Survival and function of rat hepatocytes cocultured with nonparenchymal cells or sinusoidal endothelial cells on biodegradable polymers under flow conditions

Survival and Function of Rat Hepatocytes Cocultured With Nonparenchymal Cells or Sinusoidal Endothelial Cells on Biodegradable Polymers Under Flow Conditions By Satoshi Kaihara, Stephen Kim, Byung-Soo Kim, David J. Mooney, Koichi Tanaka, and Joseph P. Vacanti Boston, Massachusetts; Kyoto, Japan; Chicago, Illinois; and Ann Arbor, Michigan

Background/Purpose: The authors have investigated hepatocyte transplantation using biodegradable polymer scaffolds as a possible treatment of end-stage liver disease. The purpose of this study was to investigate the survival rate and function of hepatocytes alone or cocultured with other cell types on 3-dimensional biodegradable polymers for 7 days under continuous flow conditions in vitro. Methods: Hepatocytes (group 1, n ⫽ 8), hepatocytes with nonparenchymal cells (group 2, n ⫽ 7), or hepatocytes with sinusoidal endothelial cells (group 3, n ⫽ 6) were isolated from Lewis rats and seeded onto the polymer scaffolds. The polymer devices subsequently were placed under continuous flow conditions for 7 days. Albumin production from the constructs was measured each day, and urea nitrogen synthesis was examined on day 7. The devices also were examined by histology at day 7. Results: Histology results showed the presence of numerous

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ESPITE MAJOR PROGRESSES in the treatment of patients with end-stage liver disease, liver transplantation currently is the only successful and established method of therapy. Unfortunately, the critical scarcity of donor organs especially in pediatric populations continues to be a problem and major limitation to this form of treatment.1,2 For this reason, there has been great interest in the fields of cell transplantation and tissue engineering. We have investigated hepatocyte transplantation using synthetic biodegradable polymer devices as a novel approach for the treatment of endstage liver disease. In the past we have shown the survival of the heterotopically transplanted hepatocytes on biodegradable polymer discs as well as partial recovery of enzyme deficiency after hepatocyte transplantation.3-6 However, one major limitation in these studies has been the insufficient survival rate of transplanted cells to permanently correct the liver function caused by the poor oxygen and nutrient supply and waste exchange simply by diffusion. To address this issue, we have fabricated highly porous 3-dimensional polymer scaffolds, in which large numbers of hepatocytes can be engrafted because of its large surface area and 3-dimensional structure.7 These cells may be able to survive because the high porosity of the polymer will allow adequate amounts of oxygen and nutrition delivery and waste exchange. We

viable hepatocytes on polymer devices, with no differences in hepatocyte viability between the 3 groups. Albumin secretion in the culture medium gradually decreased by day 7. There also were no significant differences in albumin production or urea nitrogen synthesis between the 3 groups at day 7.

Conclusions: Hepatocytes could survive on the 3-dimensional polymer scaffolds under flow conditions for 7 days, and albumin secretion and urea synthesis of hepatocytes were seen at day 7. Nonparenchymal cells and sinusoidal endothelial cells had no measurable effect on hepatocyte function in our continuous flow culture system. J Pediatr Surg 35:1287-1290. Copyright © 2000 by W.B. Saunders Company. INDEX WORDS: Hepatocyte transplantation, nonparenchymal cell, sinusoidal endothelial cell, flow culture.

also hypothesize that in vitro conditioning of hepatocytes in polymer scaffolds may improve their survival and function after implantation, and we have developed a flow culture system for the polymer-hepatocyte constructs in vitro. We have shown that hepatocytes can attach onto the 3-dimensional polymer devices in large numbers, and conditioning these cells in a flow system provides an environment more conductive for hepatocyte survival and function than standard static culture conditions.8 Many groups have shown that hepatocytes can survive

From the Department of Surgery, Harvard Medical School and Massachusetts General Hospital, Boston, MA; the Department of Transplant Immunology, Faculty of Medicine, Kyoto University, Kyoto, Japan; the Department of Surgery, University of Chicago Hospital, Chicago, IL; the Department of Urology, Harvard Medical School and Children’s Hospital, Boston, MA; and the Department of Chemical Engineering, University of Michigan, Ann Arbor, MI. This work was funded by generous grants from The Holly Ann Soulard Research Fund of Department of Surgery, Children’s Hospital in Boston and the Thomas Pappas Charitable Foundation. Address reprint requests to Joseph P. Vacanti, MD, Department of Surgery, Massachusetts General Hospital, Warren 1157, 55 Fruit St, Boston, MA 02114. Copyright © 2000 by W.B. Saunders Company 0022-3468/00/3509-0003$03.00/0 doi:10.1053/jpsu.2000.9298

