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Cell Transplantation, Vol. 15, pp. 921–927, 2006 Printed in the USA. All rights reserved. Copyright 2006 Cognizant Comm. Corp.
Self-Assembling Peptide Nanofiber as a Novel Culture System for Isolated Porcine Hepatocytes Nalu Navarro-Alvarez,* Alejandro Soto-Gutierrez,* Jorge D. Rivas-Carrillo,* Yong Chen,* Tsuyoshi Yamamoto,* Takeshi Yuasa,* Haruo Misawa,† Jiro Takei,‡ Noriaki Tanaka,* and Naoya Kobayashi* *Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan †Department of Orthopedic Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama 700-8558, Japan ‡3-DMatrix Japan, Ltd., Tokyo 102-0083, Japan
Freshly isolated porcine hepatocytes are a very attractive cell source in the cell-based therapies to treat liver failure because of unlimited availability. However, due to the loss of hepatocyte functions in vitro, there is a need to develop a functional culture system to keep the cells metabolically active. Here we compared the effect of a self-assembling peptide nanofiber (SAPNF) as an extracellular matrix (ECM) with collagen type I on hepatocyte metabolic and secretion activities following hepatocyte isolation. Isolated porcine hepatocytes were cultured in SAPNF and collagen type I. Morphological assessment at different time points was performed by using SEM and phase contrast microscope. Metabolic and secretion activities were comparatively performed in the groups, by means of ammonia, lidocaine, and diazepam as well as albumin. Hepatocytes cultured on SAPNF revealed a three-dimensional spheroidal formation, thus maintaining cell differentiation status during 2 weeks of culture. On the other hand, hepatocytes in collagen revealed a spread shape, and by day 14 no hepatocyte-like cells were observed, but cells with long shape were present, thus revealing a degree of dedifferentiation in collagen culture. Hepatocytes in SAPNF were capable of drug-metabolizing activities and albumin secretion in higher ratio than those cultured on collagen. The present work clearly demonstrates the usefulness of SAPNF for maintaining differentiated functions of porcine hepatocytes in culture. Key words: Porcine hepatocytes; Self-assembling peptide nanofiber; Extracellular matrix; Hepatocyte culture
hepatocytes has inevitable drawbacks, because the cells lose their polarity during the isolation procedure and the functions decrease rapidly in the subsequent in vitro culture (26). Importantly, hepatocytes are anchorage dependent and sensitive to environmental factors such as cell density and nature of the neighboring cells (9). Therefore, in the development of hepatocyte-based therapies, the provision of an in vivo microenvironment where can be attained cell-to-cell contact as well as cell-to-matrix interactions is of extreme importance to maintain the liver-specific functions (26). Toward that goal, we have focused on the use of a novel biomaterial through molecular self-assembling peptide nanofiber RADA-16 (arginine–alanine–aspartic acid) (30), composed by four repeats of alternating hydrophilic (aspartic acid negatively charged, and arginines positively charged) and hydrophobic amino acids (alanines) [COCH3]-RADARADARADARADA-[CONH2]
Mortality of acute liver failure (ALF) remains high despite intensive care treatment (19). Thus, there is a compelling need to develop alternative therapies to treat this life-threatening condition. Bioartificial liver (BAL) can be used in order to bridge the liver function until a suitable donor organ is available for transplantation or the patient’s own organ has recovered (7,16). Healthy human hepatocytes are an ideal source for BAL; however, their relative scarcity is one of the major disadvantages, compounded by the competing demands of orthotopic liver transplantation (OLTX). Therefore, researchers have focused on the use of porcine hepatocytes, because of unlimited availability, as a xenograft for temporary support (20) and extracorporeal perfusion (4), as well a biological component in BALs (22). However, independent of the cell source, the use of primarily isolated
Received July 7, 2006; final acceptance August 3, 2006. Address correspondence to Naoya Kobayashi, M.D., Ph.D., Department of Surgery, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama, 700-8558, Japan. Tel: (+81) 86-235-7255; Fax: (+81) 86-235-7485; E-mail: [email protected]
