Micropatterned Primary Hepatocyte Co-Culture (HEPATOPAC) for Fatty Liver Disease Modeling and Drug Screening

doi:10.21203/rs.3.rs-2370782/v1

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Micropatterned Primary Hepatocyte Co-Culture (HEPATOPAC) for Fatty Liver Disease Modeling and Drug Screening

https://doi.org/10.21203/rs.3.rs-2370782/v1

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published 22 Sep, 2023

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Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, progressive disorder and growing public health concern. To date, no treatments exist for NAFLD. To address this issue, considerable research has been undertaken in pursuit of new NAFLD therapeutics. Development of effective, high-throughput in vitro models are an important aspect of drug discovery. Here, a micropatterned hepatocyte coculture (MPCC) was used to model liver steatosis and NAFLD. The MPCC model (HEPATOPAC) involves cells patterned onto a standard 96-well plate, increasing throughput and allowing the cultures can be handled and imaged like 2D cultures. Treatment of MPCC with free fatty acids (FFA), high glucose and fructose (HGF), or a combination of both induces hepatic steatosis. Additionally, inclusion of Kupffer cells to generate a tri-culture (MPTC) increased lipid loading induced by steatotic media. MPCC treatment with ACC1/ACC2 inhibitors, as either a preventative or reversal agent showed efficacy against FFA induced hepatic steatosis. Drug induced steatosis was also evaluated. Treatment with valproic acid showed steatosis induction in a lean background, which was significantly potentiated in a fatty liver background. Additionally, these media treatments changed expression of fatty liver related genes. Treatment of MPCC with FFA, HGF, or a combination reversibly altered expression of genes involved in fatty acid metabolism, insulin signaling, and lipid transport. These changes were largely consistent with data from other clinical or in vivo fatty liver studies. Together, these data demonstrate that MPCC and MPTC are easy to use long-term functional in vitro models of NAFLD. MPCC and MPTC are able to replicate numerous clinical and in vivo NAFLD observations, allowing their broad utility for compound screening, drug toxicity evaluation and assessment of gene expression changes.

Biological sciences/Biotechnology/Assay systems

Biological sciences/Cell biology

Biological sciences/Drug discovery

Biological sciences/Biological techniques/Biological models

Biological sciences/Biological techniques/Imaging

Health sciences/Gastroenterology/Hepatology/Liver diseases/Non alcoholic fatty liver disease

Nonalcoholic fatty liver disease (NAFLD) is a common disorder characterized by hepatic steatosis ¹. Simple steatosis, the first phase of NAFLD, has been considered generally benign, however, several studies have demonstrated that NAFLD can develop as a result of- or in tandem to- more serious metabolic disorders including hypertension, hypercholesterolemia, insulin resistance, and type 2 diabetes ^2–4. Moreover, NAFLD will progress to nonalcoholic steatohepatitis (NASH) in approximately 25% of patients which, in addition to hepatic steatosis, involves liver inflammation and injury, hepatocellular ballooning, and fibrosis ⁵. Unlike NAFLD, NASH is significantly tied to liver-related mortality. NASH can eventually advance to severe liver cirrhosis, which potently increases the risk of hepatocellular carcinoma (HCC) development ^6,7. Although NAFLD is extremely common, with an estimated prevalence of 25% in the general population, very few diagnostic tests exist and there are no dedicated treatments for NALFD or NASH. Presently, the gold standard for NAFLD and NASH diagnosis is liver biopsy, though imaging-based techniques such as ultrasound, magnetic resonance spectroscopy (MRS), and magnetic resonance imaging (MRI) are often employed as non-invasive alternatives ⁸. Since no specific therapeutics have been approved for NAFLD and NASH, treatment typically revolves around controlling underlying disorders such as obesity, diabetes, and inflammation by both lifestyle adjustments and pharmaceutical interventions ⁹.

NAFLD and NASH progress over time; based on numerous signaling events. Initially, sustained consumption of excess fat and sugar contribute to lipid accumulation in the liver. Often this occurs indirectly due to release of fatty acids from visceral fat stores into circulation which overloads the fatty acid oxidative capacity of the liver ¹⁰. Eventually, excess lipid in hepatocytes can lead to hepatic damage and release of inflammatory mediators by liver immune cells. Monocytes and macrophages in the liver, including the liver resident Kupffer cells, have been shown to play a particularly important role in inducing and perpetuating the inflammatory environment found in fatty liver disease ¹¹. One previous study demonstrated that fatty acids influence Kupffer cell polarization. In this study palmitic acid (PA), a saturated fatty acid, promoted an M1 pro-inflammatory phenotype in Kupffer cells; while oleic acid, an unsaturated fatty acid, induced an anti-inflammatory M2 Kupffer cell phenotype ¹². Furthermore, in vitro and in vivo studies suggest that Kupffer cell mediated inflammation increases lipid accumulation in hepatocytes ^13,14. In addition to increasing steatosis, Kupffer cell mediated inflammation can also contribute to insulin resistance, oxidative stress, and further perturbation of nutrient metabolism ¹¹. Combined, these factors along with others can cause chronic liver damage leading to progressive fibrosis and contribute to NASH development.

The lack of approved therapeutics for NAFLD and NASH underscores the need for continued preclinical research and screening. In vivo models have been useful in improving the mechanistic understanding of fatty liver disease, as well as in observing long term effects of disease and potential drug treatments. On the other hand, higher throughput in vitro models are also needed for screening purposes as well as to facilitate studies that may be difficult or costly to perform in vivo. Simple in vitro models use hepatic cell lines or primary cells plated in monoculture. Typically, these cells are treated with free fatty acids (FFA) and/or sugars such as glucose and fructose ^15,16. Studies have demonstrated the ability of monoculture cells to accumulate lipid in response to excess nutrients or steatosis inducing drugs ^16,17. Although consistent and easy to use, these simple models lack long term hepatic function and do not recapitulate interactions with other nonparenchymal cells (NPC) of the liver. Three dimensional models, including spheroids and organ on a chip, have demonstrated long-term hepatic functions and recapitulated hepatocyte-NPC interactions in fatty liver disease models, however, difficulty in handling, low-throughput, and challenging imaging protocols can limit their use ¹⁶.