Journal of Pediatric Surgery, Vol 35, No 9 (September), 2000: pp 1287-1290

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for long periods and increase in specific function when they are cocultured with other cell types such as nonparenchymal cells (NPCs), sinusoidal endothelial cells (SECs), fibroblasts, and liver epithelial cells.9-12 In this study, we evaluate survival rate and function of hepatocytes on 3-dimensional biodegradable polymers under continuous flow conditions and show the effects of cocultured NPCs or SECs. MATERIALS AND METHODS

Animals Male Lewis rats weighing 150 to 200 g were used for cell harvesting. All animals were purchased from Charles River Laboratories (Wilmington, MA) and were housed in the Animal Research Facility of Children’s Hospital, Boston, Massachusetts, in accordance with the National Institutes of Health guidelines for the care of laboratory animals. They were maintained under a 12-hour light-dark cycle and given rat chow and water ad libitum.

Polymer Fabrication Microporous biodegradable polymer tubes, 5 mm in length and 5 mm in outer diameter with an internal diameter of 1 to 2 mm, were created from sheets of a nonwoven mesh of polyglycolic acid (PGA) fibers (Smith and Nephew, Heslington, York, UK) sprayed on the outer surface with 5% polylactide-co-glycolide (PLGA; 85L:15G, Ethicon Inc, Somerville, NJ). Each fiber was 15 ␮m in diameter, and the average pore size of the fiber mesh was 250 ␮m. The polymer was coated with 100 ⫻ diluted collagen solution (Vitrogen 100, Collagen Corp, Palo Alto, CA), kept under ultraviolet light overnight and washed for 3 times with phosphate-buffered saline (PBS) before seeding.

Culture Media William’s E media supplemented with 1 g sodium pyruvate (Sigma, St Louis, MO) and 1% glutamine-penicillin-streptomycin (Gibco, Gaithersburg, MD) was used during the cell isolation process. Hepatocytes and NPCs or SECs were cocultured in DMEM (Gibco BRL) supplemented with 0.2% bovine albumin, 0.2% D⫺(⫹) galactose, 30 ␮g/mL L-proline, 5 mmol/L niacinamide, 0.1 g/L ornitine, 0.1 ␮mol/L dexamethasone, 1% penicillin/streptomycin (Sigma), 1% glutamine (Gibco BRL), insulin-transferrin-sodium selenite (5 mg/L-5 mg/L-5 ␮g/L; Roche Molecular Biomedicals, Indianapolis, IN), trace metals, and 20 ng/mL epidermal growth factor (Collaborative Biomedical Products, Bedford, MA).13

Cell Isolation and Seeding Hepatocytes were isolated using a modified 2-step collagenase perfusion.14,15 Liver was perfused with a calcium-free buffer to wash out the blood followed with a collagenase solution to digest the extracellular matrix. The liver was excised and agitated mechanically to produce a single-cell suspension. NPCs were isolated by differential centrifugation.16,17 Both the number and viability of hepatocytes and NPCs were determined using a trypan blue exclusion test. SECs were isolated using a 2-step Percoll graduation.18 Cells were characterized as SECs based on the existence of fenestration under scanning electron microscopy (SEM). Cell seeding was performed using a dynamic seeding method because more cells would be uniformly seeded onto the polymer compared with static seeding using a pipet.19 Hepatocytes were resuspended to William’s E and seeded onto the PGA polymer (group 1, n ⫽ 8). NPCs (group 2, n ⫽ 7) or SECs (group 3, n ⫽ 6) were mixed with

Fig 1. Schema of flow culture system.

hepatocytes at the same cell density. The cell suspension was pumped through the polymer at a flow rate of 1.5 mL/min for 4 hours. The entire seeding unit was maintained at 37°C with 5% CO2.

Flow Culture System Cell-polymer constructs were cultured under continuous flow conditions (Fig 1). The culture medium was pumped at a flow rate of 1.0 mL/min from 100 mL of reservoir; through the oxygenation tubing, air trap, and cell-polymer construct housing unit; and recirculated back to the reservoir. Medium was exchanged every day, and the entire unit was maintained at 37°C with 5% CO2. The cell-polymer constructs were harvested after 7 days and examined by histology and scanning electron microscopy.

