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Maintenance of hepatocyte functions in coculture with hepatic stellate cells
Biochemical Engineering Journal 20 (2004) 113–118
Maintenance of hepatocyte functions in coculture with hepatic stellate cells Shinji Higashiyama a , Megumi Noda a , Satoko Muraoka a , Naoki Uyama b , Norifumi Kawada c , Takeshi Ide d , Masaya Kawase a , Kiyohito Yagi a,∗ a
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan b Department of Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, 54-Kawaracho, Shogoin, Sakyoku, Kyoto 606-8507, Japan c Department of Hepatology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno 545-8585 Osaka, Japan d Department of Chemistry, Nara Medical University, Kashihara, Nara 634-8521, Japan Received 26 March 2003; accepted after revision 24 July 2003
Abstract Hepatic stellate cells (HSCs) are a type of nonparenchymal liver cells (NPCs) and are present in the perisinusoidal space of Disse. Hepatocytes were cocultured with HSCs isolated from the NPC fraction with the aim of maintaining differentiated liver functions in vitro. Hepatocytes inoculated directly onto the HSC layer (Co-mix) exhibited lower activity of albumin secretion and higher DNA synthesis activity than hepatocytes of the monoculture control. On the contrary, hepatocytes cocultured with HSCs but separated by a semipermeable membrane (Co-sep) maintained the activities of albumin secretion and urea synthesis. The soluble factor(s) secreted from HSCs had the maintenance effect. Subcultured HSCs were activated to myofibroblast-like cells (MFBs) and decreased the maintenance effect on hepatocyte function. However, the MFBs were found to resume the ability to maintain the hepatocyte function by cultivation on type I collagen. The coculture of hepatocytes and HSCS/MFB could be applied to the development of bioartificial liver support system and liver regenerative medicine. © 2003 Elsevier B.V. All rights reserved. Keywords: Nonparenchymal liver cells; HGF; Bioartificial liver; Urea synthesis; Albumin secretion
1. Introduction Recent experimental clinical studies of bioartificial liver support (BAL) systems indicate that they are potential new therapeutic approaches for use as liver supports [1]. An effective BAL, which incorporates liver cells, is expected to perform the multiple synthetic, metabolic and detoxifying functions of the liver. The clinical trials in the USA have been carried out using porcine hepatocytes cultured on collagen-coated dextran microcarriers and packed in hollow-fiber-type bioreactors. We call it the first-generation BAL for the treatment of patients with severe hepatitis. For the construction of future-type BAL with high performance, which can be used for wide range of patients, it is necessary to develop effective scaffolds for liver cells and to maintain liver functions for long time in vitro. For the cellular component, most systems employ primary hepato∗ Corresponding author. Tel./fax: +81-6-6879-8195. E-mail address: [email protected] (K. Yagi).
1369-703X/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2003.07.002
cytes [2]. However, hepatocytes lose functions following isolation within 3–4 days of culture [3]. Therefore, there have been many attempts to maintain and improve liver functions expressed in hepatocytes [4–6]. Many groups have shown that hepatocytes can survive for long periods and maintain specific functions when they are cocultured with other cell types, such as nonparenchymal liver cells (NPCs) [7,8]. We previously reported that formation of multicellular spheroids consisting of hepatocytes and NPC in a hierarchical coculture, in which both cell-types were separated by a collagen layer [9], was very effective for the maintenance of liver functions, such as albumin secretion, urea synthesis and induction of tyrosine aminotransferase. However, NPCs consist of several cell-types, such as Kupffer cells, sinusoidal endothelial cells and stellate cells. It is considered necessary to choose the most effective cell-type as a partner for the maintenance of differentiated functions of the hepatocytes. Since much attention has been focused on the role of hepatic stellate cells (HSCs) for hepatocyte growth and
