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Article Cite This: ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

Establishment of an ex Vivo Model of Nonalcoholic Fatty Liver Disease Using a Tissue-Engineered Liver Qiao Wu,§,†,‡ Juan Liu,§,‡ Lijin Liu,‡ Yu Chen,† Jie Wang,‡ Ling Leng,‡ Qunfang Yu,‡ Zhongping Duan,*,† and Yunfang Wang*,‡ †

Artificial Liver Center, Beijing Youan Hospital, Capital Medical University, Beijing 100069, China Tissue Engineering Lab, Institute of Health Service and Transfusion Medicine, Beijing 100850, China

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ABSTRACT: The prevalence of nonalcoholic fatty liver disease (NAFLD), a common cause of chronic liver disease, continues to increase in parallel with that of obesity. Currently, there are no preclinical models to study its complex pathogenesis nor to assess candidate therapies. We have established a tissueengineered (TE) liver by seeding cells into liver-derived matrix scaffolds and then perfusing the scaffolds with a medium that dynamically provides requisite nutrients, vitamins, minerals, and hormones. Liver-specific biomatrix scaffolds, comprised of almost all of the liver’s known extracellular matrix (ECM) components and matrix-bound soluble signals (e.g., growth factors/cytokines), were recellularized with human hepatic cell line HepG2 and perfused with a complete medium enabling the cells to form functioning liver tissue. By perfusing the system with medium with a high fat content, the cells established a TE fatty (TEF) liver model paralleling that of livers in NAFLD patients. The high fat medium containing 500 μM of free fatty acids (FFAs) (oleic acid:palmitic acid = 2:1) caused the TEF livers to accumulate 2-times more fat than those in the control medium over an 8 day culture period and significantly influenced the capacity of fatty acid synthesis and metabolism. PDK4, CYP2E1, and CYP7A1 genes associated with NAFLD and other liver diseases were all up-regulated, and the metabolic activity of CYP3A4 was significantly impaired. Excess FFAs also induced alterations in transporters and key enzymes in the lipid biosynthesis pathway. The TEF liver was used to test if an antisteatotic drug, Metformin, used in patients with NAFLD, would be able to provide effects paralleling those observed in some patients. Metformin treatment of the TEF liver model caused reduced cellular triglycerides, activated AMPK molecule, inhibited mTORC1 signaling pathway, which thus affected the synthesis and metabolism of FFAs. Overall, the TEF liver offers a stable and reproducible model to study the NAFLD development process and antisteatotic drug effects. KEYWORDS: nonalcoholic fatty liver disease (NAFLD), perfused culture system, tissue-engineered liver, Metformin, mTORC1

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INTRODUCTION

stages of NAFLD. However, a major obstacle for studying NAFLD is that accurate ex vivo models are lacking.6 Rodent models of NAFLD are used for studying high-fat diet and genetic defects.7 Although these animal models can simulate both the occurrence and development of NAFLD, they are unable to incorporate the contributions of individual differences in populations and the difficulties in controlling the experimental conditions.6 Cynomolgus monkeys have also been used in NAFLD studies,8,9 and they have the advantages of animals with a relatively long life and close similarities in biological characteristics to humans.8 However, investigations using monkeys are prohibitively costly, and these animals are well-known to be difficult ones to handle. The establishment of ex vivo NAFLD models has been pursued for a long time. In the past few decades, a variety of

Nonalcoholic fatty liver disease (NAFLD) is a clinical manifestation of liver conditions occurring without significant alcohol consumption but with symptoms of hepatic steatosis, nonalcoholic steatohepatitis (NASH), and cirrhosis.1,2 The initial stage of NAFLD is steatosis, referred to as fatty liver, and then develops further into NASH and liver failure.1,3 The prevalence of NAFLD is between 6 and 35% worldwide (median at 20%).1,4 Its complexity in pathogenesis hinders present studies and limits our understanding of its mechanisms.5 There is still no effective drug developed to treat NAFLD successfully. Previously, a variety of therapeutic strategies have failed during the drug developmental process. Recently, the relationships among metabolic state (intracellular triglyceride), environmental stress (cytokine and oxidative stress), transcriptional regulation, and cell fate have been studied for consideration of their importance in understanding the progression from steatosis to the late severe © XXXX American Chemical Society

