Hepatic Maturation of Human Fetal Hepatocytesin Four-Compartment Three-Dimensional Perfusion Culture

Alexander Ring, M.D.,1 Jorg Gerlach, M.D., Ph.D.,2 Grant Peters,2 Benjamin J. Pazin,2

Crescenzio F. Minervini, Ph.D.,2 Morris E. Turner, M.D.,3 Robert L. Thompson, M.D.,3

Fabio Triolo, Ph.D.,4 Bruno Gridelli, M.D.,4 and Toshio Miki, M.D., Ph.D.2

Bio-artificial liver support systems have been utilized as bridging devices to support acute and chronic liverinjury. However, prolonged function of adult hepatocytes has not been achieved due to compromised prolifer-ation and long-term survival of adult cells in vitro. As an alternative cell source, we investigated the potential ofhuman fetal hepatocytes (hFH) in a four-compartment hollow fiber–based three-dimensional (3D) perfusionculture system. hFH were isolated from 17- to 19-gestational-week livers and cultured in the 3D perfusionbioreactors for 14 days. Metabolism activity, hepatocyte-specific gene expression, protein expression, and hepaticfunction were investigated. Increased glucose consumption and lactate production indicated cell proliferationin the bioreactor. The ratio of cytochrome P450 3A4 to 3A7 gene expression and the increase of the number ofasialoglycoprotein receptor-positive cells indicated cell differentiation into mature hepatocytes. Histological andimmunohistochemical analysis revealed reorganization of fetal liver cells. Hepatic function was further examinedfor ammonia metabolism and for albumin production using colorimetric assays and enzyme-linked immuno-sorbent assay, respectively. In contrast to conventional 2D culture, the 3D perfusion culture system inducedfunctional maturation to hFH; these cells may be useful as an alternative cell source for extracorporeal liversupport.

Introduction

Extracorporeal liver support systems with bio-artificial liver (BAL) bioreactors have been proposed to

replace some of the hepatic functions of the failed liver andsupport patients to recovery or bridge them to orthotopicliver transplantation.1,2 Initial clinical applications usingeither xenogeneic or allogeneic adult hepatocytes had beenreported.3–9 Although the concept is accepted and safety,feasibility, and improvement of some clinical parametershave been demonstrated, the technology is not a standardtherapy yet in clinics.10

One remaining key issue is identifying the right cell sourceto charge BALs with. There are several candidate cell types:adult human hepatocytes, xenogeneic hepatocytes (mainlyporcine), and human hepatoblastoma cell lines, or immortal-ized human hepatocytes. Although porcine hepatocytes aremore readily available,7,11,12 the concerns for potential zoo-notic transmission, species differences, and immunological

problems reduce enthusiasm for their clinical application.13,14

Human hepatoblastoma cell lines15 and immortalized humanhepatocytes16 were suggested to overcome the shortage ofadult human hepatocytes; however, their safety and biologi-cal efficiency are still major concerns.17

Human fetal hepatocytes (hFH) are also considered asan alternative cells source,18 as their intrinsic proliferativecapacity and functional authenticity are attractive for BALapplications. There is no risk of zoonotic transmission andimmunological problems such as those associated with xe-nogeneic hepatocytes. Since cells are not genetically modi-fied, there is reduced risk of tumorigenicity by contrast tohepatoma cell lines or immortalized human cell lines. Onthe other hand, cell number availability and functional ma-turity represent issues of concern for fetal cells. Our goalwas to investigate the culture behavior of hFH in a three-dimensional (3D) perfusion bioreactor developed for liversupport. In this study, a downscaled (8-mL cell compartmentsize) 3D perfusion bioreactor was used to explore culturing

1Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charite-Universitats Medizin Berlin, Division of Experimental Surgery,Berlin, Germany.

2Department of Surgery, McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania.3Allegheny Reproductive Health Center, Pittsburgh, Pennsylvania.4Regenerative Medicine and Cell Therapy Unit, ISMETT—Mediterranean Institute for Transplantation and Advanced Specialized

Therapies, Palermo, Italy.

TISSUE ENGINEERING: Part CVolume 16, Number 5, 2010ª Mary Ann Liebert, Inc.DOI: 10.1089=ten.tec.2009.0342

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17–19-week-gestational-age hFH. As various authors havedescribed, 3D culture and physical parameters such as flowimprove the culture conditions for hepatocytes in vitro.19,20

Also, it has been previously described that cell density forhepatocyte culture in bioreactor culture plays a crucialrole.21,22 Previous studies using the 4-compartment hollowfiber membrane bioreactor developed by our group dem-onstrated that the system is capable to maintain hepaticfunction of adult hepatocytes for a prolonged period oftime.19 In a recent study using mouse embryonic stem cellswe demonstrated that these cells spontaneously differentiateand organize into in vivo–like structures under 3D perfusionculture conditions.23 We hypothesized that a high-density3D perfusion bioreactor system may support in vitro expan-sion of hFH while allowing maturation.