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functions [10], we chose HSCs for the partner of hepatocytes. HSCs are NPC with a characteristic stellate morphology, which are present in the perisinusoidal space of Disse. HSCs contain variable amounts of lipid droplets mainly containing vitamin A [11]. When there is an injury to the liver, HSCs undergo activation, which also occurs when HSCs are cultured on polystyrene plates. Activation is a characteristic transformation from a quiescent vitamin A-storing cell type to a myofibroblastic-like cell-type (activated stellate cells) eliciting active proliferation, increased extracellular matrix production and enhanced contractility. Activated HSCs also produce various biologically active mediators such as transforming growth factor-␣ and -, interleukin-6 and tumor necrosis factor-␣, but they lose the ability to express hepatocyte growth factor (HGF) found in quiescent HSCs [12–14]. Several studies have recently been carried out to regulate hepatocyte proliferation, when cocultured with HSCs. Uyama et al. reported that hepatocytes proliferation was stimulated in the presence of quiescent HSCs through HGF, extracellular heparan sulfate (HS) and HS proteoglycan [15]. Uemura and Gandhi reported that DNA synthesis of hepatocytes was inhibited in a conditioned medium prepared from endotoxin-activated HSC culture [16]. In this study, we focused on the maintenance of liver-specific functions in hepatocytes, when cocultured with HSCs, particularly in the absence of direct contact between the two types of cell. This coculture system, which incorporates hepatocytes and HSCs, is considered to be important in the development of high-performance BAL. 2. Materials and methods 2.1. Media The basal medium (BM) used consisted of 100 U/ml penicillin G, 100 g/ml streptomycin, 50 ng/ml amphotericin B, and 100 ng/ml aprotinin (Nacalai Tesque, Kyoto, Japan) in William’s medium E (WE, ICN Biochemicals, Costa Mesa, CA, USA). Medium A contained 10% fetal bovine serum (FBS, ICN Biochemicals) in BM. Medium B contained 1 nM insulin (Sigma Chemicals, St. Louis, MO, USA) and 1 nM dexamethasone (Nacalai Tesque, Kyoto, Japan) in Medium A. 2.2. Isolation and culture of hepatocytes and HSCs Hepatocytes were isolated from male Sprague-Dawley rats weighing 150–200 g by perfusing the liver with collagenase (from Clostridium histolyticum Type I; Sigma Chemicals, St. Louis, MO, USA) according to the method of Seglen [17]. HSCs were isolated from male Sprague-Dawley rats weighing 300–400 g by digesting the liver with Pronase-E (Merck Darmstadt, FRG) and collagenase (from C. histolyticum Type I; Wako Pure Chemical Co., Osaka, Japan) as
previously described [18]. These animals were housed in an air-conditioned room at 22± ◦ C before the experiment. Hepatocytes were seeded at a density of 1 × 105 cells/cm2 onto 12-well polystyrene culture plates (Nippon Becton Dickinson, Tokyo, Japan). After 6 h cultivation in Medium B, the cells were cultivated in BM. Isolated HSCs were seeded at a density of 2×105 cells/cm2 onto 12-well polystyrene culture plates or cell culture inserts (12-well format, 3.0 m pore size, PET track-etched membrane, Nippon Becton Dickinson, Tokyo, Japan). 2.3. Coculture of hepatocytes with HSCs Freshly isolated hepatocytes suspended in Medium B were inoculated onto the culture plates in which isolated HSCs had already been cultured for 4 days. After 6 h, the culture medium was replaced with BM. The medium was replaced with a fresh one every 24 h for 2 days of cultivation and culture was continued up to 6 days (mixed coculture (Co-mix)). For the separated coculture (Co-sep), hepatocytes and HSCs were cocultured without any cell-to-cell contact using a cell culture insert. Freshly isolated HSCs were plated on the cell culture insert. After 4 days, hepatocytes were inoculated on the culture plate below the cell culture insert in BM. The medium was replaced with a fresh one every 24 h for 2 days of hepatocyte cultivation and the culture continued up to 6 days. 2.4. Cell count Adherent cells were treated with 0.25% trypsin solution containing 0.02% EGTA in Ca2+ and Mg2+ free phosphate-buffered saline at 37 ◦ C for 5 min, and the viable cell number was determined using a hemocytometer after trypan blue staining. 