Received: June 6, 2018 Accepted: June 20, 2018

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DOI: 10.1021/acsbiomaterials.8b00652 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Franklin Lakes, NJ; Bethyl Laboratories, Montgomery, TX, USA; BioRad Laboratories, Hercules, CA, USA; BioAssay Systems, Hayward, CA, USA; Carl Zeiss Microscopy, Oberkochen, Germany; ColeParmer, Court Vernon Hills, IL; Fisher Scientific, Pittsburgh, PA; Gatan, München,Germany; GE Healthcare, Pittsburgh, PA, USA; GIBCO Life Technologies Corp., Grand Island, NY, USA; Leica, Washington, DC; Merck Millipore, Billerica, MA, USA; Nanjing Jian Cheng, Nanjing, China; PerkinElmer, Waltham, MA, USA; Qiagen, Germantown, MD; R&D Systems, Minneapolis, MN, USA; Santa Cruz Biotechnology, Inc., Dallas, TX; Sigma-Aldrich, München, Germany and also St. Louis, MO; Sircol-Biocolor, Carrickfergus, UK; TOYOBO, NY, NY, USA; Vector Laboratories, Burlingame, CA, USA. Animals. Sprague−Dawley rats (weights 180−220 g) were obtained from the animal center of the Military Academy of Medical Sciences, Beijing, China. They were fed ad libitum until used for experiments. All experimental work was approved by and performed in accordance with the Animal Use and Care Committee guidelines. Preparation and Characterization of Rat Liver Biomatrix Scaffolds. Our protocol for isolation of rat liver biomatrix scaffolds has been described previously.22 Briefly, 2-month-old male rats were anesthetized with ketaminexylazine, and their abdominal cavity was opened. The portal vein was inserted with a 22-gauge catheter (383407, BD) to provide a perfusion inlet to the vasculature of the liver. The delipidating buffer comprised of 36 U/L of phospholipase A2 in 1% sodium deoxycholate (Fisher) was perfused through the liver for ∼30 min (up to 1 h) or until the tissue became transparent after the blood was removed by perfusing the tissue with PBS for ∼15 min. This was followed by perfusion for 30 min with 3.4 M NaCl buffer, which keeps almost all of the ECM components insoluble but depletes nucleic acids and cytosolic components. The liver was rinsed for 15 min with PBS to eliminate the delipidation buffer and then perfused with 100 mL of DNase (1 mg per 100 mL; Fisher) and RNase (5 mg per 100 mL; Sigma-Aldrich) to remove any residual nucleic acid from the scaffold. Finally, the scaffolds were rinsed with PBS for 1 h to eliminate any residual salt and nucleases. The remaining right lobe, cannulated through the portal vein with all the vascular structures intact and weighing ∼3 g, was used for recellularization after being sterilized with a dose of 1.5 M rad of γ radiation by a cobalt-60 gamma irradiator. The efficiency of the decellularization was confirmed by quantification analysis of nuclear acids, collagen, and glycosaminoglycans (GAGs). The residual DNA was quantified by using a Fluorescent DNA Quantitation Kit (Bio-Rad Laboratories) according to the manufacturer’s recommended protocol. Collagen content was quantified with Sircol dye (soluble collagen assay, Sircol-Biocolor) by reading the absorbance at 555 nm. The GAG content was measured by absorbance at 625 nm using the Blyscan assay (Blyscan, Sulfated glycosaminoglycan assay, Sircol-Biocolor) and normalized with a heparin sodium standard curve. To further examine scaffold ultrastructures, we performed scanning electron microscope (SEM) observations of the scaffolds. Images were taken using a Zeiss Supra 25 FESEM operating at 5 kV, working distance of 5 mm, and 20 μm aperture (Carl Zeiss Microscopy). Cell Culture in Normal vs Fat-Supplemented Medium. The human hepatoma cells HepG2 were maintained in DMEM medium (GIBCO) supplemented with 10% fetal bovine serum (FBS), 100 IU/ mL penicillin, and 100 μg/mL of streptomycin. Passaging of cells was performed every 2−3 days. A mix of free fatty acids (FFAs) was prepared and used as a fat-supplemented medium. Oleic acid and palmitic acid (Sigma-Aldrich) were dissolved in 100% ethanol to 1 mmol/L. Then, 33.3 μL of oleic acid, 16.7 μL of palmitic acid, 10 μL of NaOH, and 800 μL of DMEM were mixed and placed in an ultrasonic bath at 60 °C for 30 min. Finally, 3.1 mL of fatty acid-free bovine serum albumin was added and adjusted to pH 7.4 with concentrated HCl. These prepared FFAs were added to 100 mL of complete medium and used as the fat-supplemented medium; the control medium was the same medium but without the fats. For the effect of Metformin to be tested, the 2DF models were treated with

model systems have been established with several types of hepatocytes, both in vitro and ex vivo, and used primarily for evaluations of safety and metabolic applications. However, these model systems have been inadequate for understanding the variables in the pathogenesis of NAFLD. The spectrum of disease progression involves complexities and disparaties in interrelated cellular processes. Usually, stable enzyme activities associated with drug and xenobiotic metabolism are critical for assessing the drug responses in the cell models. Traditionally, in vitro models with hepatocytes cultured on static rigid substrata dedifferentiate rapidly and lose the key metabolic enzyme activities and eventually survival.10 The extremely high, nonphysiological concentrations of hormones, enzymes, and growth factors used in cell culture media to keep hepatocyte viabilities and differentiated functions provided conditions distinct from those in vivo. For example, hepatocytes in simple monolayer cultures and in media containing excess free fatty acids (FFAs) only last for short periods of time, generally 12−48 h.11 Therefore, only transient cellular changes can be analyzed, but the longer lasting effects of triglyceride accumulation could not be studied. In recent years, there has been a transition to floating aggregates of cells conferring polarity and 3-dimensionality (3D) features, ones essential for many aspects of differentiated functions.12−14 The aggregates comprised of only one cell type are called spheroids, and those with more than one cell type (e.g., epithelial-mesenchymal aggregates) are called organoids; in all cases, the cells in such aggregates are early lineage stages that are dominated by stem/progenitors.15−17 The spheroid/ organoid culturing systems have been used recently to develop NAFLD models. As disease models for drug discovery, spheroids/organoids are 3D models and have proven far better than classic monolayer (2D) culture models. However, they still have many drawbacks such as lack of proliferative capacity in the conditions used at present, the use Matrigel, and conditions devoid of dynamic perfusion.18−20 Liver tissue from NASH patients is typically collected when confirming the diagnosis of NASH in patients and is confirmed by histological scores (e.g., NAFLD activity scores) of liver biopsy tissues. This approach ideally incorporates the full spectrum of human cells presenting in the liver within its native architecture as well as genetic and epigenetic factors associated with the donor tissue. Limitations existing in this approach include that the tissue function will begin to decline within only a couple days, limiting the treatment duration (e.g., 24 h).21,22 In this study, a novel ex vivo NAFLD model was established through recellularization of rat liver biomatrix scaffolds with HepG2 cells and perfused with medium supplemented with fats (the experimental) versus with normal medium (the control). The model established incorporates epithelialmesenchymal interactions, a system-level matrix framework, and dynamic perfusion to elicit biological responses mimicking NAFLD. Metabolic, transcriptomic, and phenotypic changes were measured in this model and proved to be an approximate model to mimic the situations observed in patients. This novel NAFLD model also provides potential applications in drug discovery and other therapies newly developed for NAFLD.

