Development of Dual-Compartment Perfusion Bioreactor for Serial Coculture of Hepatocytes and Stellate Cells in Poly(lactic-co-glycolic acid)-Collagen Scaffolds F. Wen,1–4 S. Chang,2,5 Y. C. Toh,3,4 T. Arooz,3 L. Zhuo,3 S. H. Teoh,1,4,6 H. Yu2–4,6–8 1
Department of Mechanical Engineering, Faculty of Engineering, National University of Singapore, Singapore
2
Department of Physiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore
3
Institute of Bioengineering and Nanotechnology, Agency for Science, Technology and Research, Singapore
4
Graduate Program in Bioengineering, NUS Graduate School for Integrative Sciences and Engineering, Singapore
5
Department of General Surgery, Xiang Ya Hospital, Central South University, Changsha, Hunan, China
6
National University of Singapore Tissue Engineering Programme, Singapore
7
Singapore-MIT Alliance, Singapore
8
Department of Haematology-Oncology, National University Hospital, Singapore
Received 7 February 2007; revised 7 December 2007; accepted 18 December 2007 Published online 22 April 2008 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jbm.b.31086
Abstract: An important challenge in liver tissue engineering is to overcome the rapid loss of hepatocyte functions. In vivo, hepatocytes are compact polyhedral cells with round nuclei; however, they readily loss many of their differentiated functions in vitro. To overcome this challenge, we have established a new perfusion bioreactor that consists of two compartments which enabled the serial coculture of hepatocytes and hepatic stellate cells-T6 without direct contact between each other. Three dimensional scaffolds were utilized in the bioreactor as physical anchors for cells. The scaffolds consist of collagen grafted poly(lactic-co-glycolic acid) microfibers and cross-linked collagen sponges between microfibers for additional cellular support and adhesion. The advantages of this new bioreactor are enabling cell culture in three dimensional organization and controlling the culture parameters of the supporting cells independently from the hepatocytes. The results showed that the hepatocytes exhibited much higher levels of the differentiated functions such as albumin secretion, urea synthesis, and cytochrome P450 enzymatic activity when compared with the monoculture system where hepatocytes alone were cultured. This perfusion bioreactor system has potential applications in the development of bioartificial liver devices or cell-based tissue constructs transplantation therapies. ' 2008 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 87B: 154–162, 2008 Keywords: poly(lactic-co-glycolic acid); hepatocytes; hepatic stellate cell; extracelluar matrix; perfusion bioreactor
INTRODUCTION Correspondence to: H. Yu (e-mail:
[email protected]) and S. H. Teoh (e-mail:
[email protected]) Contract grant sponsor: Institute of Bioengineering and Nanotechnology, Biomedical Research Council, Agency for Science, Technology and Research of Singapore; contract grant number: R185-001-045-305. Contract grant sponsor: Ministry of Education; contract grant number: R-185-000135-112. Contract grant sponsor: National Medical Research Council Grant; contract grant number: R-185-000-099-213. Contract grant sponsor: Singapore-MIT Alliance Computational and Systems Biology Flagship Project. ' 2008 Wiley Periodicals, Inc.
154
Liver transplantation is currently the only viable treatment for metabolic liver diseases1,2; however, there is an acute shortage of donor organs.2,3 This critical shortage of donor liver catalyzed research on liver tissue engineering such as the development of bioartificial liver (BAL) devices or cell-based tissue constructs transplantation therapies. BAL device is a cell-based life-support device intended to enhance detoxification and protein synthesis functions of a patient’s liver before or after transplantation.4 Cell-based
DEVELOPMENT OF DUAL-COMPARTMENT PERFUSION BIOREACTOR
tissue constructs transplantation therapies aim at stimulating the patient’s liver regeneration potentials.5 However, both developments are still in ‘‘infancy to adolescence’’ stage.6 The most significant challenge in the success of developments is the maintenance of viable and functional hepatocytes outside of the native liver environment.2 Isolated hepatocytes start to spread and proliferate under suboptimal culture conditions, losing most of the characteristic differentiated hepatic functions within 3–4 days.7,8 To maintain viable and functional hepatocytes in vitro, several tissue-engineering strategies for modulating the culture parameters to mimic microenvironment in vivo have been adopted. The strategies are (a) culturing isolated hepatocytes on three-dimensional (3D) biocompatible substrate (scaffold),9 (b) enhancing mass transport of oxygen and nutrients through porous structure formation or perfusion culture,10,11 and (c) coculturing hepatocytes with supporting cells including liver derived and non-liver derived cells.12,13 In particular, enhancement of hepatocyte functions can occur through paracrine signaling via TGF-b113 or static coculture of hepatocytes and hepatic stellate cell without heterotypic cell–cell contact.14–16 We hypothesize that the differentiated functions of hepatocytes can be maintained by a combination of soluble chemical clues released by the supporting cells (hepatic stellate cell, HSC-T6) without heterotypic cell–cell contact, and stable 3D biocompatible scaffold support; therefore, we have developed a new bioreactor which allows serial perfusion coculture of hepatocytes and HSC-T6 in individual 3D scaffold support. Collagen grafted poly(lactic-co-glycolic acid) (PLGA) microfibers embedded in collagen sponge (PLGA-collagen scaffold), which provides adequate mechanical strength, 3D cell culture environments and stable cell–substrate interactions, was used as a 3D scaffold support in our bioreactor. Hepatocytes and HSC-T6 cells were seeded in individual scaffolds in two separate compartments of bioreactor for perfusion culture. The differentiated functions of hepatocytes in the bioreactor such as albumin secretion, urea synthesis, and cytochrome P450 enzymatic activity were investigated. The results suggest that this serial perfusion bioreactor system has potential applications for BAL devices development and cell-based tissue constructs transplantation therapies.
