Membrane Bioreactor for Expansion and Differentiation of Embryonic Liver Cells

Membrane Bioreactor for Expansion and Differentiation ofEmbryonic Liver CellsSimona Salerno,† Antonella Piscioneri,† Sabrina Morelli,† Mohamed B. Al-Fageeh,‡ Enrico Drioli,†,§,⊥

and Loredana De Bartolo*,†

†Institute on Membrane Technology, National Research Council of Italy, ITM-CNR, c/o University of Calabria, Via P. Bucci, cubo17/C, 87030 Rende (CS), Italy‡National Centre for Biotechnology, King Abdulaziz City for Science and Technology, Riyadh 11442 Saudi Arabia§Department of Chemical Engineering and Materials, University of Calabria, via P. Bucci cubo 45/A, 87030 Rende (CS) Italy⊥WCU Energy Engineering Department, Hanyang University, Seoul, S. Korea

ABSTRACT: There is a growing demand for the expansion and differentiation of stem cells for cell therapies, tissue engineering,and model systems for drug screening. Current methods for stem cell production are based on the use of batch tissue cultureflasks, which have several drawbacks. In this paper we report on the use of a crossed hollow fiber membrane bioreactor for theexpansion and differentiation of embryonic liver cells, which have been used as an alternative model of human liver progenitorcells. The bioreactor is based on two bundles of fiber (PEEK-WC HF and PES-HF) which are cross-assembled in an alternatingmanner. This bioreactor geometry ensures high mass exchange of nutrients and metabolites, which is important for cellproliferation and differentiation. The membrane bioreactor, thanks to its optimized fluid dynamics and mass transport and theadequate surface properties of hollow fibers, was able to guide the expansion and differentiation of liver progenitor cells in maturehepatocytes as demonstrated by their expression of liver specific functions in terms of urea synthesis, albumin production, anddiazepam drug biotransformation.

■ INTRODUCTION

The growing demand for replacement tissues and organstructures due to aging of the population and increase ofseveral pathologies lead to development of new tissueengineering methods and technologies. Research efforts havebeen focused in this past decade on the use of embryonic andadult stem cells because of their ability to either self-renew ordifferentiate into multiple cell lineages.1 These characteristicsmake stem cells attractive as a cell source for cell therapies,tissue engineering, and model systems for drug screening.2

Stem-cell-based technologies can be successfully implementedby developing culture systems that are able to generate largenumbers of cells with well-defined characteristics and/or topromote controlled, reproducible differentiation into selectedmature cell types. Membrane system has a great potential in thestem-cell based technologies because it is able to act as aninstructive extracellular matrix (ECM). In fact it exhibits likeECM microscale to nanoscale of chemistry and topography andis able to provide cells physical, chemical, and mechanicalsignals, which are important for the differentiation process.Several strategies are aimed at engineering membrane systemswith specific physicochemical, topographical, mechanicalproperties, and configuration able to drive the differentiationof stem and progenitor cells (e.g., neurons, liver, cartilage,bone).3−5 Recently the expansion and the functional differ-entiation of rat embryonic liver cells were observed on asynthetic polymeric membrane of a modified polyetheretherke-tone (PEEK-WC) and on a biodegradable membrane ofchitosan.5 However, most of the studies concerning theexpansion and differentiation of stem or progenitor cells were

performed under static conditions. There are a few reports inwhich fetal liver cells were cultured under perfusion conditionsin a packed bed reactor,6 three-dimensional four compartmentbioreactor,7 polyurethane foam bioreactor,8 radial flowbioreactor,9 nonwoven polyester matrix bioreactor,10 or roatingwall vessel bioreactor.11 Nevertheless, there are still challengessuch as to provide the cells adequate nutrients, growth factors,and metabolites and to efficiently remove catabolites withoutcausing shear stress or affecting cell differentiation. Membranebioreactors have a significant advantage to perfuse cells throughmembranes that serve also as supports for cell adhesion,offering in the case of hollow fibers a large surface area in asmall volume. The mass transfer can be enhanced byminimizing the distance between cells and perfusion membranelumen.12