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EXPERIMENTAL SECTION

Companies (and Their Location) Providing Equipment, Reagents, and/or Supplies. Abcam, Cambridge, MA; BD, Becton Dickenson and BD Pharmingen, San Jose, CA; Becton Dickenson, B

DOI: 10.1021/acsbiomaterials.8b00652 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

Figure 1. Construction of the NAFLD model based on decellularized liver scaffold. (A) Representative images of rat liver during the decellularization process at 15 min (a), 30 min (b), 1 h (c), and 2 h (d). (B) H/E staining of native rat liver and decellularized liver scaffold. (C) Scanning electron microscopy (SEM) images of a cross section of the decellularized liver scaffold. (D) Schematic representation of recellularization to prepare tissue-engineered liver (TE) and TE fatty liver (TEF). (E) Photograph of circulating perfusion system and the TE liver after recellularization with ∼30 million hepatocytes. (F,G) H/E and oil red staining of recellularized TE and TEF livers. (H) Transmission electron microscopy (TEM) images of hepatocytes in TE and TEF livers. The white arrow means lipid. The red arrow means the tight junction between cells. The blue arrow refers to ECM. The purple arrow means glycogen particles. Metformin at different concentrations (10, 40, 100, and 500 μM) for 3 days. Proliferation Assays. For the proliferation of HepG2 cells cultured under different conditions to be accurately tested, the alamar blue assay (Cell Viability Reagent, Invitrogen) was performed after 1, 2, 3, 4, and 5 days of cultivation of the cells under control versus fatsupplemented conditions by adding 100 μL of the alamar blue reagent to the culture wells. After 2 h of incubation at 37 °C, the fluorescent intensity of samples was measured using the microplate reader (Ensight, PerkinElmer) at wavelengths of 530 nm for excitation and 590 nm for emission. Perfusion of Tissue-Engineered (TE) Livers. Bioreactor parts were assembled in a sterile hood according to the diagram in Figure 1E. Once connected, the bioreactor was placed in an incubator with 5% CO2 at 37 °C. Medium perfusion was started at 3 mL/min, and preconditioning was performed overnight. The well-differentiated

human hepatocellular carcinoma line HepG2 has been used for recellularization. A total of 30 million HepG2 cells were seeded per scaffold at 9 mL/min. The perfusion rate chosen was selected based on that for hepatic blood flow rate in an adult rat during rest (85 mL min−1 kg−1) (Figure 1D). Cell seeding was carried through multiple intervals of cell injections into the culture media. After 24 h of seeding, the normal complete medium (DMEM + 10% FBS) and the fat-supplemented medium (complete medium containing 500 μM FFAs) were perfused to establish the TE and TE fatty (TEF) livers, respectively. The medium was changed every day. For the testing of antisteatotic drug, the TEF livers were treated with Metformin at different concentrations (10, 40, and 100 μM) for 3 days after 5 days of recellularization, and vehicle controls were used for comparation. The TE and TEF livers obtained were subjected to fixation for histology studies and transmission electron microscopy (TEM) observation. Samples were examined using a LEO EM910 transC

DOI: 10.1021/acsbiomaterials.8b00652 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering mission electron microscope operating at 80 kV (Carl Zeiss Microscopy), and digital images were acquired using a Gatan Orius SC1000 CCD Digital Camera with Digital Micrograph 3.11.0 (Gatan). Hematoxylin and Eosin (H/E) Staining, Immunofluorescence (IF), and Immunohistochemistry (IHC). Samples were fixed in 4% formaldehyde overnight at 4 °C and then processed by gradient dehydration. After the tissues were embedded in paraffin, sections were cut at 5 μm thickness for H/E and for IHC and IF analysis using monoclonal antibodies (Table S1). For IHC staining, the sections were deparaffinized and heated in a microwave for at least 15 min with antigen retrieval buffer. After cooling, endogenous catalase was removed from the sections using 0.3% H2O2 for 30 min. Using reagents from Vector Laboratories, sections were blocked with normal horse serum (VECTASTAIN Elite ABC-HRP Kit, Peroxidase, Standard, #PK-6100) in TBS for 1 h and then with Avidin/Biotin Blocking Kit (SP-2001) followed by incubation with antibodies overnight at 4 °C. After incubation with secondary antibodies (ABC Kit), the sections were stained with a NovaRED Peroxidase (HRP) Substrate Kit (SK-4800). For IF, sections were fixed in 4% formaldehyde for 20 min and washed with PBS. Then, they were incubated with 0.25% Triton X100 for 20 min, blocked in 10% serum for 1 h at room temperature (RT), and incubated with antibodies overnight at 4 °C. Cells were then washed with PBS and incubated for 1 h with secondary antibodies. DAPI staining was conducted for 10 min. Finally, sections were sealed with Fluor-Gel in Tris Buffer and photographed. The negative control was incubated with secondary antibodies alone. Pictures of each stained section were taken randomly with Vectra (PerkinElmer) at 20×/40× magnification and were analyzed by Volocity Demo ×64 (PerkinElmer). Oil Red O Staining. After the TE and TEF liver samples were fixed in 4% paraformaldehyde for 24 h and treated with 30% sucrose, 5 μm cryosections were made. The slides were washed twice with 70% isopropanol for 5 min and then stained for 30 min in a 3:2 mix of oil red O solution (Sigma-Aldrich) and dH2O. Following washing the slides three times in dH2O and twice in 70% isopropanol to remove nonspecifically bound stain, bright field images of stained scaffolds were taken using a Vectra (PerkinElmer). RNA Extraction and Real-Time PCR. Total RNA was extracted from a small sample of recellularized liver scaffolds using the Allprep DNA/RNA Mini Kit (#74106; Qiagen) followed by DNase treatment. Samples were reverse-transcribed into cDNA using ReverTra Ace qPCR RT Master MIX (TOYOBO) with the following conditions: 65 °C × 5 min, 0 °C × 2 min and 37 °C × 15 min, 50 °C × 5 min, 98 °C × 5 min. The primers used are shown in Table S2. RT-qPCR was performed with SYBR qPCR Mix without ROX (#QPS-201; TOYOBO) with a total volume of 20 μL. Data were collected using Bio-Rad CFX Manager software (Bio-Rad), and the expression of genes within a sample was normalized to GAPDH expression by the 2ΔΔCt method. Albumin Secretion and Urea Synthesis Assays. Hepatic functional assays include albumin secretion and urea secretion. Cells were cultured in DMEM medium without phenol red (31053036, GIBCO). Culture supernatants were collected 24 h after the medium was changed. Albumin and urea concentrations in the supernatants were analyzed using the human albumin ELISA kit (Bethyl Laboratories) and the QuantiChrom Urea Assay Kit (BioAssay Systems) according to the manufacturers’ instructions. Triglycerides Measurements. Cells were added to 1 mL of PBS and sonicated according to the protocol of the measurement kit (Triglyceride assay kit, A110-1, Nanjing Jian Cheng). Then, 2.5 μL of the cell homogenate was added to a 96-well plate and mixed with 250 μL of working solution followed by incubation at 37 °C for 10 min. The OD was measured at 510 nm, and the concentration was calculated. Western Blot Analyses. TE liver tissue was digested by collagenase IV (0.25 mg per 100 mL, C5138, Sigma-Aldrich) through the retaining portal venous perfusion, and then cells were isolated from liver tissue. Protein was extracted with RIPA Protein Extraction