Here, we demonstrate the utility of a micropatterned primary human hepatocyte co-culture (MPCC)- the HEPATOPAC® system- for modeling steatosis and aspects of NAFLD. HEPATOPAC contains primary hepatocytes in optimized co-culture with 3T3-J2 stromal cells which provide both soluble and contact-based signals that allow the hepatocytes to retain hepatic function (i.e., albumin, urea, and drug metabolizing enzyme activity) for at least 28 days ^18,19. The HEPATOPAC system also allows the addition of Kupffer cells to form the HEPATOMUNE® model, which is a micropatterned tri-culture (MPTC) of primary human hepatocytes, 3T3-J2 stromal cells, and primary human Kupffer cells. which will further improve the translatability of the system. Importantly, the model utilizes standard 24-well and 96-well plate formats allowing for incorporation into normal handling and imaging workflows.. In these studies, we show that MPCC load lipids in response to FFA, high glucose and fructose (HGF), or a combination of FFA and HGF and that these media induce fatty liver related gene changes. Furthermore, we demonstrate the influence of Kupffer cells on lipid loading and cell viability under inflammatory and lipid loading conditions. Using these model conditions, we showed function of this model for examining lipid lowering effects of therapeutics, drug induced steatosis, and steatosis induced gene changes. Together, these studies demonstrate the utility of the MPCC and MPTC systems in examining hepatic steatosis under a variety of conditions. Notably, since this system is handled like traditional 2D hepatocyte culture, the longevity and high functionality of this system is comparable to other complex culture systems such as spheroid co-cultures or organ on a chip system; while adding ease of use, higher throughput, and simplicity in imaging and sample collection.

Materials

HEPATOPAC (MPCC) and HEPATOMUNE (MPTC) cultures, primary hepatocytes and Kupffer cells were sourced from BioIVT. Sodium oleate, sodium palmitate, PF-05175157 were purchased from Sigma-Aldrich. Firsocostat and MK-4074 were purchased from Medchem Express. BODIPY 493/503, formalin, Lipofectamine RNAi MAX transfection reagent purchased from Thermo-Fisher Scientific. Valproic acid was purchased from Cayman Chemicals. Cell Titer Glo→ 2.0 cell viability reagents were purchased from Promega.

Micropatterned hepatocyte co-culture

MPCC (HEPATOPAC) cultures were created using patented microfabrication processes to generate an organized co-culture containing cryopreserved primary human hepatocytes (PHH) from BioIVT (Baltimore, MD) and 3T3-J2 murine embryonic fibroblasts. Briefly, cryopreserved primary human hepatocytes from one or more donor lots: TWJ, EUJ, LFQ were thawed at 37°C, counted, and plated on collagen-patterned 96 or 24 well plates. These cells were wildtype for PNPLA3 I148M (rs738409) mutation and the source tissue was assessed as not having existing NASH by NAS score. The cells were allowed to adhere until the following day, when 3T3-J2 fibroblasts were introduced to the culture plates. The resulting culture contained multiple hepatocyte islands having a 500 µm diameter spaced 1200 µm apart, center-to-center; with 3T3-J2 murine fibroblasts filling the area surrounding the hepatocyte islands. MPCC cultures were stabilized in serum containing medium for 5–7 days. At maturity, approximately 3,100 hepatocytes and 15,000 fibroblasts were present/well in a 96 well plate and about 21,000 hepatocytes and 90,000 fibroblasts were present/well in a 24 well plate. If applicable- MPTC (HEPATOMUNE) were manufactured following MPCC maturation. Cryopreserved primary human Kupffer cells from BioIVT were thawed, counted, and added to the mature cultures on day 7 at a hepatocyte: Kupffer cell ratio of 1:0.4.

Lipid Loading

Hepatocyte steatosis was induced following MPCC and MPTC culture stabilization. Cells were treated with media containing 0.5 mM free fatty acid (FFA) (Oleate/Palmitate 1:2, Sigma), 10g/L glucose and 1.0g/L fructose (HGF), or a combination of FFA and HGF with media exchanges every other day. To assess the impact of steatosis and/or inflammation on cell viability MPCC and MPTC cultures were incubated in +/- 0.5mM FFA + high glucose and +/- 50 ng/mL LPS for 4 days.

Drug treatment

For steatosis prevention studies, drug treatment of MPCC cultures with Firsocostat (6.0 µm), PF-05175157 (5.0 µm), and MK-4074 (300 nM) was begun 72 hours prior to lipid loading and continued for an additional 4 days under lipid loading conditions. For steatosis rescue studies, MPCC cultures were treated with lipid loading media for 7 days with drug treatment initiated on Day 7 either with or without continued treatment with the steatosis inducer (FFA or HGF) for 3 days. For Firsocostat concentration-response steatosis rescue experiments, MPCC cultures were treated with FFA medium for 5 days and treated with Firsocostat (11.7 nM, 23.4nM, 46.8 nM, 93.8 nM, 187.5 nM, 375 nM, 750 nM, 1.5 µM, 3.0 µM, 6.0 µM, 12.0 µM, and vehicle control) for 3 days in continued FFA medium. For drug induced steatosis experiments MPCC cultures were treated with escalating concentrations of valproic acid (0.39 mM, 0.78 mM, 1.56 mM, 6.25 mM, 12.5 mM, and 25.0 mM) in the presence or absence of FFA or HGF for 8 days. At the end of each experiment the cells were fixed, stained, and imaged.

Cell viability assessment

ATP as a measure of cell viability was assessed using the Cell Titer Glo→ 2.0 (PromegaⓇ) according to manufacturer’s protocol. The luminescence was measured using a Perkin Elmer Wallac 1420 Victor2 microplate reader. All luminescence values were normalized and presented as percent of control.

