Scalable Spheroid Model of Human Hepatocytes for Hepatitis C

Publication Date (Web): April 24, 2014 .... Hee Kim , Supriya K. Kumar , Hitomi Shirahama , Jeongeun Seo , Jae-Ho Lee , Vladimir P. Zhdanov , Nam-Joon...
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Scalable Spheroid Model of Human Hepatocytes for Hepatitis C Infection and Replication Abhishek Ananthanarayanan,†,‡ Bramasta Nugraha,‡,§,∥ Miriam Triyatni,∥ Stefan Hart,⊥ Suryanarayana Sankuratri,# and Hanry Yu*,†,‡,¶,△ †

Institute of Bioengineering and Nanotechnology, A*Star, Singapore 138669 Department of Physiology, Yong Loo Lin School of Medicine, National University Health System, Singapore 117597 § Department of Biosystem Science and Engineering, ETH Zurich, 4058 Basel, Switzerland ∥ F. Hoffmann-La Roche Ltd, 4070 Basel, Switzerland ⊥ Roche Translational Medicine Hub, Singapore 169612 # Roche Pharma, 340 Kingsland Street, Nutley, New Jersey 07110, United States ¶ Mechanobiology Institute, 10-01 T-Lab, 5A Engineering Drive, Singapore 117411 △ Department of Biological Engineering, Massachusetts Institute of Technology, NE47-220, Cambridge, Massachusetts 02139, United States ‡

S Supporting Information *

ABSTRACT: Developing effective new drugs against hepatitis C (HCV) virus has been challenging due to the lack of appropriate small animal and in vitro models recapitulating the entire life cycle of the virus. Current in vitro models fail to recapitulate the complexity of human liver physiology. Here we present a method to study HCV infection and replication on spheroid cultures of Huh 7.5 cells and primary human hepatocytes. Spheroid cultures are constructed using a galactosylated cellulosic sponge with homogeneous macroporosity, enabling the formation and maintenance of uniformly sized spheroids. This facilitates easy handling of the tissue-engineered constructs and overcomes limitations inherent of traditional spheroid cultures. Spheroids formed in the galactosylated cellulosic sponge show enhanced hepatic functions in Huh 7.5 cells and maintain liver-specific functions of primary human hepatocytes for 2 weeks in culture. Establishment of apical and basolateral polarity along with the expression and localization of all HCV specific entry proteins allow for a 9-fold increase in viral entry in spheroid cultures over conventional monolayer cultures. Huh 7.5 cells cultured in the galactosylated cellulosic sponge also support replication of the HCV clone, JFH (Japanese fulminant hepatitis)-1 at higher levels than in monolayer cultures. The advantages of our system in maintaining liver-specific functions and allowing HCV infection together with its ease of handling make it suitable for the study of HCV biology in basic research and pharmaceutical R&D. KEYWORDS: 3D culture, tissue engineering, drug discovery, hepatitis C, hepatocytes

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INTRODUCTION Hepatitis C virus (HCV) affects over 170 million people worldwide and is a major health problem in both developed and developing countries.1,2 It is a leading cause of chronic hepatitis, hepatocellular carcinoma, and end stage liver failure.1,2 Lack of effective vaccines or therapeutic options to combat the disease is hindered by host specificity of HCV and the inability of in vitro models to support robust viral infection and replication.3 The initial understanding of HCV viral−host interactions came from studies involving replication of genomic and subgenomic replicons.4 However, this did not enable the study of viral entry and assembly. Replication of full-length HCV in human hepatoma cells required viral adaptive mutations, rendering such models not suitable for translational research. These drawbacks were remedied by the identification © 2014 American Chemical Society

of JFH-1, a full-length clone of the virus that does not require adaptive mutations to propagate in culture.5 JFH-1 infection and entire life cycle can be observed in hepatoma cell lines Huh 7 and Huh 7.5 and have helped to gain crucial insights into HCV host−pathogen interactions.6 Huh 7 and Huh 7.5 are functionally comparable to fetal hepatocytes, but they display aberrant interferon signaling pathways and exhibit deregulated gene expression compared to Special Issue: Engineered Biomimetic Tissue Platforms for in Vitro Drug Evaluation Received: Revised: Accepted: Published: 2106