MATERIALS AND METHODS Materials
PLGA suture with a lactic acid: glycolic acid ratio of 10:90 was purchased from Ethicon. N-Hydroxysuccinimide (NHS) was supplied by Pierce Chemical. The 2-(N-morpholino)ethanesulfonic acid (MES), 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC), epidermal growth factor, ethoxyresorufin O-deethylase (EROD), and resorufin were obtained from Sigma-Aldrich Chemical. Dulbecco’s modified eagle medium (DMEM), Williams’ E medium, insuJournal of Biomedical Materials Research Part B: Applied Biomaterials
155
Figure 1. Surface modification schematics of PLGA fiber.
lin and Pico-Green dsDNA Quantitation Kit were purchased from Invitrogen, Singapore. Vitrogen collagen solution (3 mg/ mL) dissolved in 0.012N HCl was purchased from Cohesion Technologies, CA. Urea synthesis kit was purchased from Stanbio Laboratory, Boerne, TX; albumin ELSA kit was purchased from Bethyl Laboratories, Montgomery, TX. All other reagents are purchased from Sigma-Aldrich unless otherwise specified. PLGA-Collagen 3D Scaffold
The PLGA-collagen scaffolds were fabricated as described previously.17 The process includes grafting of collagen onto the surface of PLGA fibers, lyophilization, and crosslinking (Figure 1). Before cell seeding in scaffolds, all scaffolds were sterilized by exposure to a 60Co-source irradiator (Gamma cell 220 Excel, Canada) until a total dose of 25 kGy was reached. Surface Characterization by X-ray Photoelectron Spectroscopy. The surface chemistry of PLGA or the modified surfaces were characterized by X-ray photoelectron spectroscopy (XPS). All samples were washed in distill water with 0.5% Tween 20 for 2 days and then lyophilized. A VG ESCALAB MkII spectrometer with a Mg Ka X-ray source (1253.6 eV photons) at a constant dwell time of 100 ms; pass energy 20 eV and anode current 20 mA was used to measure samples. The XPS N 1s core-level signal was used as an indicator for the analysis of the relative amount of collagen immobilized on the surface of PLGA fibers. Porosity Measurement by Mercury Porosimeter. The internal structure of PLGA-collagen scaffolds-pore size distribution, total pore area, and porosity were measured by PASCAL 140 mercury porosimeter (Thermo Finnigan, Italy, S.p.A.) with S-CD6 dilatometer. Sample preparation and measurements were performed according to instructions from the manufacturer. Perfusion Bioreactor
Bioreactor Assembly. The perfusion bioreactor consists of two polycarbonate compartments, one peristaltic pump, gas
156
WEN ET AL.
exchangeable tubings, and one culture medium reservoir (Figure 4). The dimensions of the bioreactor were as follows: 1. The inner dimensions of the upper compartment: length 20 mm, width 10 mm, height 6 mm; 2. The inner dimensions of the lower compartment: length 20 mm, width 10 mm, height 9 mm; 3. The dimensions of the scaffold holder plate: length 20 mm, width 10 mm, thickness 3 mm, diameter for distribution hole is 3 mm; 4. The thickness of the bioreactor wall: 6 mm; 5. The thickness, pore size and open area of separation membrane: 90 lm, 6 lm, and 5% (PETEX-07-6/5, Sefar AG). All components were connected with SILASTIC tubing (Dow Corning Corporation, Midland) with 2 mm inner diameter. Compartments were separated with a scaffold holder plate. The culture medium was perfused from the lower compartment to the upper compartment via medium distribution holes in the scaffold holder plate. A separation membrane prevents cross-contamination of cells in the two compartments. Before perfusion culture, all components of the bioreactor were sterilized by gamma irradiation with a total dose of 25 kGy. Flow Rate Optimization. Flow rates were optimized using albumin secretion by hepatocytes as determined by the albumin sandwich enzyme-linked immunosorbent assay (ELISA) Kit, which was described in detail in The Differentiated Functions Characterization section. Lactate Release and Glucose Consumption Measurement. Lactate and glucose levels were measured by analyzing 0.5 mL of the culture medium in a BioProfile 400 Analyzer (Nova Biomedical, Waltham). The glucose consumptions were quantified by measuring the difference in glucose concentrations between the fresh media and perfused media (after 24 h of perfusion). The releases of lactate were quantified by measuring the perfused media (after 24 h of perfusion).
