Membrane Bioreactor for Expansion and Differentiation of Embryonic

Mar 28, 2013 - National Centre for Biotechnology, King Abdulaziz City for Science and ... of Calabria, via P. Bucci cubo 45/A, 87030 Rende (CS) Italy...
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Membrane Bioreactor for Expansion and Differentiation of Embryonic Liver Cells Simona 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, cubo 17/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 culture flasks, which have several drawbacks. In this paper we report on the use of a crossed hollow fiber membrane bioreactor for the expansion and differentiation of embryonic liver cells, which have been used as an alternative model of human liver progenitor cells. The bioreactor is based on two bundles of fiber (PEEK-WC HF and PES-HF) which are cross-assembled in an alternating manner. This bioreactor geometry ensures high mass exchange of nutrients and metabolites, which is important for cell proliferation and differentiation. The membrane bioreactor, thanks to its optimized fluid dynamics and mass transport and the adequate surface properties of hollow fibers, was able to guide the expansion and differentiation of liver progenitor cells in mature hepatocytes as demonstrated by their expression of liver specific functions in terms of urea synthesis, albumin production, and diazepam drug biotransformation.

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INTRODUCTION The growing demand for replacement tissues and organ structures due to aging of the population and increase of several pathologies lead to development of new tissue engineering methods and technologies. Research efforts have been focused in this past decade on the use of embryonic and adult stem cells because of their ability to either self-renew or differentiate into multiple cell lineages.1 These characteristics make 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 implemented by developing culture systems that are able to generate large numbers of cells with well-defined characteristics and/or to promote controlled, reproducible differentiation into selected mature cell types. Membrane system has a great potential in the stem-cell based technologies because it is able to act as an instructive extracellular matrix (ECM). In fact it exhibits like ECM microscale to nanoscale of chemistry and topography and is able to provide cells physical, chemical, and mechanical signals, which are important for the differentiation process. Several strategies are aimed at engineering membrane systems with specific physicochemical, topographical, mechanical properties, and configuration able to drive the differentiation of stem and progenitor cells (e.g., neurons, liver, cartilage, bone).3−5 Recently the expansion and the functional differentiation of rat embryonic liver cells were observed on a synthetic polymeric membrane of a modified polyetheretherketone (PEEK-WC) and on a biodegradable membrane of chitosan.5 However, most of the studies concerning the expansion and differentiation of stem or progenitor cells were © 2013 American Chemical Society

performed under static conditions. There are a few reports in which fetal liver cells were cultured under perfusion conditions in a packed bed reactor,6 three-dimensional four compartment bioreactor,7 polyurethane foam bioreactor, 8 radial flow bioreactor,9 nonwoven polyester matrix bioreactor,10 or roating wall vessel bioreactor.11 Nevertheless, there are still challenges such as to provide the cells adequate nutrients, growth factors, and metabolites and to efficiently remove catabolites without causing shear stress or affecting cell differentiation. Membrane bioreactors have a significant advantage to perfuse cells through membranes that serve also as supports for cell adhesion, offering in the case of hollow fibers a large surface area in a small volume. The mass transfer can be enhanced by minimizing the distance between cells and perfusion membrane lumen.12 Previously we developed a crossed hollow fiber membrane bioreactor to support the long-term maintenance and differentiation of primary human hepatocytes.13 The bioreactor consists of two bundles of hollow fiber (HF) membranes with different molecular weight (MW) cutoff and physicochemical properties cross-assembled in alternating manner: PEEK-WC and polyethersulfone (PES), which perform different functions. PEEK-WC HF membranes provide cells nutrients and metabolites whereas PES HF removes catabolites from the Special Issue: Enrico Drioli Festschrift Received: Revised: Accepted: Published: 10387