Staining

For all assays, culture plates were fixed with 10% formalin for 10 minutes at room temperature (RT). And washed 3 times with 1X PBS. Fixed plates were stained with BODIPY 493/503 (2 µM in PBS) for 1 hour RT and washed with PBS. Fixed cells were then incubated with DAPI (0.5 µg/mL in 1X BD permeabilization solution) for 10 minutes at RT and washed with PBS prior to imaging. Fluorescent imaging was performed using a Perkin Elmer Operetta CLS high content imager. Images were taken at 20X (35 fields/well) and 5X (whole well).

Lipid quantification using HCI

Lipid staining was quantified using the Harmony 4.8 software included with the Operetta CLS high content imager. An Alexa 488 (BODIPY) global image (full well) was generated, and an image region was defined with specific intensity thresholds to only identify lipid staining. The sum of area (area that stained with BODIPY 493/503 lipid dye) was determined for each well. For drug induced steatosis assessment, the protocol was modified to account for cell loss due to drug toxicity. In that case, full cell islands or cell clusters were identified and the ratio of the sum intensity of BODIPY stain to the sum of area of the cells was measured.

RNA isolation, RT Profiler PCR Array, and analysis

RNA was extracted from cells treated with FFA, HGF, FFA + HGF and controls using the RNAeasy kit (Qiagen, Germany) as per manufacturer’s instructions. RNA was quantified spectrophotometrically, and all samples were normalized to the lowest concentration in the group. cDNA synthesis was performed using the RT2 First Strand Kit (Qiagen, Germany). The PCR reaction was setup on the RT² Profiler™ PCR Array Human Fatty Liver, PAHS-157Z (Qiagen, Germany). PCR was performed and analyzed using the QuantStudio™ 5 Design & Analysis Software v1.5.1 (Applied Biosystems, USA). A melt curve analysis was performed to verify PCR specificity.

Statistical analysis

The mean + SEM was graphed for each group based on global area of at least n = 3 wells. A one-way ANOVA with Tukey post-test was performed, when relevant, using GraphPad Prism 5.0 to determine difference between each group. For assessment of differences between multiple groups and concentrations a two-way ANOVA with Bonferroni post-test was performed in GraphPad Prism 5.0. To determine slope, R² value, and to test statistical difference between slopes when multiple groups and concentrations were assessed a linear regression was performed using GraphPad Prism 5.0. PCR array data was analyzed using the Qiagen GeneGlobe data analysis tool. Cycle count data were uploaded and normalized to the intrasample arithmetic mean of multiple housekeeping genes (ACTB, B2M, GAPDH, HPRT1, and RPLP0). Significantly up-regulated genes were determined from a composite of 3 separate samples where fold regulation was at least 2 and p value was at least 0.05. Where relevant, * = p < 0.05, **=p < 0.01, and ***=p < 0.001 as compared to the relevant control. # = p < 0.05, ## =p < 0.01, and ### =p < 0.001 as compared to the relevant experimental group.

Free Fatty Acid treatment induces lipid loading in MPCC and MPTC cultures.

In vitro models of fatty liver disease often involve inclusion of common dietary fatty acids, such as oleate and palmitate, in the culture media. Previous studies have demonstrated differences between oleate and palmitate in regard to their cytotoxicity, with palmitate showing greater toxicity that is reduced when it is used in combination with oleate ²⁰. Based on these prior observations, steatosis was induced in MPCC cultures by treating with 0.5 mM FFA, which contained oleate and palmitate at a 2:1 ratio. Cells were treated with 0.5 FFA for 4 days. Lipid loading was assessed by high content imaging and quantification of BODIPY493/593 dye staining (Fig. 1A, B). For quantification, a threshold was set to identify lipid staining in hepatocytes, but not background autofluorescence (Fig. 1C). Following threshold establishment, lipid loading in the entire well was quantified (Fig. 1D). These data demonstrate the methods for identifying and quantifying lipid by high content imaging and show the ability of the MPCC model to respond to a steatogenic stimulus.

Treatment with FFA, High Glucose and Fructose (HGF), or a combination of both induce reversible hepatic steatosis in MPCC and MPTC.

Multiple dietary factors including consumption of a diet high in fat and sugar contribute to NAFLD development ¹. To determine if media composition of fat and/or sugar plays a role in PHH steatosis induction and reversal MPCC or MPTC cultures were treated with either 0.5 mM FFA (FFA), 10g/L glucose + 1.0g/L fructose (HGF), or a combination of both (FFA + HGF) for 4 days, followed by either a return to control medium or continuation of steatotic media for an additional 3 days. In MPCC cultures, all media induced significant hepatic steatosis as compared to control wells (Fig. 2A-D, H-J). Wells treated with HGF developed less steatosis than those treated with FFA (Fig. 2B-C, H-I). Differences in the potency of the selected concentrations, as well as the distinct mechanisms by which each forcing agent induces steatosis (active lipogenesis vs. passive loading respectively) may underlie these discrepancies. When the MPCC cultures were treated with FFA + HGF the amount of lipid loading was additive (Fig. 2D, J) (NT area 8.15x10⁵ ± 1.89x10⁵, FFA 3.16x10⁶ ± 1.66x10⁵, HGF 2.06x10⁶ ± 3.42x10⁵, FFA + HGF 6.01x10⁶ ± 2.30x10⁵). In MPTC cultures, where primary human Kupffer cells were also included in the culture, FFA and HGF induced lipid loading to a larger extent than in MPCC cultures, which did not contain Kupffer cells. In control wells, and those treated with FFA + HGF, there was no significant difference between lipid loading with or without Kupffer cells. This may be due to saturated loading in the FFA + HGF treated wells, nearly the entire hepatocyte island was consumed by steatosis with this treatment (Fig. 2A-D, H-J). As in the MPCC cultures, the HGF treatment causes less hepatic steatosis as compared to the FFA (Fig. 2B-C, H-I) (NT area 1.51 x10⁶ ± 8.98x10⁴, FFA 4.69x10⁶ ± 1.25x10⁵, HGF 2.94x10⁶ ± 1.77x10⁵, FFA + HGF 5.72x10⁶ ± 1.42x10⁵).