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Cell Culture Conditions. Huh 7.5 Cells. Huh 7.5 cells were purchased from ATCC (American Type Culture Collection) and were propagated in Dulbecco’s modified Eagle medium (DMEM) (Gibco, Singapore) supplemented with 1× minimal essential amino acids (MEAA) and 10% fetal bovine serum (FBS). Cells were passaged at 80% confluence. Human Hepatocyte. Cryopreserved human hepatocytes (Lots 8110, 4248, and 4239) were purchased from BD Biosciences and Invitrogen. Human hepatocytes were selected based on their ability to form spheroids in cellulosic scaffold. The cells were maintained in Williams E medium supplemented with 1 mg/mL BSA, insulin transferrin, and selenium, 50 ng/mL linoleic acid, 50 nM dexamethasone, and 100 U/mL of penicillin/streptomycin. Cell Seeding. For both Huh 7.5 and primary human hepatocytes, 0.1 million cells (6 mm diameter sponge) were reconstituted in 16 μL of medium and 8 μL was added on either side of the sponge (performed on a 48-well plate). After 30 min incubation to allow the cells to enter the sponge, 300 μL of medium was added to each well and cells were incubated at 37 °C. To measure the total number of spheroids in the sponge, the sponge was disrupted using an 18 G needle (BD, Singapore) and the spheroids were released into the medium. The total number of spheroids in a given volume of medium was counted using a cytometer, and this was extrapolated to the total number of spheroids per 1 mm3 of the sponge. The volume of the sponge was calculated using the formula πr2h (where r denotes the radius and h the height of the sponge). The diameter of each spheroid was estimated by estimating the geometric mean of the spheroids using the formula (d1d2d3)1/3 where d1, d2, and d3 are diameters estimated from different edges of the spheroid. Synthesis of Galactosylated Cellulosic Sponge. The synthesis and detailed characterization of the cellulosic sponge have been adapted from previous reports by Nugraha et al.15 Briefly, hydroxypropyl cellulose (HPC), Mw = 80 000 g/mol and ∼3.4 degree of etherification was dehydrated by azeotropic distillation in toluene at 70 °C. Four grams of dried HPC was dissolved in anhydrous chloroform (100 mL), to which 2.095 mL of allyl isocyanate 98% and 1 mL of dibutyltin dilaurate 95% were added dropwise. The mixture was stirred vigorously for 48 h at room temperature, after which it was precipitated in an excess amount of anhydrous diethyl ether. Following vacuum drying, the product was dissolved in deionized water (DI H2O), purified by dialysis for 3 days, and finally lyophilized to the end product, HA (hydroxypropyl cellulose allyl). HA was dissolved in deionized water to a final concentration of 10 wt %/vol, after which the solution was inserted into glass tubes (diameter 6 mm, length 3 cm) (VWR, Singapore). The tubes were heated in a water bath (40 °C) until phase separation occurred and then cross-linked by γ irradiation for 1 h at a dose of 10 kGray/h (Gammacell 220, MDS Nordion, Canada). The sponge monoliths were obtained by breaking tubes subsequent to freezing in dry ice. A Krumdieck tissue slicer (Alabama Research & Development USA) was used to cut the sponges in 6 mm diameters uniformly (50 rpm for 1 mm thickness), denoted as HA sponges. The sponges were further modified with galactose by activating the hydroxyl groups with the addition of 1,1′carbonyldiimidazole (20 mM in acetone) for 24 h. D(+)-Galactosamine HCl (2 mg/mL in carbonate−bicarbonate buffer) was added to the activated sponges. The reaction was carried out for 24 h at 4 °C. To remove impurities, the sponges