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

cell compartment mimicking in this way the in vivo arterious and venous blood vessels. The combination of these two fibers creates three compartments: two intraluminal compartments of PEEK-WC HF and PES HF in which the medium flows and one extraluminal compartment represented by extracapillary network formed on both type of fibers in which cells are cultured. This geometry would ensure a high mass exchange through the cross-flow of culture medium.13 In this paper we demonstrate that crossed HF membrane bioreactor guides the expansion and the functional differentiation of embryonic liver cells, providing physical, chemical, and mechanical cues through the different membrane properties, and governing mass transfer of molecules. The perfusion conditions and the optimized mass transport through each type of fiber ensure the creation of a homogeneous environment and the possibility to monitor and control critical culture parameters. This system represents a valuable bioengineered platform for a mass production of “guided differentiated” cells to be used for cell therapy, bioartificial devices, or drug screening. Rat embryonic liver cells (17 day embryos) were used in this study as an alternative model of human liver progenitor cells14−16 since using cells from the fetal human liver is limited by a major ethical issue. Embryonic liver cells have many advantages over primary hepatocytes for proliferation in vitro to transplantation in vivo. They exhibit spontaneous proliferation and the ability to differentiate into hepatocyte and biliary duct cells representing an ideal hepatocyte source.17−20

τ=

(2)

where Q is the perfusion flow rate and V is the volume of the bioreactor.21 The agreement of the experimental F(t) with that of a continuous stirred tank reactor (CSTR) was assessed by plotting the experimental data with the theoretical curve for CSTR obtained by the equation Cout = 1 − e −t / τ C in

(3)

Membrane Preparation and Characterization. PEEKWC HF membranes were prepared according to the wellknown dry−wet spinning method. To prepare highly porous membranes, poly(vinylpyrrolidone) (PVP K17 by BASF) was used as a pore forming additive. Membranes were prepared from solutions of PEEK-WC and PVP both at 15 wt % in dimetylformamide (DMF) under continuous mechanical stirring at room temperature as described elsewhere.22 The morphological properties of the PEEK-WC HF and commercial PES HF (Membrana GmbH) membranes were characterized by scanning electron microscope (SEM) (ESEM FEG QUANTA 200, FEI Company, OR, USA) in order to evaluate the cross-sectional structure and thickness, intra- and extralumen morphology and diameters, and the shape and size of the membrane pores. The hydrophobic/hydrophilic character of the investigated membranes was estimated by contact angle technique. Water contact angles were measured using the sessile drop method at ambient temperature by CAM 200 contact angle meter (KSV Instruments LTD, Helsinki, Finland), depositing the liquid on the membrane surface using an automatic microsyringe. The mass transport properties were characterized by evaluating the hydraulic permeance through measurements of pure water flux (J) at different transmembrane pressures (ΔP). The permeation of specific metabolites such as albumin, urea, and diazepam was also assessed at different transmembrane pressures. At the same time the diffusive transport of albumin and urea, which are produced by cells, from the fiber lumen to the shell of PES HF membranes and the diffusive transport of diazepam, which is metabolized by cells, from the lumen to the shell of PEEK-WC HF membranes was also evaluated as previously described.23 Cell Culture. The rat embryonic liver cells (17 days embryonic liver of Japanese albino rat) (RLC-18) were obtained from the DSMZ (Braunschweig, Germany). Cells were 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 and between the HF membranes at a density of 1 × 104 cell/cm2. The bioreactor was maintained at 37 °C in a 5% CO2:20% O2 atm (v/v) with 95% relative humidity. After 4 h cells adhered and the bioreactor was perfused with oxygenated medium. Cells were cultured for the first 24 h in medium supplemented with

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MATERIALS AND METHODS Crossed HF Membrane Bioreactor. The membrane bioreactor consists of two bundles of 40 PEEK-WC HF and 40 PES HF membranes cross-assembled in alternating manner and potted with polyurethane adhesive (Polaris Polymers, Avon Lake, OH, USA) within glass housing. PEEK-WC HF and PES HF 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 PES fibers, and an extraluminal compartment or shell outside of the fibers, which communicate through the pores in the fiber wall. The bioreactor (volume: 40 mL) is connected to the perfusion circuit consisting of microperistaltic pump, gas-permeable silicone tubing, reservoir of medium, and glass medium waste.13 The oxygenated medium enters from the reservoir to the membrane bioreactor with a flow rate Qf of 1.2 mL/min that was set on the basis of average retention time. Fresh medium was perfused in single-pass and the stream leaving the bioreactor. Qout. was collected as waste until approaching the steady state. When the system reached the steady state, the stream leaving the bioreactor was recycled (Qr) in order to obtain the accumulation of products. The fluid dynamics of the bioreactor were characterized in terms of cumulative residence time distribution (RTD), which was investigated through the introduction of tracer (step input) at the entrance of PEEK-WC fibers and recording it in time at the exit of the PES fibers. The tracer, consisting in a solution of Williams’ medium E, was sent to the bioreactor with flow rate of 1.2 mL/min, and the change of tracer concentration stepwise in the feed stream (Cin) and the outlet concentration (Cout) was continuously monitored by an online spectrophotometer (UV Cord Pharmacia, Uppsala, Sweden). The cumulative RTD to step inputs is described by the equation F(t ) = cout /c in