Previously we developed a crossed hollow fiber membranebioreactor to support the long-term maintenance and differ-entiation of primary human hepatocytes.13 The bioreactorconsists of two bundles of hollow fiber (HF) membranes withdifferent molecular weight (MW) cutoff and physicochemicalproperties cross-assembled in alternating manner: PEEK-WCand polyethersulfone (PES), which perform different functions.PEEK-WC HF membranes provide cells nutrients andmetabolites whereas PES HF removes catabolites from the

Special Issue: Enrico Drioli Festschrift

Received: January 4, 2013Revised: March 28, 2013Accepted: March 28, 2013Published: March 28, 2013

Article

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© 2013 American Chemical Society 10387 dx.doi.org/10.1021/ie400035d | Ind. Eng. Chem. Res. 2013, 52, 10387−10395

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cell compartment mimicking in this way the in vivo arteriousand venous blood vessels. The combination of these two fiberscreates three compartments: two intraluminal compartments ofPEEK-WC HF and PES HF in which the medium flows andone extraluminal compartment represented by extracapillarynetwork formed on both type of fibers in which cells arecultured. This geometry would ensure a high mass exchangethrough the cross-flow of culture medium.13

In this paper we demonstrate that crossed HF membranebioreactor guides the expansion and the functional differ-entiation of embryonic liver cells, providing physical, chemical,and mechanical cues through the different membrane proper-ties, and governing mass transfer of molecules. The perfusionconditions and the optimized mass transport through each typeof fiber ensure the creation of a homogeneous environment andthe possibility to monitor and control critical cultureparameters. This system represents a valuable bioengineeredplatform for a mass production of “guided differentiated” cellsto be used for cell therapy, bioartificial devices, or drugscreening. Rat embryonic liver cells (17 day embryos) wereused in this study as an alternative model of human liverprogenitor cells14−16 since using cells from the fetal human liveris limited by a major ethical issue. Embryonic liver cells havemany advantages over primary hepatocytes for proliferation invitro to transplantation in vivo. They exhibit spontaneousproliferation and the ability to differentiate into hepatocyte andbiliary duct cells representing an ideal hepatocyte source.17−20

■ MATERIALS AND METHODSCrossed HF Membrane Bioreactor. The membrane

bioreactor consists of two bundles of 40 PEEK-WC HF and40 PES HF membranes cross-assembled in alternating mannerand potted with polyurethane adhesive (Polaris Polymers, AvonLake, OH, USA) within glass housing. PEEK-WC HF and PESHF are used for the medium inflow and outflow, respectively.The two fiber systems establish three separate compartments:two intraluminal compartments within the PEEK-WC and PESfibers, and an extraluminal compartment or shell outside of thefibers, which communicate through the pores in the fiber wall.The bioreactor (volume: 40 mL) is connected to the perfusioncircuit consisting of microperistaltic pump, gas-permeablesilicone tubing, reservoir of medium, and glass medium waste.13

The oxygenated medium enters from the reservoir to themembrane bioreactor with a flow rate Qf of 1.2 mL/min thatwas set on the basis of average retention time. Fresh mediumwas perfused in single-pass and the stream leaving thebioreactor. Qout. was collected as waste until approaching thesteady state. When the system reached the steady state, thestream leaving the bioreactor was recycled (Qr) in order toobtain the accumulation of products.The fluid dynamics of the bioreactor were characterized in

terms of cumulative residence time distribution (RTD), whichwas investigated through the introduction of tracer (step input)at the entrance of PEEK-WC fibers and recording it in time atthe exit of the PES fibers. The tracer, consisting in a solution ofWilliams’ medium E, was sent to the bioreactor with flow rateof 1.2 mL/min, and the change of tracer concentration stepwisein the feed stream (Cin) and the outlet concentration (Cout) wascontinuously monitored by an online spectrophotometer (UVCord Pharmacia, Uppsala, Sweden). The cumulative RTD tostep inputs is described by the equation