primary human hepatocytes.7 The use of these cell lines is thus not perfectly suited to the pathophysiological study of HCV infection and replication. Primary human hepatocytes express the complete set of viral entry receptors similar to that observed in in vivo hepatocytes. Primary human hepatocytes thus represent a physiological model for the study of HCV infection and replication, but are notoriously hard to maintain with high functionality in in vitro cultures.8 Over the past decade various strategies such as microscale technologies, fluid flow bioreactors, spheroid cultures, coculture with nonparenchymal cells, and various extracellular matrices have been used to improve the differentiated functions of hepatic cell lines and to prolong the viability and functions of primary hepatocytes in culture. However, many of these systems are not scalable and do not work interchangeably with cell lines and primary hepatocytes,9−11 mainly due to the proliferative nature of hepatocyte cell lines and poorly understood heterotypic interactions of these cell lines with nonparenchymal cells such as fibroblasts.12 Recent studies using microscale culture platforms have demonstrated HCV entry and replication in primary human hepatocytes, though the levels of infectivity were low and viral replication could not be detected using real time PCR.1 We hypothesize that enhancing liver-specific functions of hepatic cells in a scalable 3D culture system might lead to increased HCV entry and replication; thus representing an improved tool for drug screening compared to other available in vitro models. Among the various 3D culture strategies, spheroid cultures allow for tight homotypic interactions, high levels of E-cadherin expression,10 and longer maintenance of hepatocellular phenotype. Cells cultured in spheroid configuration react differently to various stimuli and are morphologically and transcriptionally distinct compared to conventional monolayer cultures.10,13 Variations of spheroid cultures have been utilized to culture primary rat and human hepatocytes; however, these systems do not enable well-defined control of spheroid dimensions and are difficult to handle.14 Uncontrolled spheroid size causes hypoxia and limited access to drugs and nutrients at the spheroid core, leading to suboptimal performance and heterogeneity of responses in downstream drug testing assays.15 Moreover, difficulty in handling and scaling of these systems are major drawbacks for drug screening in scalable platforms such as multiwell plates. Here we demonstrate the utility of a 1 mm thin galactosylated cellulosic sponge with controlled macroporous structures to culture Huh 7.5 and primary human hepatocyte spheroids in multiwell plates. Huh 7.5 spheroids display enhanced hepatocyte-specific functions, while spheroids of primary human hepatocytes maintain hepatocyte-specific functions over prolonged periods of time. The spheroids form spatially segregated basolateral and apical domains and express all HCV entry receptors. The spheroids also support HCV entry at higher levels than conventional monolayer cultures. Moreover, spheroid cultured Huh 7.5 cells support higher levels of HCV replication than conventional monolayer cultures. Our system exhibits dose-dependent decrease in viral entry upon treatment with anti-CD81 antibody, demonstrating its potential utility to screen for compounds inhibiting viral entry.

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EXPERIMENTAL SECTION All chemicals and reagents were purchased from Sigma-Aldrich (Singapore) unless otherwise stated. 2107

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Figure 1. Morphological characterization of spheroids. Human hepatocytes (A) formed spheroids of predominatly 50−100 μm spheroid size and maintained over time; while Huh 7.5 (B) initially formed 80−120 μm spheroids that grew larger over time. (C) Immuofluorescence microscopy of MRP2 (red) and CD147 (green) colocalization in spheroids indicates spatial segregation of these apical and basolateral markers as a sign of cell polarization. Percentage of colocalization in primary human hepatocyte and Huh 7.5 is 0% and 13% respectively. Scale bar = 20 μm. (D) Scanning electron microscope images at magnifications of 1000× and 2000× indicate that both primary human hepatocytes and Huh 7.5 formed tight aggregates and maintained the spheroid integrity over 14 day culture; while they remain tethered to the sponge materials. There are micropores on the surface of primary human hepatocytes as seen in typical hepatocyte spheroids. Scale bar = 10 μm.