In this study, a total of six laboratory-scale high-density3D perfusion bioreactor experiments were performed. Anexhibiting 8-mL cell compartment was charged with 17–19-week-gestational-age hFH. To determine whether pro-longed culture time is important to enhance the outcome,two different culture periods were chosen, 7 and 14 days,respectively, with three individual experiments for eachculture period.

Materials and Methods

Bioreactor, tubing, and perfusion units

The bioreactor used was designed to simulate the hepaticvascular environment with hollow fiber technology.24,25 Bio-reactors (0008=BR) and perfusion units (PS=2–8 mL) wereprototyped by Stem Cell Systems (Berlin, Germany). Eachbioreactor contains two bundles of hydrophilic hollow fibermicrofiltration membranes (400 kDa cut-off) for transport

of culture medium in a decentralized pattern, interwovenwith one bundle of hydrophobic multi-laminate hollowfiber oxygenation membranes for transport of oxygen andcarbon dioxide (CO2)26,27 (Fig. 1). Cells were cultured in theinterstitial spaces between the fibers. The perfusion devicecontains two modular pump units, a heating unit, and a gas-mixing unit (Fig. 2). The perfusion tubing with bubble trapsis made of standard medical-grade dialysis PVC (B. Braun,Melsungen, Germany). Sterilization is performed with eth-ylene oxide according to clinical standards.

Human fetal cell isolation and culture

Human fetal liver tissues of 17–19 week gestational agewere obtained from the Allegheny Reproductive HealthCenter with internal review board approval of the Universityof Pittsburgh and prior written informed consent of the do-nor. To isolate hFH, fetal livers were enzymatically digested,followed by a density-based and cell-anchorage-dependenthepatocyte enrichment protocol. Briefly, the procured fetalliver tissues were placed into 0.5 M ethylene glycol tetraacetic acid solution and mechanically disrupted using a cellscraper. The tissue=cell suspension was then washed inphosphate-buffered saline with calcium (Ca2þ) and magne-sium (Mg2þ) and incubated with 0.04% collagenase Type IIplus DNase I (2000 U) (both from Sigma–Aldrich, St. Louis,MO) for 15–30 min at 378C. All washing steps used centri-fugation at 50 g.

After isolation, human fetal liver cells were seeded onEmbryoMax� ES Cell Qualified 0.1% Gelatin Solution(Millipore, Billerica, MA)–coated plates and incubated for48 h at 378C with 5% CO2, followed by phosphate-bufferedsaline washing to remove nonadherent cells and debris.Morphology confirmed that more than 90% of cells were

FIG. 1. Bioreactor design technology.(A) Smallest repetitive capillary mem-brane unit with compartments formedium perfusion (II, IV), oxygenation(III), and cells (I); the cells are not shown,the dots represent mass exchange andparticle distribution. (B) Single unitsforming capillary bundles. (C, D) Three-dimensional (3D) arrangement of capil-laries within the cell compartment. Thecells are shown between the capillaries.Compartments can be perfused indepen-dently as shown in (B), addressing thereduction of gradient distances betweencapillary units and enhancing mass ex-change. All membrane compartments areinterwoven, forming a tight network (C).The capillaries of each compartment arebundled toward the inlet and outletheads and connected to tube systems forperfusion (B). Cells are inoculated via16 open-ended tubes, allowing even celldistribution within the cell compartment.Interweaving the smallest capillary units(A) allows scale-up without changing thesmallest units (C). Color images availableonline at www.liebertonline.com=ten.

836 RING ET AL.

hepatocytes, which were subsequently dissociated by incu-bation with trypsin–ethylenediaminetetraacetic acid for3–5 min at 378C. Cell count and viability was estimated usingthe trypan blue dye exclusion method.

Cell culture

2D culture. A total of 1�106 cells were cultured on eachconventional 100 mm plastic Petri dish, precoated with type Icollagen (Biocoat; Becton Dickinson Labware, Bedford, MA).Cells were cultured in a humidified, 5% CO2, 95% air envi-ronment at 378C. Culture medium was changed every day.

Bioreactor culture. Three independent bioreactor cul-tures were performed for each period (7 and 14 days). A totalof 1�108 cells were inoculated into each bioreactor. To pro-vide extracellular matrix components’ similar to collagen-coated dishes used for 2D culture, the collagen macroporouscarrier Cultispher-S� (Sigma–Aldrich) was used. Due to awashout effect of the bioreactor, liquid collagen or gelatincoating solutions are not suitable. Preliminary experimentshad been conducted to choose Cultispher-S, which supportsthe hFH settlement in the bioreactor. Since the diameter ofCultisphere-S particles exceeds the pore size of the hollowfiber membranes, these particles remain in the cell compart-

ment. The Cultisphere-S beads were weighed and aliquoted.For each experiment, 0.2 g rehydrated beads were mixed andinoculated with 20 mL cell suspension.