=F t c c( ) /out in (1)

where t is the actual time. The theoretical mean retention timewas calculated as

τ = VQ (2)

where Q is the perfusion flow rate and V is the volume of thebioreactor.21 The agreement of the experimental F(t) with thatof a continuous stirred tank reactor (CSTR) was assessed byplotting the experimental data with the theoretical curve forCSTR obtained by the equation

= − τ−CC

e1 tout

in

/

(3)

Membrane Preparation and Characterization. PEEK-WC HF membranes were prepared according to the well-known dry−wet spinning method. To prepare highly porousmembranes, poly(vinylpyrrolidone) (PVP K17 by BASF) wasused as a pore forming additive. Membranes were preparedfrom solutions of PEEK-WC and PVP both at 15 wt % indimetylformamide (DMF) under continuous mechanicalstirring at room temperature as described elsewhere.22 Themorphological properties of the PEEK-WC HF and commercialPES HF (Membrana GmbH) membranes were characterizedby scanning electron microscope (SEM) (ESEM FEGQUANTA 200, FEI Company, OR, USA) in order to evaluatethe cross-sectional structure and thickness, intra- and extra-lumen morphology and diameters, and the shape and size of themembrane pores.The hydrophobic/hydrophilic character of the investigated

membranes was estimated by contact angle technique. Watercontact angles were measured using the sessile drop method atambient temperature by CAM 200 contact angle meter (KSVInstruments LTD, Helsinki, Finland), depositing the liquid onthe membrane surface using an automatic microsyringe.The mass transport properties were characterized by

evaluating the hydraulic permeance through measurements ofpure water flux (J) at different transmembrane pressures (ΔP).The permeation of specific metabolites such as albumin, urea,and diazepam was also assessed at different transmembranepressures. At the same time the diffusive transport of albuminand urea, which are produced by cells, from the fiber lumen tothe shell of PES HF membranes and the diffusive transport ofdiazepam, which is metabolized by cells, from the lumen to theshell of PEEK-WC HF membranes was also evaluated aspreviously described.23

Cell Culture. The rat embryonic liver cells (17 daysembryonic liver of Japanese albino rat) (RLC-18) wereobtained from the DSMZ (Braunschweig, Germany). Cellswere maintained in RPMI medium containing L-glutamine,penicillin, and streptomycin (Biochrom AG, Berlin, Germany)and supplemented with 10% FCS (Biochrom AG, Berlin,Germany) at 37 °C in a humidified CO2 incubator (95% air, 5%CO2) and subcultured twice a week by using trypsin (0.05%)/EDTA (0.025%) solution (Carl Roth GmbH, Karlsruhe,Germany).The cells were seeded at 9 passages in the extralumen

compartment of the bioreactor on the outer surface of andbetween the HF membranes at a density of 1 × 104 cell/cm2.The bioreactor was maintained at 37 °C in a 5% CO2:20% O2atm (v/v) with 95% relative humidity. After 4 h cells adheredand the bioreactor was perfused with oxygenated medium. Cellswere cultured for the first 24 h in medium supplemented with

Industrial & Engineering Chemistry Research Article

dx.doi.org/10.1021/ie400035d | Ind. Eng. Chem. Res. 2013, 52, 10387−1039510388

10% FCS (Biochrom AG, Berlin, Germany) and successively inmedium under serum-free conditions and supplemented withhepatocyte growth factor (HGF), epidermal growth factor(EGF), insulin, ascorbic acid, transferrin, hydrocortisone 21-hemosuccinate, gentamicin sulfate 50 μg/mL, amphotericin B50 ng/mL (HCM bulletkit, Lonza Sales Ltd., Basel, Switzer-land), and in presence of diazepam 10 μg/mL.Polystyrene culture dishes (PSCD) were used as reference

substrata in static culture conditions. Cells were cultured up to14 days and the medium was changed every 48 h.Cell Staining for LSCM. The morphological behavior of