Albumin Production Culture medium was collected every day and analyzed for albumin concentration by enzyme-linked immunosorbent assay (ELISA) reported by Schwerer et al.20 In brief, each sample was seeded into a 96-well microplate (Fisher Scientific, Pittsburgh, PA) coated with sheep antirat albumin antibody (ICN, Costa Mesa, CA). After a 60-minute incubation, the plate was washed and peroxidase-conjugated sheep antirat albumin antibody (ICN) was added in each well. After a 60-minute incubation, the plate was washed, and ABTS solution (Sigma) was added in each well. Extinction was measured with spectrophotometer at 405 nm of wave length.

Urea Nitrogen Synthesis As a preliminary experiment to analyze the hepatocyte function, urea nitrogen synthesis was investigated in 4 specimens in group 1, 2 in group 2, and 6 in group 3 at day 7. The other specimens were dropped out because of the assay failure. Ammonium chloride was supplemented into culture medium to final concentration at 10 mmol/L. After 4 hours incubation, urea nitrogen concentration was measured by urea nitrogen diagnostic kit (Sigma).

RESULTS

Viable hepatocytes were observed on polymer devices at day 7 by histology (Fig 2A). SEM also showed many hepatocytes attached on the polymer surface or in spheroidal formations as well as the polymer fibers covered with extracellular matrix (Fig 2B). A few NPCs or SECs were observed in group 2 and 3; however, there was no measurable effect on the survival of hepatocytes by

SURVIVAL AND FUNCTION OF RAT HEPATOCYTES

Fig 2. (A) Histology of hepatocytes on polymer scaffolds at day 7. (Original magnification ⴛ 250.) (B) SEM of polymer scaffolds at day 7. Arrow indicates hepatocytes, arrowhead is extracellular matrix, asterisk is polymer fiber. (Original magnification ⴛ100.)

histology (Fig 3A and B). By SEM, NPCs or SECs were not detected at day 7 (data not shown). Albumin synthesis from hepatocytes gradually decreased over time; however, it was still detectable in culture medium at day 7 (Fig 4). There were no significant differences in albumin synthesis between the 3 groups at day 7 (group 1, 10.3 ⫾ 3.0; group 2, 13.9 ⫾ 4.3; group 3, 14.2 ⫾ 2.7, ␮g/d; mean ⫾ SE). Mean urea nitrogen levels for each group were 21.4 in group 1, 19.8 in group 2 and 16.6 in group 3 (␮g/4 hours).

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Fig 3. Histology of hepatocytes cocultured with (A) nonparenchymal cells (arrow) and (B) sinusoidal endothelial cells (arrowhead). (Original magnification ⴛ250.)

for 2-day cultures.8,19 In this study, we cultured hepatocytes under flow conditions for 7 days and showed their survival rate and hepatocyte specific function. SEM shows that the polymers are occupied with hepatocytes

DISCUSSION

We have developed a culture system for 3-dimensional hepatocyte-polymer constructs in vitro to improve the survival rate and function of hepatocytes before implantation. In previous studies we found that these cells could attach onto the 3-dimensional polymer devices in large numbers using a dynamic seeding method. In addition, they could be conditioned in a flow system to provide an environment more conducive for hepatocyte survival and function than regular static culture systems

Fig 4. Albumin production from hepatocytes during 7 days culture (␮g/d).

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and their extracellular matrix. These results suggest that hepatocytes on the 3-dimensional polymer scaffolds can survive and function up to 7 days under continuous flow conditions. Further examination is now on going to see the long-term survival rate of hepatocytes in this culture system. In a preliminary study of hepatocytes cocultured with SECs on polymers for 2 days, SECs and NPCs survived and formed aggregates with hepatocytes between the polymer fibers under flow conditions (data not shown). However, hepatocytes cocultured with NPCs or SECs showed no significant differences in survival rate and function as compared with hepatocytes culture alone, probably because of the few numbers of NPCs and SECs

that could survive on the polymer at 7 days in this study. These findings indicate that our flow culture system may not be appropriate for the survival of NPCs and SECs in vitro. It is imperative to figure out the optimal culture condition not only for hepatocytes but also NPCs and SECs to see the effect of these cells on hepatocytes. We are now working on a new medium and also making some modification in our culture system. Rat hepatocytes survive and are functional on the 3-dimensional polymer scaffolds under flow conditions for 7 days. However, nonparenchymal cells and sinusoidal endothelial cells have no measurable effects on hepatocyte function in our continuous flow culture system as shown by albumin and urea synthesis.

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