coated with platinum for 90 s. The samples were viewed with a scanning electron microscope (JEOL JSM-5600, Japan) at 10 kV. Real Time PCR Analysis for Hepatocyte Specific Genes. RNA was extracted from hepatocytes cultured as 3D spheroids in HA Gal sponges using RNeasy Mini Kit (Qiagen, Singapore). Total RNA concentration was quantified by Nanodrop (Thermo Scientific, Singapore), and 1 μg of RNA was converted to cDNA by high capacity RNA-to-cDNA kit

were further washed three times in excess of Dulbecco phosphate buffer saline and subsequently deionized water. The washed sponges (HA Gal sponges) were lyophilized and sterilized with γ irradiation. Scanning Electron Microscopy (SEM). Hepatocyte spheroids in sponges were fixed with 3.7% paraformaldehyde overnight and stained with 1% OsO4 for 1 h. Samples were then dehydrated stepwise with ethanol (25%, 50%, 75%, 90%, and 100%) for 10 min each, dried in a 37 °C dry oven, and sputter2108

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RESULTS Characterization of Spheroids in Sponge. Both Huh 7.5 and primary human hepatocytes formed spheroids in 24 h. The

(Applied Biosystems, USA). Primers were designed using Primer 3, and real-time PCR was performed by using SYBR Green fast master mix in an ABI 7500 Fast Real-Time PCR system (Applied Biosystems, USA). Gene expression was plotted as a Log2 transcript over GAPDH. Immunostaining. Cells were fixed with 3.7% paraformaldehyde for 15 min at 37 °C followed by washing with 1× PBS and blocking with 2% Bovine serum albumin (BSA)/0.2% Triton X-100 for 2 h. Following this, the spheroids were incubated overnight at 4 °C with mouse anti-human CD81 (clone JS-81, BD Pharmingen, USA), rabbit anti-SCARB (Scavenger Receptor Class B)-1 (Novus Biologicals, USA), rabbit anti-CLDN (Claudin)-1 (Invitrogen, Singapore), rabbit anti-OCHLN (Occludin) (Invitrogen, Singapore), rabbit antiMRP2 (Sigma-Aldrich, Singapore), mouse anti-CD147 (Serotec, USA), and Luciferase (AbCam, United Kingdom). Secondary antibodies used were goat anti-mouse and goat anti-rabbit 488 and 555 (1:1000), respectively (Invitrogen, Singapore). Nucleus staining was performed using mounting medium containing DAPI stain (Vecta Shield, USA). Images were captured using Olympus FluoView FV1000 with a 60× water lens. Images were analyzed using IMARIS with colocalization package (Bitplane Scientific Solutions, United Kingdom) and images assembled using Adobe illustrator. HCVpp Synthesis. The pseudoparticles were synthesized by cotransfection of plasmids encoding (1) E1 and E2 HCV glycoproteins and (2) HIV lacking nef and env genes and containing the luciferase gene into 293T cells.16 HCVpp Entry and Inhibition. After 3 days in culture, human hepatocytes and Huh 7.5 spheroids were subjected to treatment with 100 μL of medium containing 1.5× dimethyl sulfoxide (DMSO) and 1.5× penicillin/streptomycin containing CD81 at various concentrations. To this medium were added HCV pseudoparticles with a stock concentration of 105 TU (transducing units)/mL at a multiplicity of infection (MOI) of 0.02 for monolayer and spheroid cultures. End points of viral entry were measured 3 days postinfection by immunostaining with anti-luciferase antibody (AbCam, U.K.), and luminescence measurement was performed using Promega Steady-Glo kit (Promega, Singapore) and normalized to total protein using NanoOrange protein quantitation kit (Invitrogen, USA). Viral Replication. Viral replication was carried out using Japanese fulminant hepatitis-1 (JFH-1) strain of HCV virus derived from the supernatant of infected Huh 7.5 cells. The cells were infected with the JFH-1 strain of the virus with a stock concentration of 104 FFU (focus forming units)/mL at an MOI of 0.01 for 48 h, 3 days postseeding, following which the medium was changed and the total RNA was isolated from the cells every 2 days and purified using PerfectPure RNA 96 CellVac kit (5 Prime, USA). The RNA was converted to cDNA by high capacity cDNA reverse transcription kit (Applied Biosystems, USA), and the copy number of the virus was quantified using Taqman assay performed on Viaa7 (Applied Biosystems, USA). The comparison of viral copy numbers between the monolayer and spheroid culture was calculated by normalizing it to the expression of β-actin. Statistical Analysis. Graphs and statistical analysis for gene expression and viral replication were performed using paired t test using Origin 9 (Origin Laboratories Corporation, USA). P < 0.05 was considered significant.