To ensure even cell distribution, early prototypes withdifferent cell inoculation tube configurations (distributionand numbers) were investigated by fat emulsion inoculationand subsequent observation of distribution within the cellcompartment by magnetic resonance imaging. On the basisof these results, an optimal configuration was chosen: 16-cellinoculation tubes are incorporated throughout the cell com-partment, providing even distribution of inoculated cellsuspension.

Bioreactors were continuously perfused with culture me-dium at a recirculation rate of 30 mL=min. The volumeof recirculation circuit is 50 mL. Fresh medium was con-tinuously added from an external reservoir (Fig. 2A). Feedrate was set to a starting value of 2 mL=h, which equals onecomplete medium change in 24 h. To maintain a neutral pHand sufficient supply of cell nutrition, including glucose, thecontinuous fresh medium supply rate (pump speed) wasadjusted (up to 8 mL=h) based on the daily measurements ofpH and glucose concentration in the recirculation mediumduring bioreactor culture. The total flow of compressed air(medical grade, provided from the laboratory infrastructure)and CO2 (medical grade, conventional tank) is sterile filtered

FIG. 2. Fluid circulation and gas supply. (A) Fluid circulation setup: the red lines indicate arterial lines for fresh mediumsupply; blue are out flow venous lines from the bioreactor. Medium is prewarmed to 378C and recirculated (red and bluearrows), and fresh medium supply is adjusted according to glucose and lactate concentration to maintain constant cultureconditions. Bubble traps allow buffering the medium pressure in the circuit. (B) Gas mixture (air and carbon dioxide) issupplied by and additional tube set. The total volume of air and carbon dioxide can be individually regulated; flow rates canbe freely regulated by gas rotameters. The adjusted mixed air is supplied into the bioreactor through the number 5 port andout from number 1 port to oxygenize the cell compartment via a bundle of hydrophobic multi-laminate hollow fiberoxygenation membranes. Color images available online at www.liebertonline.com=ten.

HUMAN FETAL HEPATOCYTES IN BIOREACTOR 837

and mixed in the perfusion device, which is equipped withthree rotameters to regulate gas flow (Fig. 2B). The gas flowrate was set to 35 mL=min, with a starting mixture of 95%air and 5% CO2. On the basis of the daily measurement ofpH, the CO2 flow is adjusted to optimize the pH of the bicar-bonate buffered culture medium. A handheld bedside ana-lyzer iSTAT (Abbott Laboratories, Princeton, NJ) was used tomeasure glucose, lactate, pH, and CO2. At each end-point ofthe bioreactor culture, the upper bioreactor lid was openedand the cell mass, including the capillary layers, was cutfrom the surrounding potting and transferred into a sterileglass vessel for dividing into sections for further analysis.

Culture medium. Cells were cultured in Dulbecco’s mod-ified Eagle’s medium=F-12 (Invitrogen, Grand Island, NY)supplemented with 10% fetal bovine serum (Invitrogen),0.8 mg=L insulin, 5 mg=L transferrin, 0.003 mg=L glucagon(ITG Supplement; Biochrom AG, Berlin, Germany), 10�6 Mdexamethasone (Sigma–Aldrich), 10 ng=mL human recom-binant epidermal growth factor (BD Bioscience, FranklinLakes, NJ), 2 mM L-glutamine (Invitrogen), 100 U=mL peni-cillin G, and 100mg=mL streptomycin (Invitrogen).

Metabolic activity monitoring

The metabolic activity of the cells inside the bioreactorswas evaluated every day by measuring glucose and lactateconcentrations in the culture medium with iSTAT=Glu andiSTAT=CD4þ cartridges, respectively. The concentrations wereadjusted by inoculated cell number and feeding rate to normalizeindividual bioreactor experiments. A total of six bioreactor cul-tures were performed, and the data were plotted using Graph-Pad Prism (version 5.0; GraphPad Software, La Jolla, CA).