In both MPCC and MPTC cultures, lipid loading was able to be at least partially reversed when cultures were switched to control media for 3 days post lipid loading. In MPCC cultures treated with FFA and HGF alone lipid loading was completely reversed equivalent to non-treated control levels (Fig. 2E-F, K-L). Wells treated with FFA + HGF, however, still retained a significant amount of lipid at the end of the wash-out period (P < 0.0001) (Fig. 2G, M) (NT area 8.15x10⁵ ± 1.89x10⁵, FFA reversal 1.32x10⁶ ± 4.91x10⁴, HGF 7.93x10⁵ ± 1.00x10⁵, FFA + HGF 2.67x10⁶ ± 3.27x10⁵). When cultures contained Kupffer cells, both the FFA and FFA + HGF treated cultures had significant lipid remaining after change to control media (Fig. 2E-F, K-L). Additionally, the unloaded lipid content was significantly higher in MPTC cultures compared to MPCC (Fig. 2K-M). In MPTC cultures, only the cells treated with HGF completely reversed their lipid loading (Fig. 2F, L) (NT area 1.51 x10⁶ ± 8.98x10⁴, FFA reversal 2.77x10⁶ ± 1.02x10⁵, HGF 1.76x10⁶ ± 1.27x10⁵, FFA + HGF 3.82x10⁶ ± 1.77x10⁵). Still, the absolute amount of lipid remaining in the unloaded HGF treated was higher in MPTC cultures that MPCC. These studies show that both FFA and HGF can induce hepatic steatosis in MPCC and MPTC models. Additionally, the presence of resident immune cells, such as Kupffer cells, influence not only the amount of steatosis induced, but also the ability of the cells to clear the lipid once the steatotic forcing agent has been removed.

Treatment with ACC1/2 inhibitors prevents steatosis in MPCC.

In vitro drug screening is a useful initial step to identify lead compounds for liver steatosis reduction. ACC1/2 inhibitors have been shown to reduce hepatic steatosis in vivo ^21–24. To determine if these findings could be recapitulated in MPCC, cultures were treated with FFA, and 3 different ACC1/2 inhibitors were used to either prevent hepatic steatosis or reverse existing steatosis. Control cells had little background lipid, which was greatly increased after FFA treatment (Ctrl lipid area 8.02 x10⁴ ± 1.94x10⁴, FFA 1.26 x10⁶ ± 5.13x10⁴) (Fig. 3A, B). To test steatosis prevention, treatment with ACC1/2 inhibitors Firsocostat (6.0 µM, 0.6 µM, and 0.06 µM), PF-05175157 (5.0 µM, 0.5 µM, and 0.05 µM), and MK-4074 (0.3 µM, 0.03 µM, 0.003 µM) was begun 72 hours prior to treatment with FFA and continued for an additional 4 days. For cells treated with FFA, all compounds showed an incomplete, but concentration dependent prevention of steatosis (Firsocostat (6.0 µM, 0.6 µM, and 0.06 µM) 3.51 x10⁵ ± 9.09x10⁴_, 4.02x10⁵ ± 1.19x10⁵, 5.34 x10⁵ ± 1.21 x10⁵ respectively), (MK-4074( 0.3 µM, 0.03 µM, 0.003 µM) 1.54x10⁶ ± 9.00x10⁴, 1.06 x10⁶ ± 1.85 x10⁵, 6.63 x10⁵ ± 1.61x10⁵ respectively), (PF-05175157 (5.0 µM, 0.5 µM, and 0.05 µM) 1.54 x10⁶ ± 1.58x10⁵, 8.77x10⁵ ± 1.89x10⁵, 5.11x10⁵ ± 1.30x10⁵ respectively) (Fig. 3C-E, quantified in 3F). Of the compounds tested, Firsocostat showed the most complete prevention over the three concentrations that were tested. These data demonstrate the ability of the MPCC model to perform longer term screening assays for compounds that can prevent steatosis.

Established Steatosis Can Be Reversed By Acc1/2 Inhibitors

Drug based therapies for fatty liver disease may be used to treat established disease with or without concomitant dietary changes. To examine the ability of ACC1/2 inhibitors to reverse existing steatosis in MPCC, cultures were treated with FFA for 7 days followed by an additional three days of drug treatment. For this experiment, only the highest concentration of each compound (6.0 µM Firsocostat, 5.0 µM PF-05175157, and 0.3 µM MK-4074) was tested to maximize the effect. In these studies, dietary changes were simulated by either switching the cells to control media for the three-day treatment or continuing to treat with the ACC1/2 inhibitors in the steatotic media. In cells treated with control medium, minimal lipid loading was observed (Fig. 4A). In cells treated with FFA with continuous steatosis (Ctrl Lipid area = 2.16x10⁵ ± 3.76x10⁴, FFA = 9.29x10^5 ± 1.18x10⁴), all compounds were able to remove some lipid as compared to wells treated with FFA alone (Firsocostat = 2.45x10⁵ ± 4.89x10⁴, MK-4074 = 3.50x10⁵ ± 1.41x10⁴, PF-05175157 = 6.99x10⁵ ± 460). Additionally, lipid content in cells treated with Firsocostat and MK-4074 with continuous FFA was not significantly different compared to non-treated controls (Fig. 4B, quantified in Fig. 4D). When FFA was removed for three days, cells that were initially steatotic were able to return to NT control levels without any drug treatment (FFA removed = 2.40x10⁵). When ACC1/2 inhibitors were added, lipid levels were further reduced to below control levels (Firsocostat = 9.54x10³ ± 1.14x10³, MK-4075 = 2.54x10⁴ ± 5.15x10³, PF-05175157 = 2.55x10⁴ ± 2.79x10³) (Fig. 4C, quantified in Fig. 4E). Finally, Firsocostat was selected to perform an 11-point concentration curve on PHH treated with FFA for 4 days, followed by 3 days of drug treatment in continuous FFA media. Firsocostat reversed lipid loading under continuous steatotic pressure in a concentration dependent manner (Fig. 5A). Data were normalized to no drug controls and a nonlinear fit of the log transformed concentrations was performed to obtain an EC50 for Firsocostat lipid reduction in MPCC (computed EC50 = 4.793 µM) (Fig. 5B). This concentration is similar to those shown to be efficacious in another 3D-cell culture model of fatty liver disease ²⁵. These data show the ability of the MPCC model to screen compounds for steatosis reversal ability. These experiments also demonstrated the increased efficacy of lipid lowering drugs when a dietary change was simulated (return to control media) when FFA was used to induce steatosis.