Figure 2. Gene expression profiles of (A) human hepatocytes and (B) Huh 7.5 cells in spheroids over 14 day culture. Hepatocyte-specific genes were maintained at similar or higher levels, *p < 0.05. Data expressed as mean ± SEM (n = 3 independent experiments of duplicates).

sponge supports the uniform formation of spheroids for both Huh 7.5 cells and primary human hepatocytes with a density of approximately 17 spheroids/mm3 (Supplementary Table 1 in the Supporting Information). This amounts to 1300 spheroids in a 10 mm diameter sponge and 500 spheroids in a 6 mm sponge. Each spheroid has approximately 100 cells, and the 1.3 × 105 cells per 6 mm sponge allows for sensitive detection of downstream end points and assays. Both Huh 7.5 cells and primary human hepatocytes formed tight multicellular aggregates and maintained their spheroid configuration over prolonged culture of 14 days. We characterized the spheroid size distribution of the Huh 7.5 and human hepatocyte spheroids in culture. We found that the human hepatocyte spheroids maintained a constant size distribution from day 3 until day 14 in culture with 80% of them exhibiting 50−100 μm diameter (Figure 1A). However, due to the proliferative nature of Huh 7.5 cells, there was an 8% decrease in number of spheroids with 80−120 μm diameter from day 3 to day 14. We also observed a 27% increase in the number of larger Huh 7.5 spheroids of 150−200 μm diameter from day 3 to day 14 in culture (Figure 1B). 2109

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Figure 3. Expression of viral entry markers using confocal microscopy on nuclear staining (blue) and antigen specific staining of CD-81 (green), Claudin-1 (red), Occludin (green), SCARB-1 (red), and luciferase (red) in (A) primary human hepatocytes and (B) Huh 7.5 cells. Orthogonal sections (xy, yz, and xz) were visualized. All markers were expressed in both spheroids and the control 2D monolayer cultures, while the spatial localization patterns in spheroids are distinctly different from the control but more reminiscent of the pattern seen in vivo. Scale bar = 20 μm.

Characterization of Spheroid Using Scanning Electron Microscopy (SEM). We further characterized the integrity of the spheroids and characterized surface features of the spheroids by performing SEM analysis of the spheroids in culture. Huh 7.5 spheroids were compact and tightly aggregated and maintained their morphology for 2 weeks of culture. The Huh 7.5 spheroids did not show indentations and cavities (micropores) on their surface as those observed on primary human hepatocytes.17 This might be the result of lower maturity, cell polarization, and number of bile canaliculi in Huh 7.5 cells compared to the primary human hepatocytes. Primary hepatocytes formed tightly aggregated spheroids within 3 days in culture and remained tightly aggregated for 2 weeks in culture. Primary hepatocytes displayed distinct surface cavities/ micropores, which are possibly channels extending to the interior of the spheroid17 (Figure 1D). Characterization of Hepatocyte-Specific Gene Expression. Primary human hepatocytes rapidly lose their differentiated liver-specific function and hepatocyte-specific gene expression in monolayer cultures.13 In addition, Huh 7.5 cells express very low levels of mature hepatocyte functions such as albumin and cytochrome P450 (CYP) 3A4, similar to those in fetal hepatocytes.18 We investigated the ability of our