Cells Isolation and Culture
Adult male inbred Wistar rats (Animal Holding Unit, National university of Singapore, Singapore) weighing 200 to 300 g were used in all experiments. Housing and feeding were done according to NIH and NACLAR guidelines for the care of laboratory animals. The studies had been approved by the IACUC Committee of the National University of Singapore. Hepatocytes were harvested by a two-step in situ collagenase perfusion as described previously.7 The hepatocyte viability was determined to be 90–95% based on the conventional trypan blue exclusion test. The rat HSC-T6 stellate cell line used in this study was obtained as a gift from Dr. Scott Friedman (Mount Sinai School of Medicine, New York, NY). The HSC-T6 stellate cell line was established from male retired-breeder Sprague Dawley rats18 at passage number 56.
Dulbecco’s modified eagle medium was used during the cell isolation process. Hepatocytes and HSC-T6 cells were cocultured in Williams’ E medium supplemented with 10 mmol/L nicotinamide, 0.2 mmol/L ascorbic acid 2-phosphate, 20 ng/mL epidermal growth factor, 20 mmol HEPES, 0.5 lg/mL insulin, 44 mmol/L NaHCO3, 0.1 lmol/L dexamethasone, and 0.1 % bovine serum albumin. Hepatocytes and HSC-T6 Seeding and Culture
Scaffolds were placed in culture dishes on ice for 10 min before commencement of cell seeding. Hepatocytes (4 3 106 cells/scaffold) or HSC-T6 cells (2 3 106 cells/scaffold) were resuspended in 0.4 mL culture medium and were injected into scaffolds using a 1 mL syringe with 18 G needle. Static Culture. Cell-seeded scaffolds were kept in 6-well culture plates with 5 mL of culture medium; and incubated at 378C, 95% humidity, and 5% CO2, with culture media changed every 2 days. Albumin secretion and urea production in the culture medium were measured every 48 h. The duration of culture was 9 days. Perfusion Culture. For monoculture, a hepatocyte-seeded scaffold was placed into the upper compartment of the bioreactor; the lower compartment was kept empty. For coculture, hepatocyte- or HSC-T6-seeded scaffolds were placed into the upper and lower compartments of bioreactor, respectively, and perfusion-cultured at 378C, 95% humidity, and 5% CO2, with a peristaltic flow rate of 1.2 mL/min. Culture medium was changed every 2 days. Albumin secretion and urea production in the culture medium were measured every 48 h. The duration of culture was 9 days. Cell Morphology Visualized by Scanning Electron Microscopy. The morphology of hepatocytes in the scaffolds was characterized by scanning electron microscopy (SEM). Samples were fixed with glutaraldehyde (3% in PBS) for 30 min. After repeated rinsing, samples were further fixed with an aqueous solution of osmium tetraoxide (OsO4) (1%) at 48C for 30 min. Samples were then freeze-dried overnight and cut with a razor blade and gold-coated with an ion sputter coater (Jeol JFC-1200) at 15 mA for 80 s. Photomicrographs were acquired by SEM (JSM-7400M). The Differentiated Functions Characterization
DNA Quantification and Cell Number. The differentiated functions of hepatocytes examined in this study were normalized by the total numbers of hepatocytes attached on scaffolds, and those numbers were evaluated by quantifying the DNA content in scaffolds using Pico-Green dsDNA Quantitation Kit. Cells were lysed by immersion of scaffolds in 3 mL of 1M NaOH, followed by incubation for 30 min at 708C. After cell lysis, the pH of suspension was adjusted back to 7.0 with 3M potassium acetate, 100 lL of 200 times dilution Pico-Green dye in buffer was added to 100 lL of the suspension and the mixture was incubated for 5 min. The fluorescence signal was measured by microplate reader (Safire2, Tecan Austria GmbH) with 480 nm excitation Journal of Biomedical Materials Research Part B: Applied Biomaterials
DEVELOPMENT OF DUAL-COMPARTMENT PERFUSION BIOREACTOR
157
One-Way ANOVA was used to determine whether the differences on functions of hepatocytes at each time point among three culture conditions (cocultured under perfusion, monocultured under perfusion, and monocultured under static) were statistically significant. In case of statistically significant differences were shown by ANOVA results, multiple comparison using independent-samples t tests were carried out to determine whether the functions of hepatocytes cocultured under serial perfusion are significantly different from that of hepatocytes cultured under two other culture conditions. Differences were considered significant if p \ 0.05 for ANOVA and p \ 0.01 for multiple comparison. RESULTS Figure 2. XPS N 1s core level spectra for PLGA fiber surface with different treatments. (A) Pristine PLGA fibers. (B) PLGA fiber treated by 0.1M NaOH for 30 min. (C) PLGA fiber physically coated with collagen. (D) PLGA fiber grafted with collagen.