Reagent (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1% NP-40, 0.1% SDS) solution containing protease inhibitor cocktail (1:50 Roche). Then, protein extracts were tested for protein concentration according to the BCA Protein Quantification Kit Instructions (TE, 3.4 ug/uL; TEF, 2 ug/uL; TEF + MET, 2.7 ug/uL). SDS-PAGE was performed using a precast acrylamide gel (Bio-Rad) with 20 μg of total protein load for the TE, TEF, and TEF treated with Metformin (TEF + MET) samples, respectively. Following transfer, polyvinylidene fluoride membranes were incubated with the primary antibodies to pAMPKα, mTOR, pRaptor, and raptor GAPDH (details in Table S1) according to the manufacturer’s instructions. An Image Quant LAS 4000 mini system (GE Healthcare) was used to produce digital images of the chemiluminescent membranes. Statistical Analysis. The results from two experiments are expressed as the mean ± SEM. The data were analyzed by one-way ANOVA followed by the Student−Newman−Keuls method. A value of P < 0.05 was considered statistically significant.

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RESULTS Chemical and Ultrastructural Characterizations of Liver Biomatrix Scaffolds. After sequential perfusions with various prepared buffers (Figure 1A), a transparent biomatrix scaffold devoid of cells was obtained. H/E staining of the scaffold revealed an absence of cells (Figure 1B). Further analyses using DNA quantification confirmed that DNA levels were negligible (Figure S1A). By contrast, most of the ECM components (collagens and GAGs) were retained (Figure S1B, C). Histological analyses showed that the biomatrix scaffolds retained the anatomical features of the liver and the appropriate anatomical location of known ECM components. Collagens (III, IV, and V) and laminin were found in periportal zones, whereas fibronectin was found throughout the matrix and across all acinar zones (Figure S1D). Matrix molecules (e.g., collagen I and IV) were also identifiable by SEM (Figure 1C). Fat-Supplemented Perfusion Media Transformed TE Livers into TEF Livers. Tissue-engineered (TE) livers were established by multiple infusions of HepG2 cells (3 × 107 in total) via the matrix extracts of the portal veins. After 24 h of seeding, the established TE livers (Figure 1D) were perfused with complete medium. By contrast, the experimental TE livers were perfused with fat-supplemented medium and so established the TEF livers. A diagram of the setup of the perfusion of the bioreactors is shown in Figure 1E. Results of histological examination showed the wide distribution of the seeded cells within the scaffold of the recellularized tissue sections after perfusion culture with lean or fat medium for 8 days in a bioreactor (Figure 1F). Results indicated that cell polarity was well established in the TE livers and the control group, whereas the cells in the TEF livers showed a large number of vacuoles. This finding was further confirmed by analyses with oil red O staining (Figure 1G) and TEM (Figure 1H). Thus, a TEF liver model was successfully established with cells seeded into biomatarix scaffolds and supplied by perfusion with medium with high levels of lipids. The TEF Liver Model Mimicked Clinical NAFLD. Secretion of albumin and urea was measured in the medium for both TE and TEF livers. Both were higher in TE and TEF livers than in HepG2 cells maintained in standard monolayer (2D) cultures. Interestingly, albumin and urea secretions were higher in TEF livers than those in controls, and the extent of the increase was larger for secretion of urea than that for albumin (Figure 2A). Analyses with staining of proliferating D

DOI: 10.1021/acsbiomaterials.8b00652 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering

increase significantly, resulting in triglyceride accumulation in the TEF livers (Figure 3A). FFAs are usually stored in the form

Figure 3. Changes of hepatocellular lipid metabolism in TEF liver. (A) Levels of triglyceride (TG) of HepG2 cells in TE and TEF liver cultures. (B) Insulin resistance marker Pyruvate dehydrogenase kinase isozyme 4 (PDK4) gene expression of HepG2 cells in TE and TEF liver cultures examined by qRT-PCR. (C) Scheme represents the mechanisms of hepatocellular lipid metabolism and their dysregulation in NAFLD. Free fatty acid (FFA) uptake: CD36 mediates transport of nonesterified fatty acids across the plasma membrane. Once taken up into cytosol, FFAs are activated to form acyl-CoAs. De novo lipogenesis: palmitic acid is newly synthesized from citrate. ATP citrate lyase (ACLY), acetyl-CoA carboxylase (ACC), fatty acid synthase (FAS), and fatty acid desaturase 2 (FADS2) catalyze the rate-limiting and final steps, respectively. Acyl-CoAs are esterified by 1-acylglycerol-3-phosphate acyltransferase (AGPAT) to form phosphatidic acid (PA). PA is dephosphorylated to form diacylglycerol (DAG), which is esterified to form TGs. TGs are packaged together with apoB 100 into very low density lipoprotein (VLDL) in the endoplasmic reticulum and secreted into space of Disse. (D) Lipid metabolism-related gene expression in TE and TEF livers. *p < 0.05; **p < 0.01, two-tailed Student’s t tests.