Characterization of Apical and Basolateral Domains in Spheroid Cultures of Huh 7.5 and Primary Human Hepatocytes. Spheroids of both Huh 7.5 cells and primary human hepatocytes expressed markers of apical and basolateral domains such as multidrug resistant associated protein 2 (MRP2) and CD147. In Huh 7.5 spheroids, MRP2 protein was found in the apical domain, forming punctated structures between cells that do not extend to form tubelike structures resembling the bile-canaliculi in the liver. The basolateral domain was indicated by the staining of CD147 (Figure 1C). There was 13% colocalization of MRP2 and CD147 staining as determined by image analysis, which indicated the formation of spatially segregated apical and basolateral domains as in polarized cells. Primary human hepatocytes in spheroids formed defined apical domain indicated by MRP2 staining which marks the spatial localization of the apical transporter that is necessary for maintenance of hepatocyte function and viability in in vitro cultures.10 The primary human hepatocytes also exhibited spatially segregated basolateral and apical domains as indicated by the staining of CD147 and MRP2 (Figure 1C). There was no overlap or colocalization of MRP2 and CD147 markers in spheroid culture, supporting the enhanced cell polarization in these spheroids. 2110

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Figure 4. HCV entry and its inhibition. Comparison of HCVpp entry between 2D monolayer control and spheroid cultures of (A) primary human hepatocytes and (B) Huh 7.5 cells indicates respectively 4- and 9-fold higher levels of HCVpp entry into spheroids than the control. (C, D) Dosedependent inhibition of HCVpp entry by JS-81 antibody against CD81 receptor in human hepatocytes (C) and (D) Huh 7.5 cells, *p < 0.05. Data expressed in relative light units (RLU) and normalized RLU as mean ± SEM (n = 3 independent experiments of duplicates).

CLDN-1, OCLN, and SCARB-1 were localized at the tight junction, which is associated with the apical domain. Strikingly, viral entry receptors in spheroids of both cell types displayed cellular localizations similar to that observed in vivo, in contrast to conventional monolayer cultures (Figure 3), where localization of these receptors are not representative of the in vivo liver.16 In order to ascertain whether the spheroids can be infected by HCV, we tested their susceptibility to glycoproteinmediated HCV entry. We infected cells with HCV pseudoparticle (HCVpp), an in vitro system used to investigate HCV entry and neutralization in hepatocytes.19 HCVpp consists of the HCV envelope proteins displayed on a retroviral core and contains the luciferase gene that is expressed in the infected target cells. We observed that spheroids of both Huh 7.5 cells and primary human hepatocytes are susceptible to glycoprotein-mediated entry (Figure 4A,B). The spheroid cultures also supported 4-fold higher levels of HCVpp entry in human hepatocyte spheroids (Figure 4A) compared to conventional monolayer cultures and 9-fold higher levels of HCVpp entry in Huh 7.5 spheroids compared to conventional monolayer cultures (Figure 4B), thus demonstrating the advantages of our system in studying HCV infection in vitro. Compound screening for new drugs against HCV infection is essential for the identification and development of new candidate HCV therapeutics. We therefore tested for suitability in potential screening compounds that can inhibit HCV entry in hepatocytes. To this end, we incubated spheroid cultures

spheroid culture system to maintain gene expression of primary human hepatocytes and enhance the expression of mature hepatocyte-specific genes in Huh 7.5 cells. Spheroid cultures of primary human hepatocytes preserved all the tested hepatocytespecific gene expression, namely, alpha antitrypsin (AAT), CYP 1A1, CYP 3A4, HNF4α, and albumin (Alb) for 2 weeks in culture with minimal dedifferentiation when compared to the cryopreserved freshly thawed hepatocytes (Figure 2A). The spheroid culture of Huh 7.5 elevated CYP3A4 levels by 184-fold from day 3 to day 7 in culture, after which the levels stabilized until day 14 in culture. Expression of albumin increased by 29-fold from day 5 to day 7 in culture, after which the albumin levels stabilized until day 14 in culture. Expression of AAT, CYP 1A1, and HNF 4α were maintained at similar levels from day 3 to day 14 (Figure 2B). These results indicate that our spheroid culture system can maintain hepatocytespecific gene expression in both primary human hepatocytes and Huh 7.5 cells over a prolonged culture. Expression of Viral Entry Markers and Pseudoparticle Entry. HCV entry into hepatocytes is mediated by interplay between 4 receptors: CD81, Claudin 1 (CLDN-1), Occludin (OCLN), and Scavenger receptor class B member 1 (SCARB1).1 We performed immunostaining to determine their expression and the localization in in vitro culture and found that spheroid cultures of both Huh 7.5 cells and primary human hepatocytes expressed all the viral entry markers (Figure 3). CD81 was localized along the basolateral domain, while 2111