wavelength and 520 nm emission wavelength. A standard curve was established by measuring DNA content from known number of cells and the cell numbers in samples were determined from the standard curve. All results represent triplicate from three independent experiments. Albumin ELISA Assay. The albumin secretion from hepatocytes was determined by ELISA. Samples were harvested from culture supernatant 24 h after changing medium. The medium samples were frozen at 2808C. The daily albumin production was measured using the rat albumin ELISA Quantitation Kit. Urea Synthesis Assay. To induce urea synthesis, ammonium chloride was added into fresh culture medium to a final concentration of 1 mM and circulated through system for 2.5 h before changing back to fresh culture medium. Samples of the ammonium chloride containing medium after completing one circulation cycle were taken as the starting points. Samples after 2.5 h-culture were centrifuged at 6000 rpm for 4 min, and the supernatant was subjected to the urea analysis using Urea Synthesis Kit. Cytochrome P450 Function Assay. Cytochrome P450 enzymatic 1A1/2 activity in terms of EROD was measured by microplate reader according to the protocol adapted from Jiang et al. 2004. Ethoxyresorufin (ER) concentration was independently determined from both the absorbance at a wavelength of 480 nm and fluorescence of resorufin (R). Fluorescence was measured at an excitation wavelength of 543 nm and at an emission wavelength of 570 nm, the measured fluorescence was corrected by subtraction of contribution of the remaining ER calculated, and net R production was determined by a calibration curve.
Statistical Analysis
All statistical analyses were performed using SPSS version 15. Data are expressed as mean 6 standard deviation (SD) Journal of Biomedical Materials Research Part B: Applied Biomaterials
PLGA-Collagen 3D Scaffolds
We have characterized the surface chemistry of the modified PLGA using XPS. The N 1s core level spectra of pristine PLGA fibers (a); PLGA fiber treated with 0.1M NaOH for 30 min (b); PLGA fiber physically coated with collagen (c); and PLGA fiber grafted with collagen (d) were shown in Figure 2. A peak corresponding to N 1s (binding energy, 400 eV) appeared on the spectra of PLGA fibers physically coated with collagen or grafted with collagen. The level of the nitrogen peak contributed by collagen was a good indicator of the amount of collagen on the fiber surface.19 There was more collagen on the surface of the collagengrafted PLGA fibers than the collagen-coated PLGA fibers and no collagen was present on the surface of the pristine PLGA fibers or hydrolyzed PLGA fibers (Figure 2). The microstructure of the scaffolds such as pore size distribution, total pore area, and porosity of the scaffolds were characterized by PASCAL 140 mercury porosimeter and SEM. The porosity of the cross-linked PLGA-collagen scaffold was 81%, indicating that the structure was highly
Figure 3. Scaffold microstructure. A typical pore size distribution of the cross-linked PLGA-collagen scaffold measured by mercury porosimeter is represented by relative pore volume (h) which is defined as the percentage of the volume for the pore with a specific radius to total pore volume; and cumulative volume ( ) which is defined as summarized volume of pores with pore sizes beyond a specific radius.
158
WEN ET AL.
Figure 4. Schematic drawing of the components of the perfusion bioreactor system.