Liver-specific gene expression

Cells were harvested before inoculation into the bioreac-tor (Pre-BR), 7 or 14 days after bioreactor (Post-BR), and after2D static culture (2D culture). Total RNA was extractedfrom cells using the RNeasy� Mini Kit (Qiagen, Valencia,CA). Two micrograms of total RNA was used to synthesizecomplementary DNA with conventional reverse transcrip-tion (Promega, Madison, WI). Quantification of mRNA usingreal-time polymerase chain reaction analysis was performedusing an ABI PRISM 7000 system (Applied Biosystems,Foster City, CA). The predesigned TaqMan probe and primersets for albumin (Hs00609411_m1), alpha-1 antitrypsin (A1AT)(Hs00165475_m1), cytochrome P450 (CYP)3A4 (Hs00430021_m1), CYP3A7 (Hs00426361_m1), HNF4a (Hs00230853_m1),tyrosine aminotransferase (Hs00356930_m1), G6PC (Hs00609178_m1), and b-actin (Hs99999903_m1) were selected fromTaqMan Gene Expression Assays (Applied Biosystems). Re-lative gene expression was analyzed using delta-delta-Ct(ddCt) methods. All values were then normalized to b-actinmRNA expression and are presented as relative increase ofgene expression compared to freshly isolated hFH (day 0).Two-way analysis of variance test and Bonferroni posttestwere performed to calculate p-value.

Flow cytometric analysis

To determine functional maturation of hFH, flow cytome-tric analysis was performed with an anti-asialoglycoprotein

receptor (ASGPR) antibody. Before inoculation and after14 days in culture, a total of 1�106 cells were harvested from2D static culture or 3D perfusion culture and incubated withmonoclonal anti-ASGPR antibody (Cell Sciences, Canton,MA) for 1 h, followed by Alexa 488–conjugated goat anti-mouse IgG secondary antibody (Invitrogen, Carlsbad, CA)for 30 min. Mouse IgG1 isotype control antibody (Dako,Carpinteria, CA) was used as negative control.

Analyses were performed on a FACSCalibur system usingCell Quest software (Becton Dickinson, San Jose, CA). For-ward and side scatter gates were set to include all viablecells. The percentage of positive cells was measured from acut-off set using an isotype-matched control antibody usingFlowJo software (Tree Star, Ashland, OR). Student’s t-testswere performed using the Mann–Whitney U-test with asignificance level of 95%.

Measurement of liver-specific functions

To determine the production of albumin, recirculationmedium samples were taken every day and stored at �1508Cuntil analysis. Human albumin concentration in the culturemedium was determined by Human Albumin ELISA Quan-titation kit (Bethyl Laboratory, Montgomery, TX) accordingto the manufacturer’s instructions.

To evaluate the hepatic detoxification function, ammoniachallenges were performed on day 4, 7 (n¼ 3), and 11 (n¼ 1)of the bioreactor culture cells. In brief, ammonium chloride(NH4Cl; Sigma–Aldrich) was injected into the medium cir-cuit to a final concentration of 5 mM. After 30 min to allowfor equimolarity, the cells were incubated for 4 h. During thisperiod the system was operated without fresh medium sup-ply to eliminate dilution. After incubation, samples werecollected and urea concentrations were measured by using acolorimetric assay kit (Urea Colorimetric Assay Kit, Quanti-Chrom; BioAssay Systems, Hayward, CA).

Histological and immunohistochemical analysis

Bioreactor samples were harvested at each end-point ofbioreactor culture (day 7 and 14) and immediately embed-ded into 2% low-melting agarose gel, after fixation in 10%formalin. The paraffin-embedded samples were cut into4-mm-thick sections, and hematoxylin and eosin staining wasperformed to evaluate tissue architecture and organization.Immunohistochemistry (IHC) and immunofluorescence (IF)were performed on the paraffin-embedded samples. Theantibodies used were polyclonal rabbit anti-human albumin(IHC; DakoCytomation, Carpinteria, CA), polyclonal rabbitanti-human a1 antitrypsin (IHC; Dako), monoclonal mouseanti-human cytokeratin (CK) 19 (IHC; CK19; Chemicon,Temecula, CA), and polyclonal goat anti-Ki67 antibody (IHC;Santa Cruz Biotechnology, Santa Cruz, CA), monoclonalmouse anti-human albumin (IF; Sigma–Aldrich), monoclo-nal rabbit anti-human CK8 (IF; Abcam, Cambridge, MA),monoclonal mouse anti-human CK19 (IF; Santa Cruz Bio-technology), and polyclonal rabbit anti-human CYP3A4 (IF;Cypex, Dundee, Scotland). Adjacent sections served as neg-ative controls and were incubated with appropriate isotypecontrol antibodies and secondary antibodies. For IHC, the3,30-diaminobenzidine Substrate Kit (Vector Laboratories,Burlingame, CA) was used to develop the slides and hema-toxylin staining was performed as a nuclear counter staining.

838 RING ET AL.

Secondary antibodies used for IF were polyclonal goat anti-mouse Alexa Flour 488–conjugated (Invitrogen) and poly-clonal goat anti-rabbit Cy3–conjugated (Dianova, Hamburg,Germany). Nuclei were observed with 40-6-diamidino-2-phenylindole staining.