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conventional monolayer cultures on day 4 and day 8 respectively (Figure 5B).

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DISCUSSION We have described an in vitro spheroid culture system of Huh 7.5 cells and primary human hepatocytes, constructed using a galactosylated cellulosic sponge.15,20 The cells cultured in the cellulosic sponge form spheroids of uniformly sized multicellular aggregates, whose size is limited by the pore size in the sponge. It provides appropriate chemical cues via galactose conjugation, and mechanical cues via controlled mechanical stiffness of the material. Spheroids cultured in the galactosylated cellulosic sponge support the maintenance of Huh 7.5 cells to exhibit a more hepatocyte-specific phenotype and prevent the dedifferentiation of primary hepatocytes over prolonged culture. This is in agreement with the previous reports on enhanced spheroid formation and maintenance on galactosylated substrates, and their differential gene expression compared to monolayer cultures.13,14,20,21 Both cell types in the spheroid configuration are entrapped in the pores of the sponge and maintain their spheroid morphology over weeks of culture while maintaining their appropriate cell polarization. This would be useful features to study various events such as chronic toxicity and progression of other hepatotropic infections.22 With regard to the utility of the system to study HCV infection and replication we found that SCARB-1, Occludin-1, and Claudin-1 were localized at the tight junction of polarized hepatocytes. This is consistent with previous reports showing that tight junctional localization of Claudin and Occludin is necessary for the cellular tropism of HCV and entry of HCV into polarized hepatocytes.23,24 The spheroid cultures also display similar patterns of expression of SCARB-1 and CD81 compared to the in vivo liver.25,26 Huh 7.5 and primary human hepatocytes in cellulosic sponge spheroids exhibit strong cell adhesion and tight junctional protein complexes and express certain functions similar to that observed in in vivo tissues.27 This allows for improved polarization of cells and higher infectivity of HCVpp compared to conventional monolayer cultures. Therefore, spheroid cultures in cellulosic sponges provide an appropriate model to investigate HCV entry, in particular to study organization, interaction, and stoichiometry of HCV receptors with tight junctional proteins.28 Importantly, we observed that primary human hepatocytes in culture are polarized, express viral entry markers, and support glycoprotein mediated HCV entry even after 2 weeks in culture (Supplementary Figure 1 in the Supporting Information). An inverse relationship between host cell polarization and HCV entry has been observed in Caco-2 and HepG2 cells expressing CD81.29,30 These cells are not known to support HCV replication. Here, we observed higher levels of HCVpp entry in spheroid cultures of Huh 7.5 and primary human hepatocytes exhibiting good cell polarization marker features. Huh 7.5 cells in spheroid configuration with good cell polarization were more permissive to HCVcc replication compared to conventional monolayer cultures with poor cell polarization. Supporting our findings, previous reports on human hepatoma cells have shown that tight junctional barriers formed in 3D constructs of human hepatoma cells are not inhibitory to HCV infection.28 Further studies would be needed to resolve these seemingly conflicting observations. We observed that Huh 7.5 spheroids supported HCVcc infection and replication as shown by the increase in intracellular viral titers over 8 days in culture. The decrease