porous.20 The total pore area was 1.41 m2/g and the mean pore radius was 51 6 23 lm. A typical plot of the pore diameter distribution is illustrated in Figure 3. Collagen appeared as integrated extensions of the PLGA fibers as interconnected channel structures, which could provide space for cell seeding and efficient mass transfer during cell culture (Figure 3, inset). Perfusion Bioreactor for Serial Coculture
Hepatocytes and HSC-T6 cells were seeded in separate PLGA-collagen 3D scaffolds and placed into the upper and lower compartment of the bioreactor, respectively (Figure 4). Overnight leakage test was performed before perfusion culture. After the cell-containing scaffolds were placed into bioreactor, all connectors, tubing, and bioreactor enclosure were sealed securely, perfusion-culture was initiated and culture medium level in the reservoir was monitored regularly. No culture medium leakage from the perfusion bioreactor was observed. Visual observations through the transparent bioreactor, tubings, and reservoir indicated good stability of the scaffolds with no signs of fungal or bacterial contamination for up to 9 days of perfusion culture. The flow rate in the perfusion bioreactor was optimized against albumin secretion by hepatocytes (Figure 5). A range of flow rates from 0.33 to 4.00 mL/min yielded reasonable cellular functions in hepatocyte perfusion culture.21 In our perfusion bioreactor, the optimal flow rate was 1.20 mL/min, which yielded the highest level of albumin secreted by hepatocytes. To investigate the nutrients and oxygen delivery in the perfusion bioreactor for both monoculture and serial coculture, we measured glucose and lactate concentrations in the culture medium over a period of 9 days [Figures 6 (A,B)]. The releases of lactate and glucose
consumptions in the media for both monoculture and serial coculture were kept below 0.70 g/L and 0.50 g/L, respectively.
Serial Coculture of Hepatocytes and Hepatic Stellate Cell (HSC-T6)
The coculture ratio of hepatocytes and HSC-T6 cells was optimized against albumin secretion by hepatocytes; and the optimum hepatocytes: HSC-T6 ratio was 2:1 (data not shown). Hepatocytes and HSC-T6 cells cocultured in 3D scaffolds after 9 days of serial perfusion in the bioreactor
Figure 5. Flow rates optimization. Effect of flow rate on albumin secreted by hepatocytes cultured in perfusion bioreactor. (n) 0.33 mL/min. (*) 0.66 mL/min. (~) 1.2 mL/min. (!) 4 mL/min. Data are mean 6 SD, n 5 3. All results represent triplicate from three different cell populations. Journal of Biomedical Materials Research Part B: Applied Biomaterials
DEVELOPMENT OF DUAL-COMPARTMENT PERFUSION BIOREACTOR
159
Figure 6. (A) Glucose consumptions and (B) Releases of lactate in the perfusion media under (~) monoculture and (~) coculture conditions are quantified at various time points during the 9-day culture with perfusion flow rate of 1.2 mL/min. Data are mean 6 SD, n 5 3. All results represent triplicate from three different cell populations.
were imaged by SEM [Figures 7(A,B)]. Both hepatocytes and HSC-T6 maintained cuboidal cell morphology. Albumin secreted by hepatocytes cultivated under all conditions (normalized by cell number as shown in Table I) increased gradually during the first 5 days of culture and then decreased afterwards till day 9 [Figure 8 (A)]. There were statistically significant differences on the albumin secreted by hepatocytes at all time points during cell culture among three culture conditions (p \ 0.001), and specially, 65.96 lg/106 /day for hepatocytes cocultured under perfusion on day 5 was 5-fold higher than that of hepatocytes under static culture (11.49 lg/106/day), and 1.5fold higher than that of hepatocytes monocultured under perfusion (37.77 lg/106 /day). The albumin secretion of hepatocytes cocultured under perfusion was maintained at high levels throughout 9 days of culture. Urea synthesis function of hepatocytes was assessed by exposing the cells to culture medium containing 1 mM NH4Cl for 2.5 h [Figure 8(B)]. There were statistically significant differences on the urea synthesized by hepatocytes at all time points during cell culture among three culture
conditions (p \ 0.001), and specially, 83.97 lg/106/2.5 h for hepatocytes cocultured under perfusion on day 6, which was 8-fold higher than that of hepatocytes under static culture (7.26 lg/106/2.5 h), and 1.5-fold higher than that of hepatocytes monocultured under perfusion (52.75 lg/106/2.5 h). The phase I metabolic activity of hepatocytes, measured by cytochrome P450-dependent EROD assay [Figure 8(C)], revealed that there were statistically significant differences on cytochrome P4501A1 functions of hepatocytes on day 9 among three culture conditions (p \ 0.001), hepatocytes cocultured under perfusion maintained relatively higher levels of cytochrome P4501A1 function (15.20 nmol/3 h/106 cells) than that of hepatocytes after 9-day static culture (5.07 nmol/3 h/106cells).
DISCUSSION Optimization of culture conditions in vitro for maintenance of hepatocytes’ survival and functions is necessary for the development of effective BAL devices or cell-based tissue
Figure 7. Scanning electron micrographs of the modified PLGA fiber scaffold with hepatocytes (A) and HSC-T6 (B) cocultured on day 9. Journal of Biomedical Materials Research Part B: Applied Biomaterials
160
WEN ET AL.