(29). The peptide forms a nanofiber scaffold of about 10 nm in diameter with pores between 5 and 200 nm, namely hydrogels with extremely high water content [>99.5% (w/v) water]. The RAD motif may function in a similar way to the ubiquitous integrin receptor binding site RGD (arginine–glycine–aspartate) (28). Within this framework, the cells are surrounded in a manner comparable to the native extracellular matrix (29). Here we report the development of a novel cell culture system by promoting spheroid formation of porcine hepatocyte using the self-assembling peptide nanofiber, RADA-16, to enable the cells to recover from isolation injury, restore their functions, and maintain differentiated functions in vitro. MATERIALS AND METHODS Isolation and Culture of Porcine Hepatocytes All procedures performed on the pigs were approved by the Okayama University Institutional Animal Care and Use Committee and thus within the guidelines of laboratory animals. Under general anesthesia, pigs (JA West, Okayama, Japan) weighing 20 kg underwent upper middle incision and the left lateral lobes, weighing 80 g, were surgically removed for hepatocyte isolation. Hepatocytes were isolated with a four-step retrograde dispase/collagenase perfusion method, as previously reported (18). Cell viability was assessed by a trypan blue dye exclusion method. In the hepatocyte isolation and culture experiments, the isolated hepatocytes with viabilities of more than 90% were used. Self-Assembling Peptide Nanofiber Preparation Self-assembling peptide nanofiber (SAPNF) hydrogel 1% (kindly provided by 3-DMatrix Japan Ltd., Tokyo, Japan) was sonicated for 30 min before use and diluted with an equal volume of sterile water; 200 µl of 0.5% SAPNF was added to a well of 12-well plates (BD Biosciences, San Jose, CA). Culture medium was added on the top of SAPNF, which resulted in instantaneous gelation of the material. The medium was changed twice every 15 min to equilibrate pH of the culture medium, and SAPNF-coated culture well was incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C for porcine hepatocyte culture. Collagen type I-coated plates (Biocoat, Becton Dickinson) were used as a control (Fig. 1). Cell Seeding and Culture Porcine hepatocytes were seeded in the wells of 12well SAPNF-coated or collagen-coated plates to a final density of 2 × 105 per well. Williams’ E medium (Sigma, St. Louis, MO) supplemented with 10% fetal bovine serum (Sigma), 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma) was used in all the experiments. The
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hepatocytes were incubated in a humidified atmosphere of 5% CO2 and 95% air at 37°C with daily medium changes. In the following experiments, hepatocyte functions were evaluated per unit of time per milligram of cellular protein at each of time points. Cellular protein concentrations were determined by Multiscan JX (Thermo Electron Co.,Yokohama, Japan). Morphological Assessment During the culture time, morphological appearance of porcine hepatocytes was observed using a phase contrast microscope (Olympus CK40-SL Japan) and scanning electron microscope (SEM) (Hitachi S-2300, Hitachi Co. Ltd., Tokyo, Japan). For SEM, the samples were washed with PBS followed by fixation with 2% glutaraldehyde for 2 h at 37°C, and gently washed with PBS. The samples were then postfixed with osmium tetraoxide for 2 h and dehydration was accomplished using a graded series of ethanol (50%, 60%, 70%, 80%, 90%, and 99%). The samples were then dried at critical point for 2 h in absolute alcohol, mounted on aluminum stub, and sputter-coated with gold before viewing under SEM. Evaluation of Metabolic Capacity and Albumin Secretion by Porcine Hepatocytes For the measurement of metabolic capacity, 2 × 105 porcine hepatocytes were inoculated in each well of a 12-well plate and ammonia-, lidocaine-, and diazepammetabolizing activities of the hepatocytes per milligram of cellular protein were evaluated over time in both groups. Ammonium sulfate (Nakarai Tesuku Co., Kyoto, Japan), lidocaine (Fujisawa Pharmaceutical Co., Osaka, Japan), or diazepam (Takeda Chemical Industries, Osaka, Japan) was added to the culture medium at the final concentration of 0.56 mM, 1 mg/ml, or 1 µg/ ml, respectively. The concentration of each reagent was measured 4 h later to compare the metabolic capacities between the groups. Ammonia concentration was determined using a FujiDri-Chem slide (FujiCo., Tokyo, Japan). Concentrations of lidocaine and diazepam were measured by SRL Co. (Okayama, Japan). Twenty-fourhour albumin secretion into the culture medium was measured over time per milligram of cellular protein by a pig albumin enzyme-linked immunoabsorbent assay quantitation kit (Bethyl, Laboratories, Inc., Montgomery, TX). Statistical Analyses Mean values are presented with standard deviations (SDs). A two-tailed Student’s t-test was used to calculate the significance of difference in mean values. A value of p < 0.05 was considered statistically significant.
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Figure 1. Schematic drawing of the experimental design. Hepatocytes were isolated from porcine livers and cultured in SAPNF or in collagen type I. They were maintained for 14 days, for an in vitro assessment of the functionality and morphology.