Figure 5. HCV clone JFH-1 replication in Huh 7.5 cells. (A) Increase in JFH-1 copy number in spheroid cultures of Huh 7.5 cells over 8 days indicates viral replication. (B) Ratio of JFH-1 titers in spheroids over 2D monolayer control cultures as measured by ΔΔCt in RT-PCR normalized to β-actin shows preferential increase in viral replication in spheroids compared to the control over time. *p < 0.05. Data expressed as mean ± SEM (n = 3 independent experiments of duplicates).

with increasing concentrations of JS-81, an antibody that targets and inhibits the HCV receptor CD81.1 We observed a dosedependent decrease in HCVpp entry upon addition of increasing concentrations of anti-CD81 antibody in both primary human hepatocyte (Figure 4C) and Huh 7.5 spheroids (Figure 4D). Therefore, our system might be a potential platform for drug screening/discovery applications and for studying HCV entry in vitro. HCV Replication. We further investigated if the galactosylated cellulosic sponge-based spheroid cultures can support the replication of live full-length virus in culture. We infected spheroids with the JFH-1 (genotype2a) strain of the virus and monitored replication over 8 days of culture. The cryopreserved primary human hepatocytes did not support HCV live viral replication as detected using real time PCR, consistent with previous reports.1,3 The spheroid cultures of Huh 7.5 cells supported HCV replication with a 3-fold increase in viral titers from day 2 to day 4 in culture, after which there was a 2-fold increase from day 4 to day 6 and a 1.5-fold increase from day 6 to day 8 (Figure 5A). The spheroid cultures also supported 4fold and 8-fold higher copy number of the JFH-1 virus than the 2112

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in viral titers on day 6 compared to day 4 and day 8 could be due to the differential viral entry and proliferation rate of monolayer cultures compared to spheroid cultures12 and the inverse relationship between confluence and viral replication as documented by Nelson et al.31 Though spheroid cultured primary human hepatocytes support higher levels of HCV entry compared to monolayer cultures, they do not support its persistent infection or replication. This could be due to the effects of cryopreservation on primary human hepatocytes or due to the activation of interferon pathways upon viral entry and consequent inhibition of robust HCV replication, which might lead to undetectable viral titers.32 More sensitive detection methods such as the one described by Jones et al.33 might help determine if our galactosylated cellulosic spongederived spheroids support replication of the full length HCV in primary human hepatocytes.1 Further modification of cellulosic sponge-based 3D culture systems will also allow us to culture other cell types such as HepaRG cells and stem cell-derived hepatocytes that support HCV and HBV infections with improved phenotype and infectivity.34,35 This will help us to overcome the current bottleneck of using primary human hepatocytes that are highly variable in their quality, and facilitate compound screening across different patient-specific populations. The system helps maintain the differentiated status of primary human hepatocytes for 2 weeks in culture and can be used to study other pharmaceutically relevant end points such as drug metabolism, drug−drug interaction, and toxicity,15 which are important parameters measured during preclinical drug development.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

Figure depicting HCVpp entry in primary human hepatocytes at multiple time points of culture normalized to uninfected controls. Table listing number of spheroids per unit volume, number of spheroids in different size scaffolds, and number of cells per spheroid. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Department of Physiology, Yong Loo Lin School of Medicine, National University Health System, MD9-04-11, 2 Medical Drive, Singapore 117597, Singapore. Tel: +65 65163466. Fax: +65 68748261. E-mail: [email protected]. Notes

The authors declare the following competing financial interest(s): HY holds equity in Ming-Mei-BBBMD and Invitrocue which license and commercialize the cellulosic sponge and related 3D culture technologies and services from the A*STAR.

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ACKNOWLEDGMENTS We would like to thank Drs. Igor Cima, Yi-Chin Toh, and Jeffrey Robbens and all members of the laboratory of Cellular and Tissue engineering. This work is supported in part by funding from the Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research (A*STAR) of Singapore; and grants from Hoffman La-ROCHE, Singapore-MIT Alliance for Research and Technology BioSyM, and Mechanobiology Institute funding to HYU. 2113

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