2.5. Quantification of urea and albumin in the culture medium The amount of urea was determined according to the method of Ormsby [19]. Albumin secretion was measured using ELISA in 96-well maxisoap microtiter plates (Nunc, Rochester, NY, USA). Rat albumin standard solutions (1, 0.5, 0.25, 0.125, 0.0625, 0 m/ml) and culture supernatant with serial dilutions were incubated overnight at 4 ◦ C. After solutions were removed, nonspecific binding was blocked by filling wells to the top with 1% gelatin in PBS for 2 h at room temperature (RT). After a washing with 0.05% Tween 20 in PBS (T-PBS), plates were incubated for 2 h at RT with horseradish peroxidase-conjugated sheep anti-rat albumin antibody (ICN Pharmaceuticals, Aurora, OH, USA). After washing with T-PBS, color was produced by addition of o-phenylenediamine substrate and reaction was stopped with the addition of hydrochloric acid. Absorbance at 492 nm was determined using Microplate Manager III software linked to a Model 550 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
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2.6. Labeling of proliferating cell nuclei with BrdU Hepatocytes cultured for 5 days was treated with 40 M BrdU for 24 h. Incorporated BrdU was immunocytochemically evaluated. The number of cells with brown-colored nuclei was counted in three microscopic fields randomly selected in each well. The BrdU labeling index (BrdU L.I.) was calculated as the number of BrdU-positive cells/number of cells in the same area × 100 (%). 3. Results 3.1. Effect of coculture on albumin secretion of hepatocytes Albumin secretion, which represents the liver-specific function, was measured in hepatocytes cocultured with HSCs. After 5 days of cultivation, the amount of albumin secreted into the culture medium for 24 h was measured using an ELISA, as described in Section 2. Fig. 1 shows that the Co-mix, in which hepatocytes were inoculated directly
Fig. 1. Maintenance of albumin secretion in hepatocytes cocultured with hepatic stellate cells. Hepatocyte monoculture (Control), hepatocyte cocultured with hepatic stellate cells without any cell-to-cell contact using a culture insert (Co-sep), hepatocyte cocultured with hepatic stellate cells on the same surface (Co-mix). Albumin secretion was measured using ELISA. Albumin secretion was expressed as the value per unit area after 6 days of cultivation. Values are mean ± S.D. of three experiments. Asterisk indicate values significantly different from values for control (∗ P < 0.05).
Fig. 2. Effect of mixed coculture on DNA synthesis of hepatocytes. BrdU labeling index (BrdU L.I.) of hepatocyte monoculture (Control) and hepatocytes cocultured with hepatic stellate cells on the same surface (Co-mix) after 6 days of cultivation. Values are mean ± S.D. of three experiments. Asterisks indicate values significantly different from values for control (∗∗ P < 0.01).
onto the preformed HSC layer, had significantly lower albumin secretion after 5 days of cultivation. Since hepatocytes in the Co-mix might enter into S-phase resulting in the decrease of differentiated function, DNA synthesis activity was examined by BrdU uptake assay (Fig. 2). A significant number of hepatocytes showed BrdU uptake in Co-mix, as expected. On the contrary, the Co-sep, in which hepatocytes and HSCs affected each other through the membrane having an average pore size of 3 m, had a significantly higher albumin secretion than the control, as shown in Fig. 1. These results indicate that the Co-sep would have an advantage over the Co-mix, when HSCs are used for the BAL system. Hepatocytes and HSCs were thus cultured in the Co-sep in the following experiments. 3.2. Effect of separated coculture on hepatocytes viability and function Hepatocytes cultured in vitro caused apoptotic or necrotic cell death and were detached from the surface of the culture substrate. The number of viable hepatocytes was counted by a trypan blue exclusion test after 6 days of cultivation (Fig. 3A). The initial density of hepatocytes was about 1.0 × 105 cells/cm2 at the start of the cultivation.
Fig. 3. Maintenance of urea synthesis in hepatocytes cocultured with hepatic stellate cells. Hepatocyte monoculture (Control), hepatocytes cocultured with hepatic stellate cells without any cell-to-cell contact using a culture insert (Co-sep). (A) After 6 days of cultivation, viable cells were counted using the trypan blue exclusion test. (B) Urea synthesis was expressed as the value per area after 6 days of cultivation. Values are mean ± S.D. of three experiments. Asterisks indicate values significantly different from values for control (∗∗ P < 0.01).