The periodic acid-Schiff (PAS) staining system (Sigma–Aldrich) was used to observe glycogen in the tissue-likestructure according to the manufacturer’s instructions.

Statistical analysis

Data were obtained from two to six independent runsunder each culture condition and are expressed as meansand standard deviations. Statistical analysis was carried outusing the statistic and graphic software Prism 5 for Mac OSX(GraphPad Software).

Results

Metabolic activity of fetal hepatocytesin a 3D perfusion bioreactor

Glucose consumption and lactate production of hFH in-creased continuously during the culture period, indicatingcell proliferation and increasing total metabolic activity (Fig.3A, B). Assuming that each individual cell consumes simi-lar basal quantity of glucose, the overall increasing glucoseconsumption would reflect fetal hepatocyte proliferation.Overall, the metabolic activity in conjunction with the belowdescribed Ki-67 proliferation marker staining results indi-cates that the hFH were actively proliferating in the 3Dbioreactor culture system.

Hepatocyte-specific gene expression indicatesspontaneous maturation

Quantitative real-time reverse transcriptase–polymerasechain reaction was performed to investigate the maturationof hFH during the 3D perfusion bioreactor culture. Expres-sion of seven hepatic genes (HNF4a, albumin, A1AT, tyro-sine aminotransferase, glucose-6-phosphatase, CYP3A4, andCYP3A7) was investigated. Overall, except for CYP3A4, ex-pression of these genes under bioreactor culture conditionsdid not differ significantly between culture conditions.However, expression of all hepatic genes investigated tendedto be higher if the cells were cultured in the 3D bioreactorthan in 2D cultures. CYP3A4 gene expression was signifi-cantly increased in the 3D bioreactor cultured cells (Fig. 4A).The expression ratio of CYP3A4 and CYP3A7 was comparedto evaluate hepatic maturation. The values of day 7 and 14CYP3A4=3A7 ratio relative to the day 0 sample are depictedin Figure 4B. Although both 2D static and 3D perfusionculture conditions increased CYP3A4 expression, indicatingprogress of CYP3A7 to CYP3A4 conversion, the ratio wassignificantly higher in the 3D bioreactor cultured cells( p< 0.05, Fig. 4B). These data suggest that hFH spontane-ously differentiated toward mature hepatocytes and that the3D perfusion culture system induced more maturation than2D static culture conditions.

Ratio of ASGPR-positive hepatocytes

The hFH show the presence of ASGPR as demonstratedby fluorescence-activated cell sorting. The anti-ASGPRantibody had been utilized to identify functional mature

FIG. 3. Time course of metabolic activ-ity in a 3D perfusion bioreactor. Metabolicparameters glucose (A) and lactate (B)were measured in daily medium samplesfrom recirculation over 14 days in thebioreactor. Concentrations were normal-ized against inoculated cell number andfeeding rate to compare each individualbioreactor experiment. Graphs (A) and(B) show metabolic activity of humanfetal hepatocytes (hFH) throughout7- and 14-day culture. The data aremean� SD, n¼ 6 for each group.

FIG. 4. Cytochrome P450 3A familygenes expression ratio. (A) Hepatocyte-specific gene expression in 2D static and3D perfusion culture was compared onday 7 (n¼ 1) and 14 (n¼ 1). All valueswere normalized to b-actin mRNA ex-pression. (B) The gene expression ratio ofmature (3A4) and fetal (3A7) forms ofcytochrome P450 was compared between3D perfusion bioreactor culture and 2Dstatic dish culture after 7 (n¼ 3) and 14

days (n¼ 3) in culture. Two-way analysis of variance test and Bonferroni posttest were performed to calculate p-value. Barsshow the average ratio relative to day 0. Values are mean� SD, *p< 0.05, **p< 0.001.

HUMAN FETAL HEPATOCYTES IN BIOREACTOR 839

hepatocytes.28,29 Data from six 3D bioreactor culture exper-iments were studied and the ratio of functional hepatocyteswas demonstrated (Fig. 5). Results were statistically ana-lyzed with the Mann–Whitney test (n¼ 3). Before culture,freshly isolated hFH contained only a small number ofASGPR-positive cells (6.9� 3.5%). This percentage was dra-matically increased after 14 days in 3D bioreactor culture(67.11� 9.88%), whereas 2D culture induced 25.0� 12.29%ASGPR-positive cells after 14 days (Fig. 5). The data confirmthat the 3D perfusion culture system is superior to inducehepatic maturation than 2D static culture conditions.