V Q

(1) 10388

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200g for 15 min at RT. Thereafter the ethyl acetate phase was evaporated and exsiccated under vacuum condition and the pellet was dissolved in 96 μL of mobile phase consisting of acetonitrile/methanol/0.04%triethylamine pH 7.04 at proportion of 25/35/40. Samples were then HPLC analyzed using a C18-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. The mobile phase was delivered at 0.8 mL/min and the column was operated at ambient temperature. The effluents were monitored with a UV detector at 236 nm. Besides diazepam and its metabolites temazepam, oxazepam and nordiazepam were detected. For all substances calibration curves were regularly run between 10 ng/mL and 10 μg/mL. Western Blotting Analysis. For Western blotting analysis the proteins were extracted after 14 days of culture in batch system and in the crossed HF membrane bioreactors. Rat embryonic 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 were resuspended in ice-cold lyses buffer (50 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100) supplemented with protease and phosphatase inhibitor cocktails, vortexed, and incubated for 40 min at 4 °C. During the incubation time, the samples had been sonicated for 30 s and centrifuged at 10 000 rpm for 20 min at 4 °C. The supernatants were transferred in new tubes and the protein concentration was determined by using the QBIT fluorometer (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 nitrocellulose membrane. Nonspecific sites were blocked with 5% nonfat dry milk in 0.1% Tween-20 in Tris-buffered saline (TBST) for 1 h at RT and incubated overnight with primary antibodies: (1:200) antirat albumin, (1:200) antirat αfetoprotein (AFP), (1:500) antirat glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology). The antigen−antibody complex was detected by incubation of the membranes for 1 h at RT with a peroxidase-coupled anti-IgG antibody (1:3000) (Santa Cruz Biotechnology) and revealed using 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 performed using Student’t-test and linear regression analysis.

10% FCS (Biochrom AG, Berlin, Germany) and successively in medium under serum-free conditions and supplemented with hepatocyte growth factor (HGF), epidermal growth factor (EGF), insulin, ascorbic acid, transferrin, hydrocortisone 21hemosuccinate, gentamicin sulfate 50 μg/mL, amphotericin B 50 ng/mL (HCM bulletkit, Lonza Sales Ltd., Basel, Switzerland), 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 to 14 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 PSCD was investigated after 8 and 14 days of culture laser scanning confocal microscopy (LSCM) after cytoskeleton protein immunostaining. Samples were rinsed with PBS, fixed for 15 min in 3% paraformaldehyde at room temperature (RT), permeabilized for 5 min with 0.5% Triton-X100, and saturated for 15 min with 2% normal goat serum. Vinculin was visualized using a mouse monoclonal antibody raised against rat vinculin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) with goat antimouse IgG Alexa Flouor 546 conjugated (Molecular Probes, Eugene, OR, USA) as secondary antibody. Primary and secondary antibodies were incubated at RT for 2 and 1.5 h, respectively. Actin was stained with Alexa 488-conjugated phalloidin (Molecular Probes). Counterstaining for nuclei was performed 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 scanning electron 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 images displaying morphological features were obtained after 8 and 14 days 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 prechilled tubes and stored at −20 °C until assayed. Albumin secretion was measured in the samples by immunoenzymatic ELISA method. Ninety-six-well plates were coated with chromatographically purified rat albumin (Sigma, Milan, Italy) 50 μg/mL and left overnight at 4 °C. After 4 washes, 100 μL of cell culture supernatant was added to the wells and incubated overnight at 4 °C with 100 μL of antirat albumin monoclonal antibody conjugated with horseradish peroxidase (Bethyl Laboratories, Inc., USA). After 4 washes, the substrate buffer containing tetramethylbenzidine and H2O2 (Sigma, Milan, Italy) was added for 7 min and the reaction was stopped with 100 μL of 8 N H2SO4. Absorbance was measured at 450 nm using a Multiskan 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 liver progenitor cells by following its elimination and the formation of its specific metabolites temazepam, oxazepam, and nordiazepam. The samples from the culture medium were alkalinized with 20% of 4 M NaOH, precipitated with isopropanol (1:10), and extracted with ethyl acetate (5:1) by gentle rocking for 10 min and subsequent centrifugation at