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Fig. 4. Phase-contrast micrographs of quiescent HSCs and subcultured MFBs. (A) HSC (magnification of 400×). (B) MFB (magnification of 100×).
Approximately 30% of hepatocytes remained viable in both the monoculture and the Co-sep. HSCs did not affect viability of hepatocytes at 6 days of culture. Urea synthesis, which represents a liver-specific function, was then measured in both the monoculture and the Co-sep. After 6 days of cultivation, hepatocytes in the Co-sep had a significantly higher urea synthesis activity than the control, as shown in Fig. 3B. These results indicate that HSCs secreted certain factor(s) that maintain liver functions, such as albumin secretion and urea synthesis, in the Co-sep. 3.3. Effect of separated coculture with MFB on hepatocytes viability and function Considering the use of HSCs for BAL, the proliferation process is necessary to obtain a sufficient number of cells. However, HSCs are known to be activated by subculture using ordinary polystyrene culture plates and to exhibit myofibroblast-like phenotypes. After the passage in the presence of fetal bovine serum the morphology of HSC changed from the round shape to fibroblastic (Fig. 4). We next examined whether cultured myofibroblast-like cells (MFB) could be an alternative source of HSCs. The viability of hepatocytes adhering to the culture plate was examined by a trypan blue exclusion test (Fig. 5A). Hepatocytes were cocultured
with MFB without cell-to-cell contact using a culture insert (MFB Co-sep). There was a significantly lower viability than the control at 6 days of cultivation. In contrast, when hepatocytes were cocultured with MFB without cell-to-cell contact using a BIOCOAT® fibrillar collagen insert that has a deposition of fibrillar rat tail collagen type I on surface of a 1 m PET membrane (MFB collagen Co-sep), the number of viable hepatocytes was equal to that in the monoculture control at 6 days of cultivation. Urea synthesis was measured in hepatocytes cocultured with MFB (Fig. 5B). After 6 days, hepatocytes cocultured with MFB inoculated on an ordinary surface showed no significant difference in urea synthesis activity from the monoculture control. However, hepatocytes cocultured with MFB on collagen showed a significantly higher activity than the control and MFB Co-sep at 6 days of cultivation.
4. Discussion Hepatocytes in the liver stably maintain viability and their functions by interacting with various kinds of NPC. In this study we showed that HSCs isolated from NPC fraction had the potential to maintain liver-specific functions in hepatocytes. The soluble factor(s) secreted from HSCs were
Fig. 5. Maintenance of urea synthesis in hepatocytes cocultured with MFBs. Hepatocyte monoculture (Control), hepatocytes were cocultured with MFB without cell-to-cell contact using a culture insert (MFB Co-sep), hepatocytes were cocultured with MFBs without cell-to-cell contact using a BIOCOAT® fibrillar collagen insert that has a deposition of fibrillar rat tail collagen type I on surface of membrane (MFB collagen Co-sep). (A) After 6 days of cultivation, viable cells were counted using the trypan blue exclusion test. (B) Urea synthesis was expressed as the value per area after 6 days of cultivation. Values are mean ± S.D. of three experiments. Asterisks indicate values significantly different from values for control (∗ P < 0.05).
