Evaluation and Optimization of a Microcavity Plate-Based Human Hepatocyte Spheroid Model for Predicting Clearance of Slowly Metabolized Drug Candidates.

In vitro clearance assays are routinely conducted in drug discovery to predict in vivo clearance, but low metabolic turnover compounds are often difficult to evaluate. Hepatocyte spheroids can be cultured for days, achieving higher drug turnover, but have been hindered by limitations on cell number per well. Corning Elplasia microcavity 96-well microplates enable the culture of 79 hepatocyte spheroids per well. In this study, microcavity spheroid properties (size, hepatocyte function, longevity, culturing techniques) were assessed and optimized for clearance assays, which were then compared with microsomes, hepatocyte suspensions, two-dimensional-plated hepatocytes, and macrowell spheroids cultured as one per well. Higher enzyme activity coupled with greater hepatocyte concentrations in microcavity spheroids enabled measurable turnover of all 17 test compounds, unlike the other models that exhibited less drug turnover. Microcavity spheroids also predicted intrinsic clearance (CLint) and blood clearance (CLb) within threefold for 53% [9/17; average absolute fold error (AAFE), 3.9] and 82% (14/17; AAFE, 2.6) of compounds using a linear regression correction model, respectively. An alternate method incorporating mechanistic modeling that accounts for mass transport (permeability and diffusion) within spheroids demonstrated improved predictivity for CLint (12/17; AAFE, 4.0) and CLb (14/17; AAFE, 2.1) without the need for empirical scaling factors. The estimated fraction of drug metabolized by cytochrome P450 3A4 (fm,CYP3A4) using 3 µM itraconazole was within 25% of observed values for 6 of 8 compounds, with 5 of 8 compounds within 10%. In sum, spheroid cultures in microcavity plates permit the ability to test and predict clearance as well as fm,CYP3A4 of low metabolic turnover compounds and represent a valuable complement to conventional in vitro clearance assays. SIGNIFICANCE STATEMENT: Culturing multiple spheroids in ultralow attachment microcavities permits accurate quantitation of metabolically stable compounds in substrate depletion assays, overcoming limitations with singly cultured spheroids. In turn, this permits robust estimates of intrinsic clearance, which is improved with the consideration of mass transport within the spheroid. Incubations with 3 µM itraconazole enabled assessments of CYP3A4 involvement in hepatic clearance.

High throughput interrogation of human liver stellate cells reveals microenvironmental regulation of phenotype.

Liver fibrosis is a common feature of progressive liver disease and is manifested as a dynamic series of alterations in both the biochemical and biophysical properties of the liver. Hepatic stellate cells (HSCs) reside within the perisinusoidal space of the liver sinusoid and are one of the main drivers of liver fibrosis, yet it remains unclear how changes to the sinusoidal microenvironment impact HSC phenotype in the context of liver fibrosis. Cellular microarrays were used to examine and deconstruct the impacts of bio-chemo-mechanical changes on activated HSCs in vitro. Extracellular matrix (ECM) composition and stiffness were found to act individually and in combination to regulate HSC fibrogenic phenotype and proliferation. Hyaluronic acid and collagen III promoted elevated collagen I expression while collagen IV mediated a decrease. Previously activated HSCs exhibited reduced lysyl oxidase (Lox) expression as array substrate stiffness increased, with less dependence on ECM composition. Collagens III and IV increased HSC proliferation, whereas hyaluronic acid had the opposite effect. Meta-analysis performed on these data revealed distinct phenotypic clusters (e.g. low fibrogenesis/high proliferation) as a direct function of their microenvironmental composition. Notably, soft microenvironments mimicking healthy tissue (1 kPa), promoted higher levels of intracellular collagen I and Lox expression in activated HSCs, compared to stiff microenvironments mimicking fibrotic tissue (25 kPa). Collectively, these data suggest potential HSC functional adaptations in response to specific bio-chemo-mechanical changes relevant towards the development of therapeutic interventions. These findings also underscore the importance of the microenvironment when interrogating HSC behavior in healthy, disease, and treatment settings. STATEMENT OF SIGNIFICANCE: In this work we utilized high-throughput cellular microarray technology to systematically interrogate the complex interactions between HSCs and their microenvironment in the context of liver fibrosis. We observed that HSC phenotype is regulated by ECM composition and stiffness, and that these phenotypes can be classified into distinct clusters based on their microenvironmental context. Moreover, the range of these phenotypic responses to microenvironmental stimuli is substantial and a direct consequence of the combinatorial pairing of ECM protein and stiffness signals. We also observed a novel role for microenvironmental context in affecting HSC responses to potential fibrosis therapeutics.