Figure 2. Characterizations of recellularized TE and TEF livers. (A) Analysis of albumin secretion and urea synthesis of HepG2 cells under 2D, TE, and TEF liver cultures. (B) Immunofluorescence staining of proliferation marker PCNA in HepG2 cells under TE and TEF liver cultures. (C) Gene expression of CYP3A4, CYP2E1, and CYP7A1 examined by qRT-PCR. *p < 0.05; ***p < 0.001, two-tailed Student’s t tests.

cell nuclear antigen (PCNA) were conducted and suggested that there was no significant difference in the proliferative state of TEF versus TE livers (Figure 2B). However, CYP3A4 enzyme activity in TEF livers had declined to a significant degree in contrast to the relatively unaffected parameters with respect to albumin, urea synthesis, and cell proliferative status. The development of NAFLD and hepatic steatosis are associated with transcriptomic and proteomic changes, particularly for genes associated with cholesterol/lipid metabolism and insulin signaling.23 Therefore, the TEF liver model was evaluated for these features and compared to known in vivo ones. At first, the expression of key genes associated with NAFLD showed upregulation as compared with cells cultured in monolayer conditions and with both exposed to elevated fat. The responders included CYP2E1 and CYP7A1 (Figure 2C). The results indicated that our TEF model mimicked the transcriptional and protein changes associated with clinical pathological changes of NAFLD. Activities of the Lipid Synthesis Pathway Changed in TEF Livers Resulting in Triglyceride Accumulation. Intracellular triglycerides were analyzed in TE and TEF livers and indicated that triglyceride TGs were 3-times higher in TEF livers than in the controls. Elevated levels of PDK4, an indicator of insulin resistance in hepatocytes,24 were observed in TEF livers. Expression of apo-lipoprotein B (ApoB) did not

of triglyceride TGs (Figure 3B). FFA desaturases (FADS) generate polyunsaturated fatty acids (PU-FFAs). Results from RT-qPCR assays indicated that there was an increase in mRNA levels of a number of genes involved in the metabolic pathway for lipids. These included fatty acid translocase (CD36, also called platelet factor 4), fatty acid binding protein 1 (FABP1), ATP citrate lyase (ACLY), acetyl coenzyme A carboxylase (ACC), fatty acid synthase (FAS), fatty acid desaturase 2 (FADS2), and 1-acylglycerol-3-phosphate O-acyltransferase 5 (APGAT5) (Figure 3C). The phenomena parallel findings in NAFLD patients.23,25,26 Steatosis in TEF Liver Model Could Be Reduced by Metformin. The fat loading inside of cells in TEF livers was analyzed as to possible mechanisms and was subjected to treatments with known antisteatotic compounds such as Metformin. Metformin has been studied for some time given E

DOI: 10.1021/acsbiomaterials.8b00652 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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Figure 4. TEF liver showed higher sensitivity to the clinic antisteatosis drug Metformin. (A) Gene expression of Metformin transporters solute carrier family 22 member 1 and member 3 (SLC22A1 and SLC22A3) of HepG2 cells in TEF and 2DF models. (B) Immunofluorescence staining of SLC22A1 in HepG2 cells under TEF and 2DF cultures. (C) Oil red staining of TEF and 2DF models with or without the Metformin treatment. (D) Analysis of TG accumulation and PDK gene expression in HepG2 cells obtained from TEF and 2DF models with or without the Metformin treatment. *p < 0.05; ***p < 0.001, two-tailed Student’s t tests.

its known antisteatotic effects and with overall outcomes of clinical efficacy. Uptake of Metformin is mediated primarily by solute carrier family members 22A1 (SLC22A1) and 22A3 (SLC22A3) found enriched on the basolateral membranes of hepatocytes.27,28 The high levels of SLC22A1 and SLC22A3 in hepatocytes resulted in preferential distribution of Metformin in hepatocytes. Results of qPCR revealed that receptors for Metformin were expressed at significantly higher levels in TEF livers than in hepatocytes in monolayer cultures (Figure 4A). Similar results were observed using IF staining of SLC22A1, a major receptor of Metformin, and showing much higher expression in TEF livers versus monolayer cultures (TEF:2DF = 1.21 ± 0.01:0.67 ± 0.02, p < 0.01) (Figure 4B), indicating that TEF livers should be more sensitive to Metformin. The TEF livers and, in parallel, cells in monolayer cultures were maintained for 5 days under conditions with high fat levels. On the basis of previous reports,29,30 Metformin at a series of concentrations from 10 to 500 μM was first used to evaluate its effect on 2DF models. The decreased oil red staining was observed only in the 500 μM Metformin-treated group with a considerable difference (Figure S3A). The triglyceride content also decreased with a statistical difference only with 500 μM Metformin treatment (Figure S3B). Further, the gene expression results of SREBP1, which is the key transcriptional factor of lipogenesis, demonstrated that Metformin at 500 μM showed the best effect, and Metformin at 100 μM could also significantly downregulate SREBP1 mRNA expression (Figure S3C), whereas Metformin used at a lower concentration did not significantly improve steatosis. Because of the higher expression levels of SLC22A1 and SLC22A3, which are receptors to mediate Metformin entry