embryonic liver cells on PEEK-WC membrane and on PSCDwas investigated after 8 and 14 days of culture laser scanningconfocal microscopy (LSCM) after cytoskeleton proteinimmunostaining. Samples were rinsed with PBS, fixed for 15min in 3% paraformaldehyde at room temperature (RT),permeabilized for 5 min with 0.5% Triton-X100, and saturatedfor 15 min with 2% normal goat serum. Vinculin was visualizedusing a mouse monoclonal antibody raised against rat vinculin(Santa Cruz Biotechnology, Santa Cruz, CA, USA) with goatantimouse IgG Alexa Flouor 546 conjugated (MolecularProbes, Eugene, OR, USA) as secondary antibody. Primaryand secondary antibodies were incubated at RT for 2 and 1.5 h,respectively. Actin was stained with Alexa 488-conjugatedphalloidin (Molecular Probes). Counterstaining for nuclei wasperformed with 0.2 μg/mL DAPI (Molecular Probes). Finally,samples were rinsed, mounted, and observed with a LSCM(Fluoview FV300, Olympus, Milan, Italy).Sample Preparation for SEM. Samples of cells grown in

the bioreactor on the membranes were prepared for scanningelectron microscopy (SEM) by fixation in 2.5% glutaraldehyde,pH 7.4 phosphate buffer, followed by postfixation in 1%osmium tetroxide and by progressive dehydration in ethanol.Samples were examined at SEM and representative imagesdisplaying morphological features were obtained after 8 and 14days of culture.Biochemical Assays. Albumin and urea synthesis of liver

progenitor cells were evaluated for the whole culture time.Samples from the culture medium were collected in prechilledtubes and stored at −20 °C until assayed. Albumin secretionwas measured in the samples by immunoenzymatic ELISAmethod. Ninety-six-well plates were coated with chromato-graphically purified rat albumin (Sigma, Milan, Italy) 50 μg/mLand left overnight at 4 °C. After 4 washes, 100 μL of cell culturesupernatant was added to the wells and incubated overnight at4 °C with 100 μL of antirat albumin monoclonal antibodyconjugated with horseradish peroxidase (Bethyl Laboratories,Inc., USA). After 4 washes, the substrate buffer containingtetramethylbenzidine and H2O2 (Sigma, Milan, Italy) wasadded for 7 min and the reaction was stopped with 100 μL of 8N H2SO4. Absorbance was measured at 450 nm using aMultiskan Ex (Thermo Lab Systems).The urea concentration was determined by the quantitative

colorimetric urea assay kit QuantiChrom (Gentaur, Brussels,Belgium).HPLC Analysis of Diazepam and Metabolites. HPLC

was used to analyze diazepam biotransformation by liverprogenitor cells by following its elimination and the formationof its specific metabolites temazepam, oxazepam, andnordiazepam. The samples from the culture medium werealkalinized with 20% of 4 M NaOH, precipitated withisopropanol (1:10), and extracted with ethyl acetate (5:1) bygentle rocking for 10 min and subsequent centrifugation at

200g for 15 min at RT. Thereafter the ethyl acetate phase wasevaporated and exsiccated under vacuum condition and thepellet was dissolved in 96 μL of mobile phase consisting ofacetonitrile/methanol/0.04%triethylamine pH 7.04 at propor-tion of 25/35/40. Samples were then HPLC analyzed using aC18-RP Purosphere Star 5 μm, 250 × 4.6 mm column,equipped with a precolumn (Merck KGaA, Darmstadt,Germany). The sample injection volume was 20 μL. Themobile phase was delivered at 0.8 mL/min and the column wasoperated at ambient temperature. The effluents were monitoredwith a UV detector at 236 nm. Besides diazepam and itsmetabolites temazepam, oxazepam and nordiazepam weredetected. For all substances calibration curves were regularlyrun between 10 ng/mL and 10 μg/mL.