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responsible for the maintenance effect because the direct contact between hepatocytes and HSCs did not exist in the Co-sep. The conditioned medium prepared from HSC monoculture also had the maintenance effect on urea synthesis activity of hepatocytes (data not shown). We are now analyzing the effective factor(s) in a conditioned medium of the Co-sep by 2D polyacrylamide gel electrophoresis. Several spots were detected only in the Co-sep sample, but not in monoculture sample in our present study. The sequence analysis of the proteins is now underway. Although the effectiveness of NPC was reported by many researchers [7,8], we solely used HSCs as the partner of hepatocytes, thereby the number of HSCs could be increased in a limited space for the BAL system. We found that hepatocytes in the Co-mix had significantly lower albumin secretion and higher DNA synthesis activities. Hepatocytes in the Co-mix might adhere firmly to the extracellular matrix, such as collagens, which are synthesized by HSCs [10]. The quiescent hepatocytes seem to proceed from G0 to G1 phase of the cell cycle on the collagen layer. The priming step is also mediated by cytokines, such as TNF-␣ and IL-6, which are known to be secreted from HSCs [14]. The initiated hepatocytes might then enter into the S phase by the action of HGF secreted from HSCs. The contact between hepatocytes and HSCs might thus play a negative role in the differentiated functions and a positive role in cell proliferation. We can use Co-mix and Co-sep properly depending on the purpose of liver tissue engineering. The Co-mix and Co-sep could be applied to liver regenerative medicine and the future-type BAL, respectively. To realize the clinical use of BAL for the coculture system, a large amount of HSCs is required. Although subcultured HSCs (MFBs) showed decreased ability to maintain hepatocyte function, we found that MFB, when cultured on collagen type I, recovered the effectiveness on hepatocyte function in the Co-sep. HSCs cultured on a polystyrene surface show a fibroblastic-like flattened shape without process. On the contrary, it was reported that the shape of HSCs cultured on collagen gel markedly changed similarly to in vivo shape with mutipolar processes [20]. MFB could be an alternative source to primary and quiescent HSCs under optimum culture conditions. The analysis of effective factor(s) present in the conditioned medium from MFB-Co-sep by 2D polyacrylamide gel electrophoresis is also necessary to clarify the mechanism underlying the maintenance effect. In our previous study, we carried out the coculture of hepatocytes with Kupffer cells or sinusoidal endothelial cells. Kupffer cells had the maintenance effect on hepatocyte function [9]. Since Kupffer cells do not proliferate in vitro so much, HSCs/MFBs seems to be the best partner for the hepatocytes among NPCs. In this study HSCs were shown to have two important effects on hepatocytes. Depending on the configuration of coculture, HSCs could promote the proliferation or maintain the differentiated functions. HSCs are thus considered to
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be very important for the development of liver regenerative medicine as well as BAL system. References [1] A.J. Strain, J.M. Neuberger, A bioartificial liver—state of the art, Science 295 (2002) 1005–1009. [2] T. Hui, J. Rozga, A.A. Demetriou, Bioartificial liver support, J. Hepatobiliary Pancreat. Surg. 8 (2001) 1–15. [3] T. Croci, G.M. Williams, Activities of several phase I, phase II xenobiotic biotransformation enzymes in cultured hepatocytes from male and female rats, Biochem. Pharmacol. 34 (1985) 3029– 3035. [4] N. Koide, K. Sakaguchi, Y. Koide, K. Asano, M. Kawaguchi, H. Matsushima, T. Takenami, T. Shinji, M. Mori, T. Tsuji, Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and under other nonadherent environments, Exp. Cell. Res. 186 (1990) 227–235. [5] T. Matsushita, H. Ijima, N. Koide, K. Funatsu, High albumin production by multicellular spheroids of adult rat hepatocytes formed in the pores of polyurethane foam, Appl. Microbiol. Biotechnol. 