RESULTS SAPNF Facilitated Spheroidal Aggregation of Porcine Hepatocytes Freshly isolated porcine hepatocytes cultured on SAPNF-coated plates started to aggregate and formed spheroids of compact morphology within 7 days (Fig. 2A, B). The formed spheroids maintained their smooth, transparent, and round shape for at least 14 days (Fig. 2C). On the other hand, porcine hepatocytes cultured on collagen type I-coated plates showed a flattened and extended shape within 24 h (Fig. 2D) and then spread out, and the appearance of cells with a long shape started to appear (Fig. 2E, F). SEM Revealed That Porcine Hepatocytes in SAPNF Culture Developed Spheroid Structures SEM at different times after cell seeding revealed that porcine hepatocytes in SAPNF aggregated into increasingly larger cell clumps that eventually formed into tightly packed spheroid structures of around 100 µm diameter, which is an adequate size to avoid central necrosis of the cells (11) (Fig. 3A, B). In contrast, porcine
hepatocytes in collagen type I culture never formed spheroid structures. The hepatocytes showed a flattened morphology (Fig. 3C, D). Porcine Hepatocytes in SAPNF Culture Efficiently Metabolized Ammonia, Lidocaine, and Diazepam and Produced Albumin Favorably Metabolic rates of ammonia, lidocaine, and diazepam of porcine hepatocytes per 24 h/mg of cellular protein were comparatively analyzed between the groups (n = 3). Under collagen type I culture conditions, the hepatocytes rapidly lost their ability to perform differentiated functions. There was a significantly higher metabolism of ammonia observed in porcine hepatocyte culture of SAPNF in comparison with the hepatocytes cultured with collagen type I (day 1: SAPNF 58.6 ± 2%, collagen 53.1 ± 2%; day 7: SAPNF 43.8 ± 2.2%, collagen 16.6 ± 1.5%; day 14: SAPNF 26.4 ± 3%; collagen 9 ± 1.3%) (Fig. 4A). Metabolism of lidocaine was significantly higher in porcine hepatocytes in SAPNF than the hepatocytes in collagen type I culture (day 1: SAPNF 40 ± 1.78%, collagen 38.8 ± 3.4%; day 7: SAPNF 31.8 ± 4%,
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Figure 2. Morphological appearance of porcine hepatocytes. Morphological study showed that the porcine hepatocytes cultured in SAPNF maintained well-developed round-like appearance, starting with dispersed cells (A), which aggregated into doublets and triplets within the first 24 h after inoculation. Larger aggregates appeared in the culture by day 2, reaching an average size of 100 µm in diameter by day 7 (B, C). In contrast, collagen type I-cultured hepatocytes revealed a flattened appearance (D). Collagen cultures on day 7 showed very few hepatocyte-like cells (E), and some cells with fibroblast-like morphology appeared thereafter (F). Original magnification: ×200. Scale bars: 50 µm; (A–C) and 20 µm (D–F).
collagen: 11.6 ± 1%; day 14: SAPNF 19.05 ± 3%, collagen 8.1 ± 1.7%) (Fig. 4B). Diazepam was significantly better metabolized in the porcine hepatocytes in SAPNF than the hepatocytes in collagen type I culture (day 1: SAPNF 39.9 ± 2.8%, collagen 37.2 ± 1.9%; day 7: SAPNF 34.6 ± 4%, collagen 13.9 ± 2%; day 14: SAPNF 24.08 ± 3.5%, collagen 5.78 ± 1.7%) (Fig. 4C). The ability to produce albumin was maintained in the porcine hepatocytes in SAPNF culture for up to 14 days (Fig. 4D), with a slight increase in the production on day 5, due to the completion of spheroidal formation by the cells, where functions were increased. However, after this, a slight decrease in albumin production was observed, but constant levels remained until day 14. In contrast, the capacity to produce albumin by the porcine hepatocytes cultured in collagen type I was impaired in a time-dependent manner, in which a dramatic decrease was observed on day 7 and by day 14 no albumin was detected. (Fig. 4D). DISCUSSION The use of primary porcine hepatocytes is increasingly favored for clinical application as a source of BAL because of the unlimited availability and functional sim-
ilarity to human hepatocytes. However, once the hepatocytes are isolated from the liver, the cells start to lose many of the differentiated functions in a considerably shorter time, mainly due to the disrupting of cell-to-cell or cell-to-matrix interactions (26). To engineer an adequate environment for hepatic function preservation in vitro, we made use of a self-assembling peptide nanofiber, SAPNF, for porcine hepatocyte culture. Different scaffolds as extracellular matrices (ECM) for hepatocyte culture have been used. Some of them are made of synthetic polymers; however, while hepatocytes retained viability using synthetic materials, such as PLLA (poly-L-lactic acid) (14), PLGA (poly-lactic acidco-glycolic acid) (10), and alginate (25), it has been demonstrated that the cells are not able to retain liverspecific gene expression for a long time (15). The fiber size of these materials is quite larger than the cells, thus failing to mimic the in vivo environment of ECM. In addition, it has been reported that these materials sometimes cause cell cytotoxicity and inflammatory responses (3). In contrast, the fiber size of SAPNF (10 nm) is small enough for the cells to mimic in vivo ECM. Natural derived matrices for the maintenance of hepatic functions, such as the two-layer collagen gels (8)