Western Blotting Analysis. For Western blotting analysisthe proteins were extracted after 14 days of culture in batchsystem and in the crossed HF membrane bioreactors. Ratembryonic liver cells were washed once with cold PBS,collected by trypsinization by using trypsin (0.05%)/EDTA(0.025%) solution (Carl Roth GmbH, Karlsruhe, Germany)and then pelletted by centrifugation. Then cell pellets wereresuspended in ice-cold lyses buffer (50 mM Tris-HCl, 150 mMNaCl, 1% Triton X-100) supplemented with protease andphosphatase inhibitor cocktails, vortexed, and incubated for 40min at 4 °C. During the incubation time, the samples had beensonicated for 30 s and centrifuged at 10 000 rpm for 20 min at4 °C. The supernatants were transferred in new tubes and theprotein concentration was determined by using the QBITfluorometer (Invitrogen, Paisley, UK).Western blotting was performed as previously described.24

Equal amounts of protein (30 μg) were boiled for 5 min,separated under denaturing conditions by SDS-PAGE on 6%polyacrylamide Tris-glycine gels and electroblotted to nitro-cellulose membrane. Nonspecific sites were blocked with 5%nonfat dry milk in 0.1% Tween-20 in Tris-buffered saline (TBS-T) for 1 h at RT and incubated overnight with primaryantibodies: (1:200) antirat albumin, (1:200) antirat α-fetoprotein (AFP), (1:500) antirat glyceraldehyde 3-phosphatedehydrogenase (GAPDH) (Santa Cruz Biotechnology). Theantigen−antibody complex was detected by incubation of themembranes for 1 h at RT with a peroxidase-coupled anti-IgGantibody (1:3000) (Santa Cruz Biotechnology) and revealedusing the ECL Plus Western blotting detection system(Amersham, USA) according to the manufacturer’s instruction.Each membrane was exposed to the film for 1 min.

Statistical Analysis. Statistical analysis was performedusing Student’t-test and linear regression analysis.

■ RESULTS AND DISCUSSIONThe bioreactor has been designed in the crossed configurationin order to optimize the distribution of medium via a networkof channels to the cells and to increase the mass transfer bycontinual exchange of media. PEEK-WC HF membranes have ahydraulic permeance of 0.758 L/m2 h mbar and supplynutrients and drug to the cells through a prevalently diffusivetransport mechanism, which has been demonstrated in previousstudies.22 PES HF membranes show high permeability (15.2 L/m2 h mbar hydraulic permeance) that allows a facilitated andefficient removal of molecules from the cells compartmentthrough a predominantly convective mechanism. PEEK-WCHF and PES HF are combined with each other at a distance of250 μm in order to enhance the mass transfer. The fluiddynamics characterization of this membrane bioreactor



performed by tracer experiments demonstrate that the systemreached a uniform condition after 60 min, then remainedconstant throughout the duration of the experiment atoperating flow rate Qf of 1.2 mL/min. The good agreementof the cumulative RTD response curve with that resulting fromthe CSTR model (eq 3) confirmed that, under the chosenoperating conditions, the bioreactor can be considered wellmixed in the central part of its body, where cells are cultured inthe extra-lumen side of crossing fibers (Figure.1). As a result

cells are exposed at a uniform concentration of metabolites andnutrients. Furthermore the crossed configuration allows a moreefficient packing of hollow fibers. Therefore, the concentrationdifference between membrane interface and the central part ofthe extracapillary space is negligible.

Hepatic stem cells or progenitor liver cells conventionally areexpanded on polystyrene culture dishes or on components ofthe extracellular matrix.25,26 The expansion of stem cellsstrongly depends on medium supplemented with FCS, aproblem which is coming under increasing scrutiny byregulatory authorities, with the risk of transmission of infectiousagents via serum.27 In our study FCS was used only for the first24 h and then cells were cultured under serum-free conditionswith medium containing growth factors. In static conditionsembryonic liver cells easily adhered, proliferated, and spread toa confluent layer covering the substrate surface in the first 5days of culture (Figure 2). Cells exhibited tight cell−cellcontact structures, reaching a confluent degree with two-dimensional layers after 14 days of culture. Alternatively,biocompatible and biofunctional polymeric membranes, with awell-defined morphological geometry and suitable physico-chemical properties, may act as a matrix able to direct the cellorganization, offering cues and instructions for cell adhesion,growth, expansion, and differentiation.24,28 Interestingly, after 8days, embryonic liver cells cultured into the bioreactor coveredthe membrane surface saturating the whole available space andassembled themselves in three-dimensional cord-like structures(Figure 3a). Cells kept the acquired morphology within theculture time as is shown in Figure 3b where cellular aggregatesin some areas of the membrane surface are visible. The samebehavior was observed on reference substrate (Figure 3c−d).The cytoskeleton organization of the cells was investigated byLSCM. Cells appeared well spread, maintaining a high nuclearto cytoplasmatic ratio with an ovoid nucleus, characteristic ofliver progenitor cell morphology29 but also with a hint and abeginning of a polyhedral shape morphology, typical of matureand differentiated hepatocytes (Figure 4a−b). An organization