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RESULTS AND DISCUSSION The bioreactor has been designed in the crossed configuration in order to optimize the distribution of medium via a network of channels to the cells and to increase the mass transfer by continual exchange of media. PEEK-WC HF membranes have a hydraulic permeance of 0.758 L/m2 h mbar and supply nutrients and drug to the cells through a prevalently diffusive transport mechanism, which has been demonstrated in previous studies.22 PES HF membranes show high permeability (15.2 L/ m2 h mbar hydraulic permeance) that allows a facilitated and efficient removal of molecules from the cells compartment through a predominantly convective mechanism. PEEK-WC HF and PES HF are combined with each other at a distance of 250 μm in order to enhance the mass transfer. The fluid dynamics characterization of this membrane bioreactor 10389

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Hepatic stem cells or progenitor liver cells conventionally are expanded on polystyrene culture dishes or on components of the extracellular matrix.25,26 The expansion of stem cells strongly depends on medium supplemented with FCS, a problem which is coming under increasing scrutiny by regulatory authorities, with the risk of transmission of infectious agents via serum.27 In our study FCS was used only for the first 24 h and then cells were cultured under serum-free conditions with medium containing growth factors. In static conditions embryonic liver cells easily adhered, proliferated, and spread to a confluent layer covering the substrate surface in the first 5 days of culture (Figure 2). Cells exhibited tight cell−cell contact structures, reaching a confluent degree with twodimensional layers after 14 days of culture. Alternatively, biocompatible and biofunctional polymeric membranes, with a well-defined morphological geometry and suitable physicochemical properties, may act as a matrix able to direct the cell organization, offering cues and instructions for cell adhesion, growth, expansion, and differentiation.24,28 Interestingly, after 8 days, embryonic liver cells cultured into the bioreactor covered the membrane surface saturating the whole available space and assembled themselves in three-dimensional cord-like structures (Figure 3a). Cells kept the acquired morphology within the culture time as is shown in Figure 3b where cellular aggregates in some areas of the membrane surface are visible. The same behavior was observed on reference substrate (Figure 3c−d). The cytoskeleton organization of the cells was investigated by LSCM. Cells appeared well spread, maintaining a high nuclear to cytoplasmatic ratio with an ovoid nucleus, characteristic of liver progenitor cell morphology29 but also with a hint and a beginning of a polyhedral shape morphology, typical of mature and differentiated hepatocytes (Figure 4a−b). An organization

performed by tracer experiments demonstrate that the system reached a uniform condition after 60 min, then remained constant throughout the duration of the experiment at operating flow rate Qf of 1.2 mL/min. The good agreement of the cumulative RTD response curve with that resulting from the CSTR model (eq 3) confirmed that, under the chosen operating conditions, the bioreactor can be considered well mixed in the central part of its body, where cells are cultured in the extra-lumen side of crossing fibers (Figure.1). As a result

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

◊

experimental

cells are exposed at a uniform concentration of metabolites and nutrients. Furthermore the crossed configuration allows a more efficient packing of hollow fibers. Therefore, the concentration difference between membrane interface and the central part of the extracapillary space is negligible.