3D perfusion bioreactor maintainedhepatic function of hFH

Albumin production was measured as an indicator ofhepatic synthetic function (Fig. 6). Albumin showed an initialincrease, which could indicate that damaged cells releasealbumin in the culture adaptation phase, leading to falsehigh levels of albumin. This was confirmed by high levels oflactate dehydrogenase (LDH) (as marker of cell death=dis-disruption) (data not shown). After 5 days the albuminconcentration was stable and did not drastically change for14 days. Total production of albumin per 1 million inocu-lated cells was on average 3.4-fold higher under 3D condi-tions compared to 2D culture. Human albumin was notdetected in the basal medium.

Urea synthesis by hFH in the 3D bioreactor was investi-gated to monitor ammonia metabolism activity (Fig. 6B). Theincrease in activity was evaluated by comparing the levels ofurea before and after a 4 h incubation with 5 mM NH4Cl(ammonia challenge test), which show an up to 4.3-fold in-crease of urea production. The results indicate that hFH inthe 3D bioreactor maintained ammonia metabolism functioneven after 11 days in culture.

Histological and immunohistochemical analysis

Hematoxylin and eosin stain and PAS staining are shownin Figure 7. Immunohistochemical analysis showed thatmost cells stained positive for albumin and A1AT (Fig. 7B, D)as well as for PAS staining (Fig. 7F), which identifies glyco-gen storage function of the hepatocytes. Fetal hepatocytesrestructured to a hepatic plate-like morphology in the bio-reactor. There were only few areas that tested negative foralbumin. Most likely, these cells represent nonparenchymalcells that can also be found in organs in vivo. To characterizethese cells, the samples were stained with anti-CK19 anti-body. CK19 is expressed exclusively in the biliary cells of theadult liver and in biliary lineage cells including bipotentialhepatic progenitor cells of the fetal liver. We observed scat-tered CK19-positive cells and very few, but clearly CK19positive, tubular structures in the cell mass (Fig. 8B, red ar-

FIG. 5. Flow cytometric demonstrationof functional hepatocyte cell surfacemaker-positive cells in 3D perfusion bio-reactor. For each analysis, hHF before andafter 2D and bioreactor culture were ali-quoted into individual tubes at a total of1�106 cells per tube. Analysis was per-formed using untreated cells, isotypecontrol, and ASGPR-1 antibody. The rep-resentative histograms show fluorescentintensity versus percentage of maximumcell number of isotype control (line) andASGPR-positive cells (shaded). ASGPR-positive cell ratios from three individualexperiments are shown in the histogram(n¼ 3). Values are mean� SD, *p¼ 0.0061.

FIG. 6. Change of parameters forhepatocyte-specific functions. (A) Timecourse of secreted albumin in the re-circulation medium of 3D perfusionbioreactor culture (n¼ 3) compared tothat of 2D static culture (n¼ 3). Humanalbumin concentration in therecirculation=culture medium was mea-sured by enzyme-linked immunosorbentassay. Concentrations were normalizedagainst inoculated cell number andfeeding rate to compare each individualbioreactor experiment. Results are pre-sented as mean� SD, p¼ 0.015. (B) Ureasynthesis by hFH in 3D perfusion biore-actor culture was investigated. Bars represent x-fold increase of urea production by hFH after incubation with 5 mMammonium chloride for 4 h on culture days 3 (n¼ 3), 7 (n¼ 3), and 11 (n¼ 1).

840 RING ET AL.

row). These data indicate that some of the dissociated biliarylineage cells reoriented or proliferated to form bile-duct-liketubular structures within the human fetal liver cells. Thesetissue formations were not observed under conventional 2Dculture.

To observe the proliferating cells, Ki-67 staining was per-formed (Fig. 8D). Ki-67 is a cellular marker strictly associatedwith cell proliferation.30 The protein is present during allactive phases of the cell cycle (G1, S, G2, and mitosis), butis absent in resting cells (G0). Supporting the metabolic

FIG. 7. Expression of hepatic proteinsafter 3D perfusion bioreactor culture.Representative histological sections of3D bioreactor cultured for 14 days.Paraffin-embedded samples were im-munostained against human albumin(20�, B), alpha-1 antitrypsin (40�, D),and histochemically stained for glycogenby periodic acid-Schiff staining (20�, F).The yellow arrow indicates gelatinCultispher-S microbeads. Serial sectionswere stained with isotype control anti-body (A, C) or with hematoxylin andeosin (E). Arrowheads indicate innerwall of the hollow fiber (A, E). Colorimages available online at www.liebertonline.com=ten.

FIG. 8. Reconstructed liver structure ina bioreactor. Immunohistological analy-sis with anti-cytokeratin (CK) 19 andKi-67 (proliferation marker) antibodies.After 14 days in culture, hFH formedductal structures in the 3D perfusionbioreactor (20�, B). The cells in the duc-tal structure were positive for biliary cellmarker CK19 (red arrows). Cells positivefor Ki-67 are actively proliferating (40�,D). Hematoxylin and eosin staining (20�,A) and isotype control staining (40�, C)were performed on the serial section ofthe sample. Color images availableonline at www.liebertonline.com=ten.