Valproic Acid Induces Lipid Loading Alone And Exacerbates Existing Steatosis In Mpcc

Drug-induced metabolic dysfunction is another method by which liver steatosis is induced. Drug induced steatosis (DIS), a type of drug-induced liver injury (DILI), occurs when a drug impairs hepatic metabolism of fatty acids. In addition to causing new steatosis, DIS can also exacerbate underlying fatty liver disease ²⁶. For example, valproic acid, a drug used in clinical practice, has been demonstrated to cause DIS. To evaluate whether the MPCC model can be used to screen for DIS, the ability of VPA to induce steatosis in the presence or absence of a fatty liver background was tested. When MPCC cultures were treated with VPA (0.39-12 µM) for 8 days under normal media conditions, VPA induced hepatic steatosis starting at 3.13 mM as compared to NT control (Fig. 6A, D). Concurrently, MPCC cultures were treated with VPA (0.39-12 µM) in the presence of 0.5 mM FFA or high glucose and fructose (HGF). On its own, treatment with 0.5mM FFA induced hepatic lipid loading as compared to NT controls (Fig. 6B, D). When VPA was added to FFA treated cultures lipid loading increased for all concentrations of VPA assessed as compared to both NT controls and FFA treatment alone (Fig. 6B, D). When VPA treatment was added to HGF treated cultures, VPA potentiated lipid loading began with the 0.78 mM treatment (Fig. 6C, D). When toxicity was testing using ATP, the TC₅₀ were similar between media and only slightly lower in the FFA treated cells (95% CI for TC50: Ctrl 3.213–4.197 mM, FFA 2.162–2.945 mM, HGF 2.580–3.314) (Fig. 6E). Notably, the clinical C_max for VPA is between 30–100 mg/mL (200–700 µmol/L) ²⁷ which encompasses the lowest concentration tested here (0.39 mM) and is just below the second lowest concentration of VPA assessed here (0.78 mM). In these studies, these low concentration only showed DIS potential in the steatotic background. These studies show not only that the MPCC system is suitable for in vitro testing for DIS, but also that testing compounds for DIS in a fatty liver background may uncover a drug’s propensity to contribute to DIS that may not have been discovered under testing in a lean background.

Treatment with FFA, HGF, or a combination of both induces reversible changes in gene expression.

NAFLD and NASH have been shown to induce numerous gene changes. In order to assess gene changes induced by different steatotic media, samples were run on a PCR array targeting 84 genes implicated in human fatty liver disease. Cell lysates were collected and prepared for analysis from cells treated with FFA, HGF, or FFA + HGF for 4 days followed by either a return to control medium or continuation of steatotic media for an additional 3 days. The cut-off value used for significant expression was set as expressed fold changes of at least 2.0 (as compared to control media treatment) and a p-value of < 0.05. Volcano plots were used to identify changes in gene expression between control and treatment groups. When MPCC cultures were loaded with FFA, IGFBP-1, CYP7A1, and GCK were downregulated and FABP1, PDK4, ACSL5, CPT1A, and CD36 were upregulated (Fig. 7A). Treatment with HGF induced downregulation of FABP1, IGF1, and CYP2E1, and upregulation of FASN, PKLR, SCD, and SLC2A4 (Fig. 7B). Lastly, when MPCC cultures were treated with FFA + HGF, IGF1, PCK2, SLC2A2, SLC27A5, and CYP2E1 were downregulated, and SERPINE1, LDLR, SLC2A1, FASN, FABP5, ACLY (Fig. 7C). When genes with differential expression are assigned a group based on their function (as designated by the array manufacturer), genes involved in insulin signaling and cholesterol metabolism and transport were commonly down-regulated among all treatment groups and genes involved in carbohydrate metabolism, and lipid metabolism and transport were commonly up-regulated. Differentially expressed gene groups in cells treated with a combination of FFA + HGF seemed to be a combination of those seen with FFA alone and HGF alone, however seemed to favor HGF induced changes (Fig. 8A). Hierarchical clustering supports this observation, showing that global gene expression levels from this array in cells treated with FFA + HGF more closely resemble those treated with HGF than FFA, suggesting the HGF effect is more dominant (Fig. 8B).

Gene expression in cells that were switched to control medium for 3 days largely resembled cells treated with control medium, corresponding with the lipid staining showing at least partial reversal of lipid loading. When MPCC cultures were treated with FFA followed by control medium no gene expression remained changed as compared to control (Fig. 7D). Cells treated with HGF followed by control medium retained more gene changes post reversal. In these cells IGFBP-1 was down-regulated and, GCK, SLC2A4, SCD, and CD36 were upregulated (Fig. 7E). When cells were treated with a combination of FFA + HGF, IGFBP-1 was down-regulated and CD36 remained up-regulated (Fig. 7F). Hierarchical clustering supports the observation that lipid loading, and related gene changes, are partially reversed upon switching back to control medium (Fig. 8B). Together these data demonstrate that treatment of MPCC cultures with steatotic inducers such as FFA, HGF or a combination of FFA and HGF elicit expression changes in genes relevant to human fatty liver disease. Furthermore, these data further support the observation that these changes are partially reversed when cultures are switched to control medium.