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×. 10390

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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) on PSCD.

of the cytoskeletal protein actin in parenchymal-like structures was observed after 8 days and maintained up to 14 days of culture on the PEEK-WC HF membranes as well as on the reference substrate (Figure 4c−d). A dot-like distribution of vinculin evidenced the numerous cell−cell and cell−substrate interactions. It is worth noting that the cell organization seems to be related to the physicochemical properties of the membrane, which strongly influence the adhesion and morphology of cells.30 The PEEK-WC membrane exhibits a hydrophilic 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 the cell surfaces and by inducing the cellular secretion of ECM proteins.31,32 Membrane in hollow fiber configuration offers a threedimensional support in the reorganization of cellular architecture. Therefore, the combination of hollow fiber configuration with the suitable selective properties in transport phenomena and in cell interaction offers interesting opportunities for the design of a bioreactor for cell culture. The crossed HF membrane bioreactor allows an adequate perfusion of nutrients, oxygen, and growth factors and a simultaneous 10391

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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) on PSCD. Cells were stained for the cytoskeleton proteins actin (green) and vinculin (red), and nuclei (blue). Scale bar 20 μm.

BAL consisting of 3-dimensional non woven polyester matrix with hydrophobic polypropylene capillaries for the culture of fetal human liver cells (∼ 2.26 μg/h × 106 cells), and by Miyoshi et al.6 in a packed bed reactor by using pig fetal liver cells (∼ 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). As comparison with other studies the time related albumin production 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 Monga et 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 Ishikawa et al.11 The expression of albumin as a mature hepatocyte marker was further detected by Western blotting analysis in cells collected after 14 days of culture in batch system and in the crossed HF membrane bioreactor. Besides the albumin the expression of the α-fetoprotein (AFP), a marker of hepatoblasts, which are bipotent cells giving rise to hepatocytes and bile duct epithelial cells,35 was also investigated (Figure 6). The initial cell suspension, before seeding, was AFP-positive and only a very slight band appeared for albumin (data not

removal of catabolites, CO2, and metabolic products. In this optimized dynamic system embryonic liver cells were cultured (at 9 passage) in a controlled and in vivo-like microenvironment and differentiated by using supplemented media with growth factors (e.g., HGF, EGF) and insulin. It is well-known that the final maturation step of fetal hepatoblasts into hepatocytes involves HGF, soluble compounds as glucocorticoid and insulin, extracellular matrix components, and cell−cell interaction.33 Interestingly, the embryonic liver cells grown on and around the fibers in the crossed HF membrane bioreactor increased their density of 97%. The perfusion system based on two bundles of fibers with different properties and functions supports high-density cell growth replacing the vascular system. Furthermore the bioreactor promotes the functional differentiation of cells inducing the expression of the liver specific metabolic functions. In particular urea and albumin were synthesized up to 14 days of culture (Figure 5). The highest rate of urea synthesis was achieved on day 2 (79.0 ± 12 μg/h × 106 cells); thereafter a slow and gradual decrease in the time was observed (Figure 5a). These values are higher with respect to those reported in literature by Ishii et al.9 in a radial flow bioreactor containing cellulose beads using fetal porcine liver cells (∼ 0.15 μg/h × 106 cells), by Poyck et al.19 in an AMC 10392

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complexity of the bioreactor analysis, thus obtaining a satisfactory and adequate control of the operational parameters. The crossed HF membrane bioreactor increased hepatic differentiation of liver progenitor cells as confirmed also by its detoxification functionality. The ability of rat embryonic liver cells to perform drug biotransformation was monitored by administering diazepam as drug model over the whole culture time. A comparison of the ability to eliminate diazepam by cells cultured in the crossed HF membrane bioreactor and in batch static system is illustrated in Figure 7. Diazepam was

Figure 5. Rate of urea (a) and albumin (b) synthesis by rat embryonic liver cells cultured in the crossed HF membrane bioreactor. The values are the mean ± standard deviation of 9 determinations from 3 independent experiments. Figure 7. Diazepam elimination by rat embryonic liver cells cultured in batch system and in the crossed HF membrane bioreactor. The values are the mean ± standard deviation of 9 determinations from 3 independent experiments. (*): data statistically significant according to Student’t-test (p < 0.01).