Figure 1. Cumulative RTD analysis of the bioreactor: ◊ experimentalpoints; solid line: CSTR model.

Figure 2. Light microscopy images of rat embryonic liver cells after 3 (a), 5 (b), 9 (c), and 14 (d) days of culture in batch system on PSCD.Magnification 100×.



of the cytoskeletal protein actin in parenchymal-like structureswas observed after 8 days and maintained up to 14 days ofculture on the PEEK-WC HF membranes as well as on thereference substrate (Figure 4c−d). A dot-like distribution ofvinculin evidenced the numerous cell−cell and cell−substrateinteractions. It is worth noting that the cell organization seemsto be related to the physicochemical properties of themembrane, which strongly influence the adhesion andmorphology of cells.30 The PEEK-WC membrane exhibits ahydrophilic character with a water contact angle of 76 ± 5.1°properly suitable in the promotion of cell adhesion and

proliferation by favoring the interactions with molecules on thecell surfaces and by inducing the cellular secretion of ECMproteins.31,32

Membrane in hollow fiber configuration offers a three-dimensional support in the reorganization of cellulararchitecture. Therefore, the combination of hollow fiberconfiguration with the suitable selective properties in transportphenomena and in cell interaction offers interesting oppor-tunities for the design of a bioreactor for cell culture. Thecrossed HF membrane bioreactor allows an adequate perfusionof nutrients, oxygen, and growth factors and a simultaneous

Figure 3. SEM images of rat embryonic liver cells after 8 (a, c) and 14 (b, d) days of culture on (a, b) PEEK-WC HF membranes and (c, d) onPSCD.



removal of catabolites, CO2, and metabolic products. In thisoptimized dynamic system embryonic liver cells were cultured(at 9 passage) in a controlled and in vivo-like microenviron-ment and differentiated by using supplemented media withgrowth factors (e.g., HGF, EGF) and insulin. It is well-knownthat the final maturation step of fetal hepatoblasts intohepatocytes involves HGF, soluble compounds as glucocorti-coid and insulin, extracellular matrix components, and cell−cellinteraction.33 Interestingly, the embryonic liver cells grown onand around the fibers in the crossed HF membrane bioreactorincreased their density of 97%. The perfusion system based ontwo bundles of fibers with different properties and functionssupports high-density cell growth replacing the vascular system.Furthermore the bioreactor promotes the functional differ-entiation of cells inducing the expression of the liver specificmetabolic functions. In particular urea and albumin weresynthesized up to 14 days of culture (Figure 5). The highestrate of urea synthesis was achieved on day 2 (79.0 ± 12 μg/h ×106 cells); thereafter a slow and gradual decrease in the timewas observed (Figure 5a). These values are higher with respectto those reported in literature by Ishii et al.9 in a radial flowbioreactor containing cellulose beads using fetal porcine livercells (∼ 0.15 μg/h × 106 cells), by Poyck et al.19 in an AMC