NAFLD and NASH are a prevalent and growing public health concern. Despite an urgent need, currently no dedicated therapeutics have been approved which can improve disease outcomes. Effective, high-throughput preclinical models, including in vitro models, are an important aspect of drug discovery. Several in vitro models have been developed for NAFLD, including those which use hepatic cell lines, PHH, or PHH in combination with liver NPCs. Of the models that use PHH, the easiest and most accessible is a standard 2D culture with or without NPCs. Unfortunately, PHH plated in this manner quickly lose hepatic specific function, such as DME activity and urea and albumin synthesis, which may be particularly important for longer-term screening assays. On the other hand, in vitro models of fatty liver disease which offer complexity and long-term function often come at the expense of throughput, ease of use, or accessible endpoint collection. In these studies, we aimed to develop a long-term model of hepatic steatosis in MPCC and MPTC, which are micropatterned co-cultures of primary human hepatocytes, 3T3-J2 stromal cells, and primary human Kupffer cells (MPTC) which can maintain the throughput and ease-of-use from standard 2D PHH cultures. In this model, steatosis was induced by FFA, specifically palmitate and oleate, high sugars (HGF), or a combination of both; strategies which parallel dietary drivers of fatty liver disease used in vivo as well as those seen clinically. All media conditions induced steatosis and were at least partially reversible either through drug treatment or by restoring cells to control medium. These observations demonstrate the utility of this model for screening studies with in vivo relevance.

Progression of NAFLD severity is, in part, driven by inflammatory signals from liver immune cells. In particular, in vivo ablation studies have demonstrated increased steatosis as a result of Kupffer cell induced inflammation ^{13,14, 28–30}. These findings were recapitulated in the MPCC model presented here, where MPTC cultures loaded more lipid in response the steatotic media than MPCC cultures. Additionally, MPTC cultures were also less capable of clearing out accumulated lipid when control medium was re-introduced. Interestingly, Kupffer cell depletion in vivo has been shown to increase expression of genes involved in fatty acid oxidation; resulting in decreased liver steatosis ²⁹. It is likely, therefore, that Kupffer cells are inducing and maintaining excess hepatic steatosis in the in vitro MPCC model presented here. These observations suggest that observations from the MPCC and MPTC models echo in vivo findings and strengthen the argument for Kupffer cells as an important component of NAFLD development and progression.

Current drug discovery strategies for NAFLD and NASH are focused on disease reversal, including fibrosis and hepatic steatosis. In vitro human models such as MPCC and MPTC can be instrumental in supporting multiple aspects of the hit-to-lead identification and confirmation processes. Here were demonstrate streamlined screening capabilities in a long-term in vivo and clinically relevant in vitro model. These studies used 96-well patterned plates that were stained with lipid dye, scanned, and analyzed using high content imaging. This format allows flexibility in examining the lipid lowering capabilities of many potential hits, generation of concentration response curves for multiple compounds at once, or examination of multiple endpoints using one or more compounds. Since the MPCC and MPTC models are able to be used long-term, steatosis can be induced and drug treatment can be continued over multiple days without a loss in hepatocyte function, including DME activity. Hepatic insulin resistance, one of the underlying molecular contributors to NAFLD and NASH development, has been shown clinically to induce de novo lipogenesis (DNL) ³¹. Based on this observation, multiple potential NAFLD and NASH drugs were designed to target DNL as lipid metabolism. One such class of drugs are inhibitors of acetyl-CoA carboxylase 1 (ACC1) and 2 (ACC2) ^21–24. ACC1 and ACC2 play different roles in lipid homeostasis. Both ACC1 and ACC2 facilitate the conversion of acetyl-CoA to malonyl-CoA: ACC1 generated malonyl-CoA is used for fatty acid synthesis in the cytosol, while malonyl-CoA generated by ACC2 in the mitochondria works to inhibit fatty acid β-oxidation ³². ACC1/2 inhibitors have been shown to reduce hepatic steatosis in vivo ^21–24. This finding was replicated here in the MPCC model, where multiple ACC1/2 inhibitors were able to both prevent, and reverse hepatic steatosis induced by FFA. These findings suggest that the MPCC model is useful for drug screening studies.

In addition to screening for NAFLD treatments, the data presented here also demonstrate the utility of MPCC and MPTC models for drug toxicity screening; in particular, assessment of DIS. Valproic acid, which is known to induce liver steatosis in some patients, was used here to validate the ability of this model to assess DIS. VPA can contribute to hepatic steatosis by inhibiting mitochondrial fatty acid oxidation (FAO) and can further liver damage by contributing to mitochondrial permeability transition pore (MPTP) opening ^33–35. When non-steatotic cultures were treated with a range of VPA concentrations, excess lipid accumulation was modest and only occurred at the highest concentration. When VPA was given in conjunction with FFA, however, even the lowest concentration showed increased lipid accumulation in comparison to those treated with FFA alone. Similar results were seen when cells were treated with VPA in the presence of HGF. Interestingly, in vivo rodent studies have also shown evidence that co-administration of HFD and VPA induced lipid accumulation and hepatotoxicity, which was greater that what was seen with either treatment in isolation ³⁶. Furthermore, the lowest concentration tested here is near the clinical C_max measurements for standard VPA doses ²⁷. This demonstrates that the MPCC model can be used to screen for DIS, and that screening in both a lean and steatotic environment can be beneficial for determining a drugs propensity to either induce liver steatosis or worsen existing NAFLD.