hepatic cells, in TEF liver models, the lower concentrations of Metformin were used to investigate its effect on improving steatosis in the TEF model. The results showed that, even at 10 μM, the same concentration with human Cmax,31 the Metformin was effective to some extent (Figure S3D). Further, the decreased triglyceride content was observed in a dosedependent way (Figure S3E). The expression level of SREBP1 showed a similar tendency (Figure S3F). In addition, Metformin at both 40 and 100 μM presented similar effects, indicating that the drug effect may have reached a stable status in the TEF liver model. Further, the TEF and 2DF models treated with Metformin at 40 μM were chosen to compare their changes. Metformin reduced fat loading of HepG2 in both TEF livers and cells in monolayer cultures. However, the differences of drug reactivity were considerable between the two model systems (Figure 4C). TEF livers showed a nearly 50% decrease in triglycerides and reduction of mRNA levels of PDK4, indicating that insulin resistance could be inhibited by Metformin (Figure 4D). In contrast, there was only an ∼16% reduction of triglycerides in the monolayer cultures and negligible change in PDK4 gene expression, indicating that insulin resistance was not alleviated. This finding has been reported previously from studies with monolayer cultures.32 Metformin Suppressed the mTORC1 Pathway, Influencing FFA Metabolism. Knowledge of the effects of Metformin on biochemical pathways and processes in liver, its primary target, is limited. One established effect of Metformin is to decrease cellular energy levels. AMP-activated protein kinase (AMPK) is a well-characterized energy sensor that couples cellular responses to changes in environmental conditions. AMPK was inhibited in TEF liver and activited F

DOI: 10.1021/acsbiomaterials.8b00652 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX

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ACS Biomaterials Science & Engineering by Metformin (Figure 5A). It is well-established that activated AMPK inhibits the mechanistic target of rapamycin complex 1

Figure 5. The AMPK/mTORC1 signaling pathway played an important role in the process of TEF formation and Metformin therapy. (A) Western blot analysis and the quantified expression of pAMPKa, mTOR, p-Raptor, and Raptor in TE and TEF livers with or without Metformin treatment. (B) Gene expression of the major elements about lipid biosynthesis. *TEF vs TE, p < 0.05; **TEF vs TE, p < 0.01, #TEF+MET vs TEF, p < 0.05; ##TEF+MET vs TEF, p < 0.01, two-tailed Student’s t tests.

Figure 6. Metformin downregulated key points in lipid synthesis by regulating the AMPK/mTORC1 pathway. (A) Schematic representation of the molecular mechanism about how AMPK/mTORC1 works in the TEF liver. (B) Immunofluorescence images of SREBP1 and PPAR-γ expression in TEF livers with or without Metformin treatment. SREBP1 is expressed in the nucleus, endoplasmic reticulum membrane, and golgi apparatus membrane, and PPAR-γ is located in the nucleus. (C) Gene expression of key transporters and synthesis enzyme of fat in TEF liver cells before or after Metformin treatment. *p < 0.05; **p < 0.01, ***p < 0.001, two-tailed Student’s t tests.

(mTORC1) signaling.29 mTORC1, which includes mTOR and raptor, are the key metabolism regulators that are activated and inhibited, respectively, in acute responses to cellular energy depletion.33,34 Both mTOR and raptor were elevated in TEF livers, suggesting that mTORC1 was activated by FFA but was significantly inhibited after Metformin treatment. After mTORC1 was activated, its downstream target molecules were upregulated. These included the S6 ribosome, the ribosomal protein S6 kinase B1 (S6KB1), the sterol regulatory element binding protein 1 (SREBP1), and peroxisome proliferator receptor gamma (PPAR-γ) (Figure 5B), whereas these downstream target molecules were inhibited after the TEF livers were treated with Metformin. We summarized how the molecular pathway of mTORC1 changed and worked in TEF livers (Figure 6A). FFA entered hepatocytes via the CD36 receptor on the cell membrane. Then, FFA activated the mTORC1 complex directly and activated downstream signaling molecules such as S6, S6KB1, PPAR-γ, and SREBP1. Metformin inhibited the expression of downstream signaling molecules by activating AMPK and inhibiting mTORC1. Further, we found by fluorescence staining that PPAR-γ and SREBP1, two transcription factors promoting lipid synthesis, were significantly reduced after treatment by Metformin (Figure 6B). These changes resulted in downregulation of key enzymes in lipid metabolism. This was confirmed by qPCR studies (Figure 6C). We showed that Metformin robustly inhibited mTORC1 in TEF livers, resulting in a decreased expression of mTOR and raptor. Additionally, the same correlation between mTORC1 activity and the fat synthesis pathway was seemingly at odds with the well-known function of mTORC1 to promote fatty acid synthesis. Finally, we showed that Metformin profoundly inhibited hepatocyte fat synthesis in a manner that was largely dependent on its ability to suppress mTORC1 signaling.

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DISCUSSION In this study, we established a novel ex vivo NAFLD model comprised of human hepatic cells seeded into scaffolds of liverspecific ECM and perfused with relevant media supporting either a tissue-engineered liver versus tissue-engineered fatty liver. As human models of an in vitro culture system, primary human hepatocytes (PHHs) are widely used and considered as the gold standard for establishing preclinical models due to their high expression levels of liver-specific gene. However, they rapidly dedifferentiate and lose their drug-metabolizing and drug transporter activities when cultured in vitro. Moreover, the low availability and genetic variability of freshly prepared PHHs highly limit in their uses in contrast to the commercially available cell lines. Therefore, several human hepatic (carcinoma) cell lines, such as HepG2, Hep3B, and Huh7, have been used for establishing preclinical models. HepG2 cells have been widely used as a model of hepatocytes because of their broad utility, stability, and ease of handling compared with PHHs. Westerink et al. showed that the CYP1A1, 1A2, 2B6, and 3A4 induction in HepG2 cells showed good correlation with earlier results observed in PHHs. Furthermore, CYP2D6 and CYP2E1 could, like in primary hepatocytes, not be induced with AhR, PXR, and CAR agonists.35 However, unfortunately, fundamental liver functions generally deteriorate over time in HepG2 cells. G