BAL consisting of 3-dimensional non woven polyester matrixwith hydrophobic polypropylene capillaries for the culture offetal human liver cells (∼ 2.26 μg/h × 106 cells), and byMiyoshi et al.6 in a packed bed reactor by using pig fetal livercells (∼ 62.6 ng/h × 106 cells).The results of functional differentiation show an increase of

albumin synthesis with time, which reaches values of 246 ng/h× 106 cells from day 7 to day 8 of culture (Figure 5b). Ascomparison with other studies the time related albuminproduction rates detected in our bioreactor were similar to,or in some cases higher than, those reported in literature: 2 ng/h × 106 cells by Miyoshi et al.,6 80 ng/h × 106 cells by Mongaet al.,7 127 ng/h × 106 cells by Poyck et al.,10 160 ng/h × 106

cells by Nibourg et al.,34 and 167 ng/h × 106 cells by Ishikawaet al.11 The expression of albumin as a mature hepatocytemarker was further detected by Western blotting analysis incells collected after 14 days of culture in batch system and inthe crossed HF membrane bioreactor. Besides the albumin theexpression of the α-fetoprotein (AFP), a marker ofhepatoblasts, which are bipotent cells giving rise to hepatocytesand bile duct epithelial cells,35 was also investigated (Figure 6).The initial cell suspension, before seeding, was AFP-positiveand only a very slight band appeared for albumin (data not

Figure 4. LSCM images of rat embryonic liver cells after 8 (a, c) and 14 (b, d) days of culture on (a, b) PEEK-WC HF membranes and (c, d) onPSCD. Cells were stained for the cytoskeleton proteins actin (green) and vinculin (red), and nuclei (blue). Scale bar 20 μm.



shown). The liver progenitor cells cultured in batch systemwere AFP-positive and very weakly albumin-positive, as a resultof incomplete differentiation as mature hepatocytes. In contrast,for cells cultured in the crossed HF membrane bioreactor theAFP band disappeared and a marked band for albuminappeared, confirming their differentiation in hepatocytes. Inline with the study performed by Monga et al.,7 cells culturedinto the bioreactor lost their progenitor characteristics reflectedby AFP expression and gained more differentiated featuresreflected by high expression of albumin, consistently with thehigh rates of albumin production. The bioreactor used in thestudy of Monga et al. had a complex geometry consisting offour compartment capillary membranes with decentralizedoxygen supply, which was proven to ensure adequate perfusionof cells. Our bioreactor is based on two different kinds of HFmembranes that facilitate the mass exchanges betweenmedium/cells compartments. This approach reduces the

complexity of the bioreactor analysis, thus obtaining asatisfactory and adequate control of the operational parameters.The crossed HF membrane bioreactor increased hepatic

differentiation of liver progenitor cells as confirmed also by itsdetoxification functionality. The ability of rat embryonic livercells to perform drug biotransformation was monitored byadministering diazepam as drug model over the whole culturetime. A comparison of the ability to eliminate diazepam by cellscultured in the crossed HF membrane bioreactor and in batchstatic system is illustrated in Figure 7. Diazepam was

metabolized with low metabolic rates by the embryonic livercells in the batch system, whereas in the bioreactor cellsexhibited a significantly higher diazepam elimination rates withvalues of 69.7 ± 1 μg/h × 106 cells on day 10 (Figure 7). Thedrug elimination rate exhibited by cells is comparable withother data reported in literature10 even if it is not related to thespecific metabolism of diazepam since the most of studiesconcerning the diazepam biotransformation of fetal liver cellsregard static culture conditions.To further elucidate diazepam biotransformation we

investigated the formation of diazepam metabolites (Figure8). It was found that embryonic liver cells in batch static culturesystem metabolized diazepam prevalently through the for-mation of oxazepam, 4-idroxydiazepam (from day 3), andnordiazepam (from day 5). The rate of formation increasedwith time reaching high and stable levels from day 10 to day 14(Figure 8a). Differently, in the crossed HF membranebioreactor all the four metabolites of phase I reaction weredetected for the whole culture time from the first day of thedrug administration (Figure 8b). In particular the diazepambiotransformation occurred with the formation of temazepamto a larger extent with respect to oxazepam, nordiazepam, and4-idroxydiazepam, exhibiting high metabolic rates (1.5 ± 0.5μg/h × 106 cells on day 13). The diazepam elimination and thefull metabolite formation in the crossed HF membranebioreactor demonstrated the induction and functional activityof all the phase I cytochrome P450 (CYP) monooxygenases.The metabolic pathway of diazepam biotransformation involvesa variety of CYP isoenzymes. Indeed, rat liver microsomesCYP2D1, CYP3A2, and CYP2C11 catalyze the p-hydroxyla-tion, 3-hydroxylation, and N-desmethylation of diazepam,respectively.36 In contrast to adult liver, fetal hepatocytes

Figure 5. Rate of urea (a) and albumin (b) synthesis by rat embryonicliver cells cultured in the crossed HF membrane bioreactor. The valuesare the mean ± standard deviation of 9 determinations from 3independent experiments.