SELF-ASSEMBLING PEPTIDE NANOFIBER FOR ISOLATED PORCINE HEPATOCYTES
and Matrigel derived from Engelberth-Holm Swarm mouse sarcoma (1), have also been used. However, the major limitations of these materials are host reaction and zoonosis. The animal origin of the products would make their clinical application very difficult (12,15,21). On the other hand, the components of SAPNF can be naturally degraded over time, producing only amino acids, which can be reused by the cells with no adverse effects. Thus, its clinical application seems to be highly possible. In addition, SAPNF has been successfully tested in animal experiments with no immunological reaction (6). Considering the clinical setting, one of the pivotal functions of BAL is the ability to eliminate ammonia from the patient’s blood to reduce the risk of encephalopathy (17,22). Remarkably, porcine hepatocytes cultured in SAPNF demonstrated a high degree of ammonia-metabolizing capacity in vitro for the first 7 days (Fig. 4A). Similar results were observed with lidocaine and diazepam in the porcine hepatocytes cultured in SAPNF (Fig. 4B, C). In contrast, porcine hepatocytes cultured in collagen type I failed to maintain the hepatic functions even for a week. In addition, we observed the presence of cells with long shape, fibroblast-like cells, probably due to dedifferentiation of original hepatocytes
by using collagen type I (Fig. 2E, F). Our findings are consistent with previous reports indicating that hepatocyte cultures on collagen type I were maintained no longer than 1 week (24). The maintenance of porcine hepatocyte functions in SAPNF could be explained by the fact that the influence of the hydrophilic/hydrophobic properties of SAPNF and the occurrence of charged functional chemical groups could reconstitute the interactions of the cells with these polypeptides (31,32). It is well known that a variety of signals for the regulation of growth and differentiation of the cells can be provided by intercellular contacts, as well as the contact with adequate ECM (13,23). Despite the fact that the hepatocytes were highly functional during the first week of culture when using SAPNF, a slight decrease in function was observed thereafter, but was still maintained up until 14 days. We assumed that the diminishment of hepatocyte functions in a time-dependent manner was partially due to the mechanical disruption of SAPNF induced by medium change. To reduce such a damage to the SAPNF, we are now investigating the use of a culture insert. Other possibilities for the decreased hepatic functions would be culture stress on the cells and dedifferentiation
Figure 3. Structural examination of hepatocytes by SEM. Porcine hepatocytes cultured in SAPNF revealed a well-developed round microscopic appearance at 24 h after inoculation (A) and formed spheroidal aggregation on day 7 (B). In contrast, hepatocytes cultured in collagen type I showed flattened microscopic appearance at 24 h (C). Then the cell number decreased and an overgrowth of fibroblast-like cells was observed by day 7 (D). Scale bars: 50 µm.
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Figure 4. Ammonia and drug-metabolizing capacities and albumin-producing ability of porcine hepatocytes. Metabolic capacity per milligram of cellular protein was comparatively analyzed among the groups. Porcine hepatocytes cultured in SAPNF significantly increased ammonia metabolism (A) 4 h after loading ammonium sulfate in the culture medium at a final concentration of 0.56 mM ammonia, in comparison with hepatocytes cultured in collagen type I (A). (B, C) After loading lidocaine (1 mg/ml) or diazepam (1 µg/ml) in the culture medium, metabolic rates were compared 4 h later. Porcine hepatocytes cultured with SAPNF significantly metabolized both lidocaine (B) and diazepam (C), in higher ratio compared to collagen. (D) Albumin production was also significantly better in SAPNF-cultured hepatocytes than in collagen. The data are representative of three independent experiments. *p < 0.01.
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