metabolized with low metabolic rates by the embryonic liver cells in the batch system, whereas in the bioreactor cells exhibited a significantly higher diazepam elimination rates with values of 69.7 ± 1 μg/h × 106 cells on day 10 (Figure 7). The drug elimination rate exhibited by cells is comparable with other data reported in literature10 even if it is not related to the specific metabolism of diazepam since the most of studies concerning the diazepam biotransformation of fetal liver cells regard static culture conditions. To further elucidate diazepam biotransformation we investigated the formation of diazepam metabolites (Figure 8). It was found that embryonic liver cells in batch static culture system metabolized diazepam prevalently through the formation of oxazepam, 4-idroxydiazepam (from day 3), and nordiazepam (from day 5). The rate of formation increased with time reaching high and stable levels from day 10 to day 14 (Figure 8a). Differently, in the crossed HF membrane bioreactor all the four metabolites of phase I reaction were detected for the whole culture time from the first day of the drug administration (Figure 8b). In particular the diazepam biotransformation occurred with the formation of temazepam to a larger extent with respect to oxazepam, nordiazepam, and 4-idroxydiazepam, exhibiting high metabolic rates (1.5 ± 0.5 μg/h × 106 cells on day 13). The diazepam elimination and the full metabolite formation in the crossed HF membrane bioreactor demonstrated the induction and functional activity of all the phase I cytochrome P450 (CYP) monooxygenases. The metabolic pathway of diazepam biotransformation involves a variety of CYP isoenzymes. Indeed, rat liver microsomes CYP2D1, CYP3A2, and CYP2C11 catalyze the p-hydroxylation, 3-hydroxylation, and N-desmethylation of diazepam, respectively.36 In contrast to adult liver, fetal hepatocytes

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

shown). The liver progenitor cells cultured in batch system were AFP-positive and very weakly albumin-positive, as a result of incomplete differentiation as mature hepatocytes. In contrast, for cells cultured in the crossed HF membrane bioreactor the AFP band disappeared and a marked band for albumin appeared, confirming their differentiation in hepatocytes. In line with the study performed by Monga et al.,7 cells cultured into the bioreactor lost their progenitor characteristics reflected by AFP expression and gained more differentiated features reflected by high expression of albumin, consistently with the high rates of albumin production. The bioreactor used in the study of Monga et al. had a complex geometry consisting of four compartment capillary membranes with decentralized oxygen supply, which was proven to ensure adequate perfusion of cells. Our bioreactor is based on two different kinds of HF membranes that facilitate the mass exchanges between medium/cells compartments. This approach reduces the 10393

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guiding the expansion and differentiation of cells. The significant proliferation of cells and expression of liver specific functions in terms of urea synthesis, albumin production, and diazepam biotransformation demonstrated the complete differentiation of embryonic liver cells in mature hepatocytes. Future challenges include the development of optimal culture conditions for various stem cell types and scale-up of the membrane bioreactor.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +39 0984 492036. Fax: +39 0984 402103. E-mail: l. [email protected] or [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge King Abdulaziz City for Science and Technology (KACST), Kingdom of Saudi Arabia for funding the project “Membrane systems in regenerative medicine, tissue engineering and biotechnology” (KACST-ITM 03), and University of Calabria and Regione Calabria for the award of the post doc fellowship DR 2718/201 Regional Operative Program (ROP) ESF 2007/2013 - IV Axis Human Capital Operative Objective.

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

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exhibit a modest detoxification function. CYP gene expression increases during liver ontogeny and is characterized by a strong secretion of CYP3A737 that decreases in the perinatal period, meanwhile its adult counterpart, CYP3A4, with other CYPs expressed in adult liver, increase.38 In the crossed HF membrane bioreactor a hepatic differentiation of liver progenitor cells occurred as a result of their enhanced detoxification 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 membrane bioreactor creates a homogeneous environment for cell culture in which the concentrations of nutrients and metabolites are monitored and controlled and differentiation signals are provided to the cells. Selective exchange of gases and metabolites through the selective HF membranes ensured a microenvironment adequate for the induction and maintenance of the activity of the CYP monooxygenases which are among the most sensitive and fragile enzymes found in hepatocytes. Also, in a 3D microenvironment the reorganization of cellular architecture increased the amount of cell−cell contacts and interestingly in freshly isolated hepatocytes the high degree of intercellular established contacts is a prerequisite for high CYP3A expression and activity.39

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CONCLUSIONS This study demonstrated the potential to expand and differentiate stem cells in a crossed hollow fiber membrane bioreactor. The geometry of the bioreactor ensured optimal perfusion conditions, which paid a crucial role together with the surface and transport properties of the HF membranes in 10394

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

Industrial & Engineering Chemistry Research

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