HUMAN FETAL HEPATOCYTES IN BIOREACTOR 841

biochemical data, most of the hepatocyte-like cell nuclei werepositive for the Ki-67 antibody (Fig. 8D).

IF analysis

IF double staining was performed with anti-humanalbumin=CYP3A4 (Fig. 9) and anti-human CK8=CK19 anti-bodies (Fig. 10) on day 14 samples. Most of the cells stainedpositive for albumin (Fig. 9A, D) and more than half of thealbumin-positive cells were also positive for CYP3A4 (Fig.9B, D). The observation indicates that hFH are functionallywell maintained and induced further maturation in the 3Dperfusion bioreactor. To further characterize the cells aftercell culture, double staining for CK8 and CK19 was per-formed. Scattered CK19-positive cells were found (Fig. 10B,D) within the cell clusters that showed predominantly CK8-positive hepatic characteristics (Fig. 10A, D). Duct-likestructures were not observed with the CK8=19 double-positive cells. Morphologically, the CK8=19 double-positivecells are small in size and have a low nucleus-to-cytoplasmratio. Most likely, these cells represent bipotential liver pro-

genitor cells that can give rise to hepatic epithelial and biliarycells.31–33 These results showed that some of the human bi-potential liver progenitor cells were still maintained duringthe 14 days of 3D perfusion bioreactor culture.

Discussion

Bioengineering developments for extracorporeal tem-porary liver support offer technology advancements andprovide improved culture conditions.34–37 One remaining bi-ological limitation is the lack of a suitable cell source for BALbioreactors meeting criteria such as safety, availability, andfunctionality. The goal of this work was to test whether the3D perfusion culture system enables expansion and inducesmaturation of hFH, and if so, how it compares with the con-ventional 2D culture system. Due to the engineering struc-ture of the bioreactor, which is designed in its clinical scale asa component of an extracorporeal liver support system toprovide metabolic functions rather than producing remov-able cells, cell retrieval is very complicated. The dense 3Dnetwork within which hepatocytes are cultured and form

FIG. 9. Immunofluorescence analysisof the human fetal liver cells in the 3Dperfusion bioreactor. Double immuno-staining for human albumin (green, 20�,A) and cytochrome P450 3A4 (red, B) ofhFH after 14 days in culture. Most of thefetal liver cells stain positive for albu-min, whereas more than half of the cellsare positive for CYP3A4 (Merged im-ages: D). Nuclei and colony morphologywere observed with 40-6-diamidino-2-phenylindole staining (blue, C, D) andphase contrast imaging (E), respectively.Color images available online atwww.liebertonline.com=ten.

FIG. 10. Hepatic progenitor cells werepreserved in the 3D perfusion bioreactor.Samples were taken from hFH after 14days of culture, and double im-munostaining for human CK8 (red, 40�,A) and 19 (green, B) was performed,revealing CK8=19 double-positive cellsscattered in the CK8-positive cell clusters(D). The double-positive cells are smallin size with a low nucleus-to-cytoplasmratio. Nuclei and colony morphologywere observed with 40-6-diamidino-2-phenylindole staining (blue, C, D) andphase contrast imaging (E), respectively.Color images available online atwww.liebertonline.com=ten.

842 RING ET AL.

tissue-like clusters makes it almost impossible to obtain exactcell numbers during and after the cell culture. Therefore,indirect methods such as monitoring of metabolic activity(glucose consumption and lactic acid production) and stain-ing of cored biopsies (analog to clinical liver diagnostics) forthe proliferation marker Ki67 were used to assess cell pro-liferation. Under the assumption that each cell’s averageglucose consumption and lactate acid production are stable,higher consumption of glucose and production of lactic acidwas correlated to augmentation of cell mass in the bioreactor.Steady increase in metabolic activity was observed, sug-gesting that cells actively proliferated throughout the cultureperiod. Staining for Ki-67, a proliferation marker, showedcell clusters that largely positive cells. Although we did notintend to induce terminal differentiation with exogenousgrowth factors, the hFH indeed spontaneously differentiated.The drug metabolism enzymes are considered as late-phasehepatocyte-specific genes7; thus, the gene expression ratio offetal and adult forms of CYP enzymes was used to evaluatehepatic maturation.38 CYP3A7 is predominantly expressed inhFH and normally extinguished in adult hepatocytes, whereCYP3A4, the adult counterpart, takes over.39,40 Although thematuration was not completed within 14 days, the tendencyappears promising for further differentiation induction byadditional culture condition optimization with exogenousgrowth factors. The spontaneous differentiation was alsoconfirmed by expression of the ASGPR. The ASGPR is spe-cific for desialylated glycoproteins and is expressed exclu-sively in hepatic parenchymal cells.41 Specific expression hasbeen used as a mature hepatocyte marker42,43 or utilized todistinguish healthy functional hepatocytes from hepatomacells or nonfunctioning cells with nuclear medical ap-proaches.44,45 Studied synthetic and excretory hepatic func-tions were also maintained. The inability of maintainingthese functions for long-term in primary human adult he-patocyte culture is well documented.15,41 It has been shownthat the 3D perfusion hollow fiber bioreactor technologyenables maintenance of the hepatic function of primary adulthepatocytes over several weeks.25,46 Interestingly, hFH ex-hibit the prolongation of hepatic synthetic function in both2D static and 3D perfusion bioreactor culture systems. Therewas no significant difference of the quantity of albuminmRNA expression between 2D and 3D culture, however, theactual quantity of secreted albumin was significantly higherin the 3D bioreactor culture. Since the albumin gene ex-presses relatively early phase of the liver development, theprotein production might not be dramatically influencedby further maturation, resulting in the relatively stable pro-duction throughout culture. Nevertheless, continuous per-fusion provides an additional advantage of the bioreactorsystem, which can easily be adjusted to the needs of culturedcells without compromising sterility by numerous mediumchanges. Future application of automated sensors, such asfor pH, could completely automatize this process.