TABLE I. The Number of Hepatocytes in Scaffolds During Cell Culture
Numbers of Hepatocytes in Scaffolds (106) Time Points Day Day Day Day Day Day Day Day Day
1 2 3 4 5 6 7 8 9
Monoculture Under Static (Mean 6 SD) 2.55 2.38 2.10 1.92 1.58 1.48 1.06 0.93 0.76
6 6 6 6 6 6 6 6 6
0.39 0.37 0.35 0.14 0.18 0.09 0.35 0.49 0.19
Monoculture Under Perfusion (Mean 6 SD) 2.76 2.75 2.63 2.60 2.55 2.49 2.44 2.35 2.29
6 6 6 6 6 6 6 6 6
Coculture Under Perfusion (Mean 6 SD)
0.49 0.46 0.51 0.34 0.25 0.38 0.29 0.23 0.34
2.97 2.94 2.90 2.89 2.86 2.79 2.73 2.72 2.64
6 6 6 6 6 6 6 6 6
0.57 0.84 0.69 0.71 0.36 0.33 0.30 0.20 0.33
All hepatocyte numbers in above table were calculated according to 2 3 1025 lg DNA/hepatocyte, and the DNA contents in every scaffold at each time point were measured by Pico-Green dsDNA Quantitation Kit.
constructs transplantation therapies. Several researchers have shown that extracellular matrix support, neighboring cells, nutrients and oxygen transfer can influence the viability and functions of hepatocytes.11,22,23 Hepatocytes are anchorage-dependent cells which are best cultured in 3D scaffolds in vitro for survival, reorganization, proliferation, and functions. These 3D scaffolds where hepatocytes anchor on not only provide a surface for cell adhesion, but
also have profound influences on modulating the cell shape and gene expression, relevant to cell growth and liver-specific functions.24 Furthermore, the intra microporous structures in these scaffolds are also important for efficient packing of sufficient cells and good mass transfer of oxygen and nutrients for intended applications.1,25,26 In this study, we have processed collagen into sponges to generate interconnected microporous structures to support hepatocyte
Figure 8. The differentiated functions of hepatocytes under various culture conditions during 9-day culture. (A) Albumin secretion, (B) urea synthesis, and (C) cytochrome P450 1A1/2 activities of hepatocytes were qualified and normalized against the cell numbers (2 3 1025 lg DNA/hepatocyte)11 in each scaffold. ( ) co-cultured under perfusion. ( ) Mono-cultured under perfusion. ( ) Mono-cultured under static. *Significant to mono-cultured under static (p \ 0.01). **Significant to mono-cultured under perfusion (p \ 0.01). N.S. not significant. Data are mean 6 SD, n 5 3. All results represent triplicate from three different cell populations. Journal of Biomedical Materials Research Part B: Applied Biomaterials
DEVELOPMENT OF DUAL-COMPARTMENT PERFUSION BIOREACTOR
culture in 3D scaffolds, whose mechanical properties are further strengthened by PLGA fibers. Furthermore, to facilitate seamless integration of collagen sponges with PLGA fibers for stable cell support in perfusion culture, PLGA surface was modified by the chemical bonds between collagen molecules and polymer chain on the surfaces of PLGA fiber. The remaining collagen amount after extensive rinsing was relatively high on the surfaces of the modified PLGA fiber, which improved collagen adhesion to the surfaces of the collagen-conjugated PLGA fibers and stabilized the microporous collagen sponges in the interfiber space,17 The stable collagen grafting onto the PLGA fibers in scaffolds resulted in a favorable cell culture environment with improved cellsurface interaction and 3D culture organization.19,23 Perfusion bioreactor has been extensively used in cell culture to enhance nutrient, oxygen supply, and waste removal for maintaining cell viability in the thick scaffolds with high mass transfer resistance.10,27 We have established a serial perfusion bioreactor here to coculture hepatocytes and HSC-T6 cells in the PLGA-collagen 3D scaffolds. The optimal flow rate (1.2 mL/min) in the bioreactor was determined against albumin secretion by hepatocytes. One interpretation of this result would be when the flow rate was too low, mass transfer of nutrients, oxygen, and metabolites cannot meet the requirements by hepatocytes for their functional maintenance; when the flow rate was too high, the hydrodynamic shear stress generated can be detrimental to the hepatocytes.28 In our perfusion culture system, glucose consumption and release of lactate in culture medium, common indicators for cell nutrient supply, cell