Dietary contributors to NAFLD include intake of high fat and high sugar, especially fructose. In vivo studies have demonstrated differences between high fat, high fructose or glucose, and a combination diet on numerous fatty liver related phenotypes including body weight, lipid and glucose metabolism, insulin resistance, and liver triglycerides^37,38. Fructose, in particular, has been demonstrated as a major contributor to insulin resistance, increased body weight, oxidative stress, and inflammation ^39–43. Additionally, in vivo data demonstrates that combination of a high fructose diet with a high fat diet augments these detrimental outcomes, further contributing to NAFLD and NASH development and progression. Furthermore, these data show that HFD, either alone or in conjunction with high fructose or glucose, can induce gene changes which are, in some cases, distinct depending on the steatotic forcing agent ³⁷. Replication of gene expression changes seen in clinical and in vivo NAFLD is an important aspect of in vitro disease modeling. In these studies, gene changes were observed in response to FFA, HGF, or FFA + HGF treatment using a PCR array targeting 84 genes shown to be important in human fatty liver disease. Gene changes varied depending on the steatotic inducer used. When genes with differential expression are assigned a group based on their function (as designated by the array manufacturer), genes involved in insulin signaling and cholesterol metabolism and transport were commonly down-regulated among all treatment groups and genes involved in carbohydrate metabolism, and lipid metabolism and transport were commonly up-regulated. When cells were treated with FFA, CD36 was highly upregulated. In NAFLD patients, in vivo, and in vitro studies, CD36 was upregulated and shown to drive hepatic steatosis by promoting DNL ^44–46. FASN, which is also involved in DNL, was also shown to be differentially regulated here. In cells treated with HGF and HGF + FFA, but not FFA alone, FASN mRNA was up-regulated. In mice, FASN is up-regulated in high fructose diets, with or without high fat diet (HFD), however was not changed in HFD alone ³⁷. Furthermore, in clinical samples FASN mRNA and protein was up-regulated in liver tissue of patients with NASH but not simple steatosis ³⁷. When MPCC cultures were treated with HGF, alone or in combination, IGF1 was downregulated which has been clinically associated with having a higher NAS score ⁴⁷. In samples treated with FFA, IGFBP-1 was downregulated, which has been shown to have an inverse relationship with insulin resistance ⁴⁷. In divergence with clinical observations ⁴⁸, downregulation of CYP2E1 was observed when MPCC cultures were treated with either HGF or FFA + HGF. A lack of CYP2E1 induction by fatty acids has been previously observed in vitro, which was also seen here when cells were treated with FFA ⁴⁹. Furthermore, in vivo, a high fructose diet was shown to reduce CYP2e1, contributing to reduced toxicity by acetaminophen ⁵⁰. In accordance with this result, a reduction of CYP2E1 was seen only in cells that were treated with HGF, either alone or with FFA. Still, most of the gene changes seen in these studies reproduce either clinical or in vivo findings. These data suggest that steatosis induction by either FFA, HGF, or a combination of both in MPCC largely represents a gene environment that replicates clinical and in vivo conditions.

In conclusion, MPCC (HEPATOPAC) and MPTC (HEPATOMUNE), the long-term culture models used here, are able to replicate numerous clinical and in vivo observations suggesting a broad utility for compound screening, drug toxicity evaluation and assessment of gene expression changes. Advantageously, the longer-term function and increased complexity does not come at the cost of throughput or ease-of-use. These systems can be handled, and endpoints can be collected, in a similar manner to simple 2D cultures, including the compatibility with automatic liquid handling systems. Furthermore, the cultures are able to be imaged using a non-confocal inverted microscope or high content imager. In summary, the studies presented here suggest the suitability of the in vitro MPCC and MPTC models, HEPATOPAC and HEPATOMUNE, for preclinical hepatic steatosis and NAFLD studies.