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and the dysregulation of cholesterol metabolism has been correlated with disease severity. In our study, the CYP7A1 expression was upregulated 3-times in the TEF model compared to that in the TE model, which was similar to the results obtained in patients.23 Moreover, PDK4 has been found to contribute to the pathogenesis of NAFLD and NASH by regulating hepatic lipid and glucose metabolism.46 Zhang et al. analyzed both the mRNA and protein levels of PDK4 in patient liver specimens. Compared with normal control livers, the mRNA level of PDK4 was upregulated 3.5 times.47 In our study, it had been increased 3.8-times in TEF livers compared with in TE liver, which is consistent with clinical reports. Therefore, this new NAFLD model mimicked the multiple pathological changes in NAFLD patients. The preliminary analyses on both the RNA level of ratelimiting enzymes and key transporters was performed for fatty acid synthesis. The results suggested that fatty acid transporters (CD36 and FABP1) and enzymes that synthesize triglycerides were upregulated, but no significant upregulation of triglyceride transporter ApoB was observed. This resulted in the intracellular accumulation of triglycerides. To further explore the mechanisms of these changes, we analyzed the mTORC1 signaling pathway. AMPK is considered a sensor of energy metabolism by “sensing” the cellular AMP:ATP ratio.48 As a major intracellular energy sensor, AMPK is considered as an important target for NAFLD.49 It has been shown that drugs that enhanced AMPK activity inhibit the mTORC1 pathway in hepatocytes.50 The mTORC1 could also be activated by crucial nutrient-derived compounds like palmitic acid added in medium.51 CD36 may induce coupled cell signal transduction and promote esterification of fatty acids.52 Recent studies have found that mTORC1 mediates the expression of CD36 in liver tissue of mice, and its specific inhibitor rapamycin can inhibit CD36 expression and adipose accumulation in small rat liver and human adipose tissue.53,54 mTORC1 can activate SREBP1 at multiple levels, such as transcriptional and post-translational modifications, playing a positive regulatory role.55,56 SREBP1 was proven to regulate genes related to lipid and cholesterol production. PPARγ is also a key regulator of fat synthesis and promotes the synthesis of fat, and mTORC1 activates PPARγ.57 mTORC1 has been shown to regulate the expression and activity of several key enzymes involved in the de novo synthesis of fatty acids. In addition, mTORC1 can also promote protein synthesis.29 In this model, we also found that the synthesis and secretion of albumin and urea increased. These mimicked the initial phase of NALFD in patients but was not able to model NASH well perhaps because of the lack of involvement of inflammatory cells (e.g., Kupffer cells).9 Further, the NAFLD situation in the TEF livers was able to be alleviated by Metformin, a drug used clinically to treat steatosis.28 There is considerable evidence of beneficial effects of Metformin resulting in an improvement in NAFLD properties through improving hepatic steatosis. We compared drug sensitivity between our perfusable TEF model and the monolayer cultures. The results suggested that neither triglyceride deposition nor insulin resistance (PDK4) in these models improved significantly more in TEF than that in 2DF. This could be related to the upregulation of SLC22A1, Metformin’s receptor in the NAFLD model.27,28 SLC22A1 is expressed primarily on the basolateral membrane of hepatocytes; when the cells establish polarity, which occurs

Therefore, HepG2 cells have not been considered useful as an effiencient cell model. However, Hewitt et al. showed that the enzyme activities of HepG2 cells depend on the source and culture conditions and that, consequently, characterization of HepG2 cells is essential.36 In our system, this model is far more effective given the complexity of the biomatrix scaffolds used, a 3D microenvironment, and dynamic perfusion effects. Liver cell functions were significantly improved because of the reestablished cell−cell and cell−ECM communications and the potential for cell polarity.22,37 In addition, the perfusion process dynamically supplied nutrients and soluble factors to all cells via the natural liver circulation system. Both albumin and urea were secreted much more in the HepG2 cells cultured in TE livers than in monolayer cultures. Moreover, the CYP enzymes of HepG2 cells cultured in perfusable TE livers were significantly induced, especially CYP3A4, which is one of the major CYP450 enzymes involved in biotransformation of more than 50% of all prescription medications. The culturing of the TE liver system provided HepG2 cells a suitable hepatic microenvironment, resulting in the dramatically increased cellular functions. Therefore, TE livers should be very helpful in drug screening because the responses detected are closer to those found in vivo. The common dietary long-chain FFAs, both palmitic and oleic acids, were combined to induce fat accumulation in TE livers to generate a novel ex vivo organ model of NAFLD. In NAFLD patients, levels of fatty acids, through both uptake and de novo lipogenesis, are enhanced, but ApoB secretion is decreased.25 Therefore, VLDL particles are not produced sufficiently, resulting in triglyceride accumulation. NAFLD is mainly characterized by excess accumulation of triglyceride in hepatocytes,38−40 which could reappear in the TEF liver model. In addition, albumin is one of the major proteins synthesized in the liver. Its production is associated with energy supply. Bea et al. followed 9029 subjects without diabetes who underwent comprehensive health check-ups annually for 5 years, and they found that albumin concentration was associated with higher levels of insulin resistance and the presence of NAFLD in nondiabetic subjects.41 Urea is the final product of mammalian protein catabolism. NAFLD patients with normal renal function had significantly higher blood urea nitrogen compared to the control cases.42 Similarly, the established TEF model showed elevated albumin and urea secretion compared with TE livers. Further, the drug metabolism activity of CYP3A4 in NAFLD is impaired. The previous report has shown that, in NAFLD patients, the mRNA level of CYP3A4 decreased to ∼30% of that of normal humans,43 whereas in the current study, the mRNA level of CYP3A4 in the TEF model was 20.6% of that in the TE model, indicating the alteration of CYP3A4 in NAFLD could be mimicked in the TEF model. CYP2E1 catalyzes the biotransformation of different fatty acids. Several investigations have reported enhanced hepatic CYP2E1 expression and activity in NAFLD and NASH.44 Mitsuyoshi et al. quantified the levels of transcripts for the CYP2E1 gene from biopsy specimens of 74 patients with NAFLD and found that CYP2E1 was significantly increased in patients with simple steatosis (8.0-times) or NASH (6.2-times).45 In our study, the mRNA level of CYP2E1 in TEF liver was higher than in TE liver by 2.9-times. Therefore, the TEF model could exhibit the upregulation of CYP2E1 to a certain extent. CYP7A1, a key gene in cholesterol metabolism, was also upregulated in NAFLD patients who have increased free cholesterol levels, H