Bioengineered Liver Models for Investigating Disease Pathogenesis and Regenerative Medicine.

Owing to species-specific differences in liver pathways, in vitro human liver models are utilized for elucidating mechanisms underlying disease pathogenesis, drug development, and regenerative medicine. To mitigate limitations with de-differentiated cultures, bioengineers have developed advanced techniques/platforms, including micropatterned cocultures, spheroids/organoids, bioprinting, and microfluidic devices, for perfusing cell cultures and liver slices. Such techniques improve mature functions and culture lifetime of primary and stem-cell human liver cells. Furthermore, bioengineered liver models display several features of liver diseases including infections with pathogens (e.g., malaria, hepatitis C/B viruses, Zika, dengue, yellow fever), alcoholic/nonalcoholic fatty liver disease, and cancer. Here, we discuss features of bioengineered human liver models, their uses for modeling aforementioned diseases, and how such models are being augmented/adapted for fabricating implantable human liver tissues for clinical therapy. Ultimately, continued advances in bioengineered human liver models have the potential to aid the development of novel, safe, and efficacious therapies for liver disease.

Microscale Collagen and Fibroblast Interactions Enhance Primary Human Hepatocyte Functions in Three-Dimensional Models.

Human liver models that are three-dimensional (3D) in architecture are indispensable for compound metabolism/toxicity screening, to model liver diseases for drug discovery, and for cell-based therapies; however, further development of such models is needed to maintain high levels of primary human hepatocyte (PHH) functions for weeks to months. Therefore, here we determined how microscale 3D collagen I presentation and fibroblast interaction affect the longevity of PHHs. High-throughput droplet microfluidics was utilized to generate reproducibly sized (∼300-µm diameter) microtissues containing PHHs encapsulated in collagen I ± supportive fibroblasts, namely, 3T3-J2 murine embryonic fibroblasts or primary human hepatic stellate cells (HSCs); self-assembled spheroids and bulk collagen gels (macrogels) containing PHHs served as controls. Hepatic functions and gene expression were subsequently measured for up to 6 weeks. We found that microtissues placed within multiwell plates rescued PHH functions at 2- to 30-fold higher levels than spheroids or macrogels. Further coating of PHH microtissues with 3T3-J2s led to higher hepatic functions than when the two cell types were either coencapsulated together or when HSCs were used for the coating instead. Importantly, the 3T3-J2-coated PHH microtissues displayed 6+ weeks of relatively stable hepatic gene expression and function at levels similar to freshly thawed PHHs. Lastly, microtissues responded in a clinically relevant manner to drug-mediated cytochrome P450 induction or hepatotoxicity. In conclusion, fibroblast-coated collagen microtissues containing PHHs display high hepatic functions for 6+ weeks and are useful for assessing drug-mediated CYP induction and hepatotoxicity. Ultimately, microtissues may find utility for modeling liver diseases and as building blocks for cell-based therapies.

Assessing the compatibility of primary human hepatocyte culture within porous silk sponges.

Donor organ shortages have prompted the development of alternative implantable human liver tissues for patients suffering from end-stage liver failure. Purified silk proteins provide desirable features for generating implantable tissues, including sustainable sourcing from insects/arachnids, biocompatibility, tunable mechanical properties and degradation rates, and low immunogenicity upon implantation. While different cell types were previously cultured for weeks within silk-based scaffolds, it remains unclear whether such scaffolds can be used to culture primary human hepatocytes (PHH) isolated from livers. Therefore, here we assessed the compatibility of PHH culture within porous silk scaffolds that enable diffusion of oxygen/nutrients through the pores. We found that incorporation of type I collagen during the fabrication and/or autoclaving of porous silk scaffolds, as opposed to simple adsorption of collagen onto pre-fabricated silk scaffolds, was necessary to enable robust PHH attachment/function. Scaffolds with small pores (73 ± 25 µm) promoted larger PHH spheroids and consequently higher PHH functions than large pores (235 ± 84 µm) for at least 1 month in culture. Further incorporation of supportive fibroblasts into scaffolds enhanced PHH functions up to 5-fold relative to scaffolds with PHHs alone and 2D co-cultures on plastic. Lastly, encapsulating PHHs within protein hydrogels while housed in the silk scaffold led to higher functions than protein hydrogel-only or silk-only controls. In conclusion, porous silk scaffolds containing extracellular matrix proteins can be used for the culture of PHHs ± supportive non-parenchymal cells, which can be further built on in the future to create optimized silk-based liver tissue surrogates for cell-based therapy.

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

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.