorated by treatment with obeticholic acid, a bile acid analogue in clinical trials for the treatment of NASH. [100] In spheroids, exposure to a mixture of free fatty acids (FFAs), insulin, and carbohydrates induced reversible accumulation of lipid droplets and was accompanied by features of insulin resistance ( Figure 4C). A reduction in the hepatocellular lipid content was observed by treatment with various antisteatotic agents, such as metformin and the antioxidant vitamin E, indicating a reversal of hepatic steatosis.…”

A Microphysiological System for Studying Nonalcoholic Steatohepatitis

“…(17) Therefore, to date, most in vitro NAFLD studies involving PHHs have focused on short-term exposure (48-72 hours) to free fatty acids (FFAs), allowing the study of transient responses to triglyceride challenge. (14)(15)(16) Some studies using advanced in vitro platforms, including micropatterned co-cultures of primary hepatocytes and murine fibroblasts, (18) a hemodynamic co-culture system (19) or bioprinted cultures of primary liver cells, (20) have started to demonstrate how exposure to glucose and FFAs can affect human hepatic cell types. Moreover, with the addition of transforming growth factor β (TGF-β), early stages of fibrosis can be detected.…”

Primary Human Hepatocytes Maintain Long-term Functions in Porous Silk Scaffolds Containing Extracellular Matrix Proteins

“…Such interactions have been long replicated in vitro by co-culturing primary hepatocytes with various liver-and non-liver-derived NPCs, including C3H/10T1/2 mouse embryo cells [38], 3T3-J2 murine embryonic fibroblasts [39], and human fibroblasts [40]. The 3T3-J2 fibroblasts, in particular, have been shown to express key liver-like molecules, such as decorin [41] and Tcadherin [42], which help these fibroblasts induce higher levels of functions in PHHs in 2D cocultures than primary human dermal fibroblasts (not shown), human liver-derived hepatic stellate cells [43], liver sinusoidal endothelial cells [44], and Kupffer cells [45]. The positive effects of 3T3-J2 on PHH functions have also been shown in 3D PEGDA-RGDS hydrogels [17].…”