Figure 6. Western blotting of albumin (ALB), α-fetoprotein (AFP)expression in embryonic liver cells in the crossed HF membranebioreactor and in batch system after 14 days of culture. The same blotwas probed for glyceraldehyde 3-phosphate dehydrogenase (GAPDH)as control.

Figure 7. Diazepam elimination by rat embryonic liver cells cultured inbatch system and in the crossed HF membrane bioreactor. The valuesare the mean ± standard deviation of 9 determinations from 3independent experiments. (*): data statistically significant according toStudent’t-test (p < 0.01).



exhibit a modest detoxification function. CYP gene expressionincreases during liver ontogeny and is characterized by a strongsecretion of CYP3A737 that decreases in the perinatal period,meanwhile its adult counterpart, CYP3A4, with other CYPsexpressed in adult liver, increase.38 In the crossed HFmembrane bioreactor a hepatic differentiation of liverprogenitor cells occurred as a result of their enhanceddetoxification functionality.The expansion and differentiation depends on controlling

key process variables: nutrient and metabolite concentrations,growth factor compositions, and physiological parameters (e.g.,temperature, pH, and oxygen). The crossed HF membranebioreactor creates a homogeneous environment for cell culturein which the concentrations of nutrients and metabolites aremonitored and controlled and differentiation signals areprovided to the cells. Selective exchange of gases andmetabolites through the selective HF membranes ensured amicroenvironment adequate for the induction and maintenanceof the activity of the CYP monooxygenases which are amongthe most sensitive and fragile enzymes found in hepatocytes.Also, in a 3D microenvironment the reorganization of cellulararchitecture increased the amount of cell−cell contacts andinterestingly in freshly isolated hepatocytes the high degree ofintercellular established contacts is a prerequisite for highCYP3A expression and activity.39

■ CONCLUSIONSThis study demonstrated the potential to expand anddifferentiate stem cells in a crossed hollow fiber membranebioreactor. The geometry of the bioreactor ensured optimalperfusion conditions, which paid a crucial role together with thesurface and transport properties of the HF membranes in

guiding the expansion and differentiation of cells. Thesignificant proliferation of cells and expression of liver specificfunctions in terms of urea synthesis, albumin production, anddiazepam biotransformation demonstrated the complete differ-entiation of embryonic liver cells in mature hepatocytes. Futurechallenges include the development of optimal cultureconditions for various stem cell types and scale-up of themembrane bioreactor.

■ AUTHOR INFORMATIONCorresponding Author*Tel: +39 0984 492036. Fax: +39 0984 402103. E-mail: [email protected] or [email protected] authors declare no competing financial interest.

■ ACKNOWLEDGMENTSWe acknowledge King Abdulaziz City for Science andTechnology (KACST), Kingdom of Saudi Arabia for fundingthe project “Membrane systems in regenerative medicine, tissueengineering and biotechnology” (KACST-ITM 03), andUniversity of Calabria and Regione Calabria for the award ofthe post doc fellowship DR 2718/201 Regional OperativeProgram (ROP) ESF 2007/2013 - IV Axis Human Capital -Operative Objective.

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Figure 8. Diazepam metabolite formation: oxazepam (□), temazepam(◊), nordiazepam (Δ), and 4-idroxydiazepam (○) by embryonic ratliver cells cultured in batch system on PSCD (a) and in the crossedHF membrane bioreactor (b). The values are the mean ± standarddeviation of 9 determinations from 3 independent experiments.



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Membrane Bioreactor for Expansion and Differentiation of Embryonic Liver Cells