growth, and aerobic cell metabolism, were analyzed.27,29–31 The low level of glucose consumption (\0.5 g/L) and release of lactate (\0.7 g/L) in monoculture or coculture conditions implied that the nutrients and oxygen delivery in culture mediums under both conditions were sufficient over the entire culture period. HSCs are a major nonparenchymal cell type in the liver, which both physically and chemically interact with hepatocytes in vivo.12 They produce various biologically active mediators to regulate the behavior of hepatocytes in liver. However, previous study on the coculture of these two cell types with direct cell–cell contact has reported a decrease of hepatocytes function.14 In our study, HSC-T6 and hepatocytes were separated in different culture chambers by a membrane. The membrane thickness and pore size were chosen to separate the bulk of the cells in the two compartments while allowing efficient mass transfer in perfusion culture without imposing exceedingly high flow rate and pressure to the cells. With this dual compartments serial perfusion culture configuration, the serial perfusion coculture of HSC-T6 cells and hepatocytes in our study sustained higher level of albumin production, urea synthesis, and cytochrome P450 enzymatic activity by hepatocytes for up to 9 days, when compared with the perfusion monoculture or static monoculture. The exact mechanism of how the serial coculture of hepatocytes and Journal of Biomedical Materials Research Part B: Applied Biomaterials
161
T6 cells maintain the hepatocyte functions remains to be investigated. Some researchers have suggested that the diffusion of soluble factor(s) from cocultured cells is responsible for the maintenance of hepatocyte functions.14,15 The dual-compartment perfusion bioreactor can also be used to coculture of other cell types for various biomedical applications.32,33
CONCLUSION We have developed a new perfusion bioreactor for the serial coculture of hepatocytes and the supporting HSC-T6 in 3D PLGA-collagen scaffolds in two separate compartments. The differentiated functions of the hepatocytes in this bioreactor were maintained at higher levels relative to the monoculture system where hepatocytes alone were perfused or statically cultured. The dual-compartment perfusion bioreactor enabled independent control of the supporting cell culture parameters; and local environments can be ‘‘tailormade’’ for different applications in BAL devices or cellbased therapies.
We thank all members of the Cell and Tissue Engineering Laboratory from Yong Loo Lin School of Medicine, the Centre for Biomedical Materials Applications and Technology from Faculty of Engineering, National University of Singapore, Cell and Tissue Engineering Laboratory of IBN, A*STAR, for their technical assistance. We thank Dr. Scott Friedman for providing HSC-T6 cell line to L.Z. We also thank Dr. Shen Liang from Biostatistics Unit of Yong Loo Lin School of Medicine to give us advices on statistic analysis. F. W. is a research scholar of the National University of Singapore, and Y. C. T. is an A*STAR Graduate Scholar.
REFERENCES 1. Ogawa K, Vacanti JP. Liver. In: Atala A, Lanza RP, editors. Methods of Tissue Engineering. New York: Academic Press; 2002. pp 977–986. 2. Strain AJ, Neuberger JM. A bioartificial liver—State of the art. Science 2002;295:1005–1009. 3. Dixit V, Gitnick G. The bioartificial liver: State-of-the-art. Eur J Surg Suppl 1998:71–76. 4. Allen JW, Hassanein T, Bhatia SN. Advances in bioartificial liver devices. Hepatology 2001;34:447–455. 5. Mooney DJ, Sano K, Kaufmann PM, Majahod K, Schloo B, Vacanti JP, Langer R. Long-term engraftment of hepatocytes transplanted on biodegradable polymer sponges. J Biomed Mater Res 1997;37:413–420. 6. Wang YJ. Literature review for artificial liver. In: Wang YJ, editor. Bioartificial liver. Beijing: People’s Medical Publishing House; 2002. pp 1–36. 7. Chia SM, Leong KW, Li J, Xu X, Zeng KY, Er PN, Gao SJ, Yu H. Hepatocyte encapsulation for enhanced cellular functions. Tissue Eng 2000;6:481–495. 8. Croci T, Williams GM. Activities of several phase I and phase II xenobiotic biotransformation enzymes in cultured hepatocytes from male and female rats. Biochem Pharmacol 1985;34:3029–3035.
162
WEN ET AL.