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in those of the biomatrix scaffolds, the receptors would certainly increase. Metformin improves the symptoms of NAFLD in both patients and our TEF model. We then further explored the effect of Metformin on the mTORC1 signaling pathway. The results illustrated that Metformin inhibits the mTORC1 signaling pathway by activating AMPK, thus affecting the lipid synthesis and improved triglyceride accumulation in the cells. Therefore, this TEF model can be used to study the pathogenesis of NAFLD to better screen for therapies of NAFLD.

Article

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Yunfang Wang: 0000-0003-4446-6059 Author Contributions §

Q.W. and J.L. contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Lola Reid for critical comments. This work was supported by the National Key Research and Development Program of China (No. 2016YFC1101305), the National Natural Science Foundations of China (No. 31370990, 31700878, and 81170388), the National Science and Technology Key Project of China (2012ZX10002004006), and the National Key R&D Program of China (2017YFA0103000).

CONCLUSIONS We established a novel ex vivo NAFLD model that provides hepatic cells in matrix scaffolds derived from decellularization of liver and perfused with medium and soluble signals. According to our previous report, the biomatrix scaffolds are tissue-specific and contain >98% of the known ECM components plus bound growth factors and biologically active cytokines.22 The TEF model mimicked the clinical pathological changes of NAFLD, making it an approximate model to investigate drug effects and other therapies that are candidates for treating NAFLD patients. It also makes possible the ability to analyze mechanisms ex vivo. However, it cannot be used as a real NAFLD model. Simple steatosis is a more benign form of NAFLD, whereas NASH is a more severe one. Hepatic steatosis is often reversible. However, it can progress to NASH due to cellular damages caused by the accumulation of lipid peroxidation products in hepatocytes accompanied by inflammatory cell infiltration and/or collagen deposition (e.g., fibrosis). Liver inflammation is a key factor leading to histologic damage and NASH development, progressing to end-stage liver diseases such as cirrhosis, hepatocellular carcinoma, and even liver failure.58 Therefore, our goal is to improve our system using different cocultures of parenchymal and nonparenchymal cells to mimic the cell−cell interactions existing in the liver. These cells will include human hepatocytes, sinusoidal endothelial cells, and Kupffer cells, which are liver-specialized macrophages that secrete important inflammatory factors like IL-6 and TNF-α,59 and hepatic stellate cells that participate in the onset of liver fibrosis through producing excess collagens.9 The biomatrix scaffolds proved to be an excellent systems-level culturing system enabling complex interactions among multiple cell types, dynamic media, and paracrine signaling phenomena and resulting in a more accurate 3D model of a tissue. Modification of this model by coculturing with other cell types in liver can further improve the liver characteristics of cells and thus predict human drug response.

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsbiomaterials.8b00652.

ABBREVIATIONS AFP, α-fetoprotein; ALB, albumin; AMPK, AMP-activated protein kinase; ApoB, apo-lipoprotein B; CD36, platelet glycoprotein 4, fatty acid translocase; CYP, cytochrome P450 mixed oxidase enzyme family that handles metabolism of xenobiotics in the body; CYP2E1, a major member of the CYP family responsible for the metabolism of most drugs; CYP3A4, a CYP that handles inactivation and clearance of many xenobiotics and toxins and also activation of some xenobiotics; CYP7A1, a CYP belonging to the oxidoreductase class and responsible for converting cholesterol to 7-α-hydroxycholesterol, the rate limiting step in bile acid synthesis; DAPI, 4′,6diamidine-2′-phenylindole dihydrochloride; DMEM, Dulbecco’s modified Eagle’s medium; ECM, extracellular matrix; FBS, fetal bovine serum; FFA, free fatty acid; GAGs, glycosaminoglycans; GAPDH, glyceraldehyde 3-phosphate dehydrogenase, an enzyme that catalyzes the sixth step of glycolysis and serves to break down glucose for energy and carbon molecules; h, hour(s); H/E, hematoxylin-eosin staining; IF, immunofluorescence; IHC, immunohistochemistry; mTORC1, mTOR complex 1, also called mammalian target of rapamycin complex 1, a protein complex that functions as a nutrient/energy/redox sensor and controls protein synthesis; NAFLD, nonalcoholic fatty liver disease; NASH, nonalcoholic steatohepatitis; PBS, phosphate-buffered saline; PCNA, proliferating cell nuclear antigen; PDK4, Pyruvate dehydrogenase lipoamide kinase isozyme 4; PPARγ, peroxisome proliferator receptor gamma, a type II nuclear receptor in humans that helps to regulate fatty acid storage and glucose metabolism; SEM, scanning electron microscopy; SLC22A1 and SLC22A3, solute carrier family, members 22A1 and 22A3; SREBP1, sterol regulatory element binding protein 1; TE liver, tissue-engineered liver; TEF liver, tissue-engineered fatty liver; TEM, transmission electron microscopy

Characterization of decellularized liver scaffold, effect of FFA on 2D HepG2cells, antisteatosis effect of Metformin at a series of concentrations on 2DF and TEF models, and lists of antibodies and primers used in this study (PDF)

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DOI: 10.1021/acsbiomaterials.8b00652 ACS Biomater. Sci. Eng. XXXX, XXX, XXX−XXX