Availability of Data

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Jr, A. M. H., Greene, H. L., Dunn, G. D. & Schenker, S. Fatty liver: biochemical and clinical considerations.
Marchesini, G. et al. Nonalcoholic fatty liver disease: a feature of the metabolic syndrome.
Gupte, P. et al. Non-alcoholic steatohepatitis in type 2 diabetes mellitus.
Lorbeer, R. et al. Association between MRI-derived hepatic fat fraction and blood pressure in participants without history of cardiovascular disease.
Zhu, B. et al. Non-alcoholic Steatohepatitis Pathogenesis, Diagnosis, and Treatment.
Simon, T. G., Roelstraete, B., Khalili, H., Hagström, H. & Ludvigsson, J. F. Mortality in biopsy-confirmed nonalcoholic fatty liver disease: results from a nationwide cohort.
Bellentani, S. The epidemiology of non-alcoholic fatty liver disease.
Caussy, C. & Johansson, L. Magnetic resonance-based biomarkers in nonalcoholic fatty liver disease and nonalcoholic steatohepatitis.
Adams, L. A. & Angulo, P. Treatment of non-alcoholic fatty liver disease.
Mirza, M. S. Obesity, Visceral Fat, and NAFLD: Querying the Role of Adipokines in the Progression of Nonalcoholic Fatty Liver Disease.
Lefere, S. & Tacke, F. Macrophages in obesity and non-alcoholic fatty liver disease: Crosstalk with metabolism.
Luo, W., Xu, Q., Wang, Q., Wu, H. & Hua, J. Effect of modulation of PPAR-γ activity on Kupffer cells M1/M2 polarization in the development of non-alcoholic fatty liver disease.
Huang, W. et al. Depletion of liver Kupffer cells prevents the development of diet-induced hepatic steatosis and insulin resistance.
Diehl, K. L. et al. Kupffer Cells Sense Free Fatty Acids and Regulate Hepatic Lipid Metabolism in High-Fat Diet and Inflammation.
Gunn, P. J. et al. Modifying nutritional substrates induces macrovesicular lipid droplet accumulation and metabolic alterations in a cellular model of hepatic steatosis.
Ramos, M. J., Bandiera, L., Menolascina, F. & Fallowfield, J. A. In vitro models for non-alcoholic fatty liver disease: Emerging platforms and their applications.
Rodrigues, R. M. et al. In vitro assessment of drug-induced liver steatosis based on human dermal stem cell-derived hepatic cells.
Khetani, S. R. & Bhatia, S. N. Microscale culture of human liver cells for drug development.
Bhatia, S. N., Balis, U. J., Yarmush, M. L. & Toner, M. Effect of cell-cell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells.
Moravcová, A. et al. The effect of oleic and palmitic acid on induction of steatosis and cytotoxicity on rat hepatocytes in primary culture.
Kim, C.-W. et al. Acetyl CoA Carboxylase Inhibition Reduces Hepatic Steatosis but Elevates Plasma Triglycerides in Mice and Humans: A Bedside to Bench Investigation.
Ross, T. T. et al. Acetyl-CoA Carboxylase Inhibition Improves Multiple Dimensions of NASH Pathogenesis in Model Systems.
Matsumoto, M. et al. Acetyl-CoA carboxylase 1 and 2 inhibition ameliorates steatosis and hepatic fibrosis in a MC4R knockout murine model of nonalcoholic steatohepatitis.
Loomba, R. et al. GS-0976 Reduces Hepatic Steatosis and Fibrosis Markers in Patients With Nonalcoholic Fatty Liver Disease.
Ströbel, S. et al. A 3D primary human cell-based in vitro model of non-alcoholic steatohepatitis for efficacy testing of clinical drug candidates.
Dash, A., Figler, R. A., Sanyal, A. J. & Wamhoff, B. R. Drug-induced steatohepatitis.
Zaccara, G., Messori, A. & Moroni, F. Clinical pharmacokinetics of valproic acid–1988.
Tosello-Trampont, A.-C., Landes, S. G., Nguyen, V., Novobrantseva, T. I. & Hahn, Y. S. Kuppfer cells trigger nonalcoholic steatohepatitis development in diet-induced mouse model through tumor necrosis factor-α production.
Su, G. L. et al. Activation of human and mouse Kupffer cells by lipopolysaccharide is mediated by CD14. Am. J. Physiol. Liver Physiol. 283, G640–G645 (2002).
Ma, K. L. et al. Inflammatory stress exacerbates lipid accumulation in hepatic cells and fatty livers of apolipoprotein E knockout mice.
Smith, G. I. et al. Insulin resistance drives hepatic de novo lipogenesis in nonalcoholic fatty liver disease.
Wakil, S. J. & Abu-Elheiga, L. A. Fatty acid metabolism: target for metabolic syndrome.
Massart, J., Begriche, K., Moreau, C. & Fromenty, B. Role of nonalcoholic fatty liver disease as risk factor for drug-induced hepatotoxicity. Journal of Clinical and Translational Research 3, 212–232 (2017).
Silva, M. F. et al. Synthesis and intramitochondrial levels of valproyl-coenzyme A metabolites.
Aires, C. C. P. et al. Inhibition of hepatic carnitine palmitoyl-transferase I (CPT IA) by valproyl-CoA as a possible mechanism of valproate-induced steatosis.
Zhang, L. et al. Combined effects of a high-fat diet and chronic valproic acid treatment on hepatic steatosis and hepatotoxicity in rats.
Softic, S. et al. Divergent effects of glucose and fructose on hepatic lipogenesis and insulin signaling.
Moreno-Fernández, S. et al. High Fat/High Glucose Diet Induces Metabolic Syndrome in an Experimental Rat Model.
Jensen, T. et al. Fructose and sugar: A major mediator of non-alcoholic fatty liver disease.
Kovačević, S. et al. Fructose Induces Visceral Adipose Tissue Inflammation and Insulin Resistance Even Without Development of Obesity in Adult Female but Not in Male Rats.
Zhang, D.-M., Jiao, R.-Q. & Kong, L.-D. High Dietary Fructose: Direct or Indirect Dangerous Factors Disturbing Tissue and Organ Functions.
Baena, M. et al. Fructose, but not glucose, impairs insulin signaling in the three major insulin-sensitive tissues.
Dhingra, R. et al. Soft drink consumption and risk of developing cardiometabolic risk factors and the metabolic syndrome in middle-aged adults in the community.
Rada, P., González-Rodríguez, Á., García-Monzón, C. & Valverde, Á. M. Understanding lipotoxicity in NAFLD pathogenesis: is CD36 a key driver?
Castro, R. Q. et al. Dietary sucrose regulates the expression of the Cd36 gene in hepatic tissue of rats with obesity and Non Alcoholic Fatty Liver Disease (NAFLD).
Zeng, H. et al. CD36 promotes de novo lipogenesis in hepatocytes through INSIG2-dependent SREBP1 processing.
Stanley, T. L. et al. Relationship of IGF-1 and IGF-Binding Proteins to Disease Severity and Glycemia in Nonalcoholic Fatty Liver Disease.
Leung, T.-M. & Nieto, N. CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease.
Aljomah, G. et al. Induction of CYP2E1 in non-alcoholic fatty liver diseases.
Cho, S. et al. Fructose diet alleviates acetaminophen-induced hepatotoxicity in mice.

Competing interest reported. All authors are currently employed by BioIVT

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Micropatterned Primary Hepatocyte Co-Culture (HEPATOPAC) for Fatty Liver Disease Modeling and Drug Screening

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Journal Publication

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Abstract

Figures

Introduction

Materials And Methods

Materials

Micropatterned hepatocyte co-culture

Lipid Loading

Drug treatment

Cell viability assessment

Staining

Lipid quantification using HCI

RNA isolation, RT Profiler PCR Array, and analysis

Statistical analysis

Results

Established Steatosis Can Be Reversed By Acc1/2 Inhibitors

Valproic Acid Induces Lipid Loading Alone And Exacerbates Existing Steatosis In Mpcc

Discussion

Declarations

References

Additional Declarations

Status:

Journal Publication

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