9. Glicklis R, Shapiro L, Agbaria R, Merchuk J, Cohen S. Hepatocyte behavior within three-dimensional porous alginate scaffolds. Biotechnol Bioeng 2000;67:344–453. 10. Powers MJ, Janigian DM, Wack KE, Baker CS, Beer SD, Griffith LG. Functional behavior of primary rat liver cells in a three-dimensional perfused microarray bioreactor. Tissue Eng 2002;8:499–513. 11. Fiegel HC, Havers J, Kneser U, Smith MK, Moeller T, Kluth D, Mooney DJ, Rogiers X, Kaufmann PM. Influence of flow conditions and matrix coatings on growth and differentiation of three-dimensionally cultured rat hepatocytes. Tissue Eng 2004;10:165–174. 12. Abu-Absi SF, Hansen LK, Hu W-S. Three-dimensional coculture of hepatocytes and stellate cells. Cytotechnology 2004;45:125–140. 13. Chia SM, Lin PC, Yu H. TGF-b1 regulation in hepatocyteNIH3T3 co-culture is important for the enhanced hepatocyte function in 3D microenvironment. Biotechnol Bioeng 2005; 89:565–573. 14. Higashiyama S, Noda M, Muraoka S, Uyama N, Kawada N, Ide T, Kawase M, Yagi K. Maintenance of hepatocyte functions in coculture with hepatic stellate cells. Biochem Eng J 2004;20:113–118. 15. Uyama N, Shimahara Y, Kawada N, Seki S, Okuyama H, Iimuro Y, Yamaoka Y. Regulation of cultured rat hepatocyte proliferation by stellate cells. J Hepatol 2002;36:590–599. 16. Saito M, Matsuura T, Masaki T, Maehashi H, Shimizu K, Hataba Y, Iwahori T, Suzuki T, Braet F. Reconstruction of liver organoid using a bioreactor. World J Gastroenterol 2006; 12:1881–1888. 17. Wen F, Chang S, Toh YC, Teoh SH, Yu H. Preparation of biocompatible poly(lactic-co-glycolic acid) fiber scaffolds for rat liver cells cultivation. Mater Sci Eng C 2007;27:285–292. 18. Vogel S, Piantedosi R, Frank J, Lalazar A, Rockey DC, Friedman SL, Blaner WS. An immortalized rat liver stellate cell line (HSC-T6): A new cell model for the study of retinoid metabolism in vitro. J Lipid Res 2000;41:882–893. 19. Cheng ZY, Teoh SH. Surface modification of ultra thin poly (epsilon-caprolactone) films using acrylic acid and collagen. Biomaterials 2004;25:1991–2001. 20. Yang F, Xu CY, Kotaki M, Wang S, Ramakrishna S. Characterization of neural stem cells on electrospun poly(L-lactic acid) nanofibrous scaffold. J Biomater Sci Polym Ed 2004;15: 1483–1497. 21. Kim SS, Sundback CA, Kaihara S, Benvenuto MS, Kim BS, Mooney DJ, Vacanti JP. Dynamic seeding and in vitro culture
22.
23. 24.
25.
26.
27.
28. 29.
30.
31. 32.
33.
of hepatocytes in a flow perfusion system. Tissue Eng 2000;6: 39–44. Bhatia SN, Balis UJ, Yarmush ML, Toner M. Effect of cellcell interactions in preservation of cellular phenotype: cocultivation of hepatocytes and nonparenchymal cells. FASEB J 1999;13:1883–900. Yamada KM, Clark K. Survival in three dimensions. Nature 2002;419:790–791. Mooney DJ, Hansen L, Vacanti JP, Langer R, Farmer S, Ingber D. Switching from differentiation to growth in hepatocytes: Control by extracellular matrix. J Cell Physiol 1992; 151:497–505. Yang F, Xu CY, Kotaki M, Wang S, Ramakrishna S. Characterization of neural stem cells on electrospun poly(L-lactic acid) nanofibrous scaffold. J Biomat Sci Polym Ed 2004;15: 1483–1497. Mikos AG, Bao Y, Cima LG, Ingber DE, Vacanti JP, Langer R. Preparation of poly(glycolic acid) bonded fiber structures for cell attachment and transplantation. J Biomed Mater Res 1993;27:183–189. Lichtenberg A, Dumlu G, Walles T, Maringka M, RingesLichtenberg S, Ruhparwar A, Mertsching H, Haverich A. A multifunctional bioreactor for three-dimensional cell (co)-culture. Biomaterials 2005;26:555–562. Sato Y, Tsukada K, Hatakeyama K. Role of shear stress and immune responses in liver regeneration after a partial hepatectomy. Surg Today 1999;29:1–9. Kurokawa H, Park YS, Iijima S, Kobayashi T. Growth characteristics in fed-batch culture of hybridoma cells with control of glucose and glutamine concentrations. Biotechnol Bioeng 1994;44:95–103. Ozturk SS, Thrift JC, Blackie JD, Naveh D. Real-time monitoring and control of glucose and lactate concentrations in a mammalian cell perfusion reactor. Biotechnol Bioeng 1997; 53:372–378. Ozturk SS, Riley MR, Palsson BO. Effects of ammonia and lactate on hybridoma growth, metabolism, and antibody production. Biotechnol Bioeng 1992;39:418–431. Yamada Y, Yokoyama S-I, Wang X-D, Fukuda N, Takakura N. Cardiac stem cells in brown adipose tissue express CD133 and induce bone marrow nonhematopoietic cells to differentiate into cardiomyocytes. Stem Cells 2007;25:1326–1333. Mori K, Le Goff B, Charrier C, Battaglia S, Heymann D, Redini F. DU145 human prostate cancer cells express functional receptor activator of NFkB: New insights in the prostate cancer bone metastasis process. Bone 2007;40:981–990.
Journal of Biomedical Materials Research Part B: Applied Biomaterials