Histological examination of cultured cells revealed liver-tissue-like formation including CK19-positive duct-likestructures, which were not observed under 2D conditions.The mixture of the nonparenchymal cells might positivelyinfluence the liver cells to reconstruct in vivo–like cell envi-ronments in the BAL system.47–49 The environment mightplay a key role to prolong fetal hepatocyte proliferation anddifferentiation.50 According to our current results with PAS

staining (glycogen storage) and anti-CYP3A4 immunofluo-rescent analysis, we did not observe zonal characteristics.Although the staining patterns were speckled, there were noclear centralized or radial distributions. In future experi-ments, more specific analysis will be conducted to charac-terize zonal functions in the 3D reconstructed liver-likestructure. Although maturation of hFH cells was demon-strated, additional studies revealed a pool of bipotentialprogenitor cells that remained even after 14 days of culturein the bioreactor. Further studies, such as coculture withlabeled cells, will be important to track their fate. However, amaintained pool of expandable progenitors could be an in-nate cell-replenishing source. In addition to the phenotypicalmaintenance, the 3D-bioreactor-cultured hFH exhibit func-tional parameter activities on detoxification function. Oneimportant aspect of BAL systems is the ability to eliminateammonia. Challenging the cells with NH4Cl dramaticallyincreased urea levels in the culture medium, indicating thathFH are capable of detoxification.51 After the longer cultureperiods of 14 days, the capability to eliminate ammonia viathe urea cycle increased significantly. Further studies, pos-sibly be reseeding a defined number of cells after 3D cultureand comparing detoxification to freshly isolated hFH, will beaimed to elucidate whether higher detoxification must beattributed rather to an increased cell number or functionalmaturation of the hFH in 3D culture.

In conclusion, the predominance of matured hepatic cellsin combination with the maintenance of bipotential progen-itor cells in the investigated culture periods indicates thatdynamic 3D perfusion bioreactors are of interest to furtherstudy expansion and maturation of hFH for potential clinicalapplications. In a recently published study, Koyama et al.combined a static 3D culture with microporous carriers withsoluble growth factors and showed a positive influence onthe maturation of mouse fetal liver cells.52 In future studiesthe combination of soluble factors with our 3D dynamicperfusion system will be of interest to investigate the possiblesynergistic effect of physical and biochemical factors on liverdifferentiation and maturation. Since the in vitro microenvi-ronment enables tissue restructuring also with fetal livercells, it appears feasible to provide bioreactors that yield amajority of functional cells desired for clinical applicationsand at the same time preserve a pool of stem cells as a sourcefor self-renewal, which could significantly prolong usabilityof such a system.

Acknowledgments

Support for this study was granted by the University ofPittsburgh Medical Center. We thank Ms. Claire Keyes, andMs. Sarah Dittoe from the Allegheny Reproductive HealthCenter, and the rest of the staff for their enthusiasm anddiligence in supporting the project.

Disclosure Statement

No competing financial interests exist.

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Address correspondence to:Toshio Miki, M.D., Ph.D.

Department of SurgeryMcGowan Institute for Regenerative Medicine

University of Pittsburgh3025 East Carson St., Suite 238

Pittsburgh, PA 15203

E-mail: toshio@pitt.edu

Received: May 22, 2009Accepted: November 2, 2009

Online Publication Date: December 31, 2009

HUMAN FETAL HEPATOCYTES IN BIOREACTOR 845