36 (1991) 324–326. [6] M. Kawase, T. Shiomi, H. Matsui, Y. Ouji, S. Higashiyama, T. Tsutsui, K. Yagi, Suppression of apoptosis in hepatocytes by fructose-modified dendrimers, J. Biomed. Mater. Res. 54 (2001) 519– 524. [7] S. Kaihara, S. Kim, B.S. Kim, D.J. Mooney, K. Tanaka, J.P. Vacanti, Survival and function of rat hepatocytes cocultured with nonparenchymal cells or sinusoidal endothelial cells on biodegradable polymers under flow conditions, J. Pediatr. Surg. 35 (2000) 1287– 1290. [8] H. Okubo, M. Matsushita, H. Kamachi, T. Kawai, M. Takahashi, T. Fujimoto, K. Nishikawa, S. Todo, A novel method for faster formation of rat liver cell spheroids, Artif. Organs 26 (2002) 497– 505. [9] K. Yagi, N. Sumiyoshi, C. Yamada, N. Michibayashi, M. Kawase, Y. Miura, T. Mizoguchi, In vitro maintenance of liver function in hierarchical co-culture of hepatocytes and non-parenchymal liver cells, J. Ferment. Bioeng. 80 (1995) 575–579. [10] M. Pinzani, Novel insights into the biology and physiology of the Ito cell, Pharmacol. Ther. 66 (1995) 387–412. [11] R. Blomhoff, K. Wake, Perisinusoidal stellate cells of the liver: important roles in retinol metabolism and fibrosis, Faseb J. 5 (1991) 271–277. [12] G. Ramadori, K. Neubauer, M. Odenthal, T. Nakamura, T. Knittel, S. Schwogler, K.H. Meyer zum Buschenfelde, The gene of hepatocyte growth factor is expressed in fat-storing cells of rat liver and is downregulated during cell growth and by transforming growth factor-beta, Biochem. Biophys. Res. Commun. 183 (1992) 739– 742. [13] P. Schirmacher, A. Geerts, A. Pietrangelo, H.P. Dienes, C.E. Rogler, Hepatocyte growth factor/hepatopoietin A is expressed in fat-storing cells from rat liver but not myofibroblast-like cells derived from fat-storing cells, Hepatology 15 (1992) 5–11. [14] P. Greenwel, J. Rubin, M. Schwartz, E.L. Hertzberg, M. Rojkind, Liver fat-storing cell clones obtained from a CCl4 -cirrhotic rat are heterogeneous with regard to proliferation, expression of extracellular matrix components, interleukin-6, and connexin 43, Lab. Invest. 69 (1993) 210–216. [15] N. Uyama, Y. Shimahara, N. Kawada, S. Seki, H. Okuyama, Y. Iimuro, Y. Yamaoka, Regulation of cultured rat hepatocyte proliferation by stellate cells, J. Hepatol. 36 (2002) 590–599. [16] T. Uemura, C.R. Gandhi, Inhibition of DNA synthesis in cultured hepatocytes by endotoxin-conditioned medium of activated stellate cells is transforming growth factor-beta and nitric oxide-independent, Br. J. Pharmacol. 133 (2001) 1125–1133.
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[17] P.O. Seglen, Preparation of isolated rat liver cells, Meth. Cell. Biol. 13 (1976) 29–83. [18] N. Kawada, T.A. Tran-Thi, H. Klein, K. Decker, The contraction of hepatic stellate (Ito) cells stimulated with vasoactive substances. Possible involvement of endothelin 1 and nitric oxide in the regulation of the sinusoidal tonus, Eur. J. Biochem. 213 (1993) 815–823.
[19] A.A. Ormsby, A direct colorimetric method for the determination of urea in blood and urine, J. Biol. Chem. 146 (1942) 595–597. [20] N. Kojima, M. Sato, K. Imai, M. Miura, Y. Matano, H. Senoo, Hepatic stellate cells (vitamin A-storing cells) change their cytoskeleton structure by extracellular matrix components through a signal transduction system, Histochem. Cell. Biol. 110 (1998) 121–128.
Maintenance of hepatocyte functions in coculture with hepatic stellate cells Shinji Higashiyama a , Megumi Noda a , Satoko Muraoka a , Naoki Uyama b , Norifumi Kawada c , Takeshi Ide d , Masaya Kawase a , Kiyohito Yagi a,∗ a
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan b Department of Gastroenterological Surgery, Graduate School of Medicine, Kyoto University, 54-Kawaracho, Shogoin, Sakyoku, Kyoto 606-8507, Japan c Department of Hepatology, Graduate School of Medicine, Osaka City University, 1-4-3 Asahimachi, Abeno 545-8585 Osaka, Japan d Department of Chemistry, Nara Medical University, Kashihara, Nara 634-8521, Japan Received 26 March 2003; accepted after revision 24 July 2003
Abstract Hepatic stellate cells (HSCs) are a type of nonparenchymal liver cells (NPCs) and are present in the perisinusoidal space of Disse. Hepatocytes were cocultured with HSCs isolated from the NPC fraction with the aim of maintaining differentiated liver functions in vitro. Hepatocytes inoculated directly onto the HSC layer (Co-mix) exhibited lower activity of albumin secretion and higher DNA synthesis activity than hepatocytes of the monoculture control. On the contrary, hepatocytes cocultured with HSCs but separated by a semipermeable membrane (Co-sep) maintained the activities of albumin secretion and urea synthesis. The soluble factor(s) secreted from HSCs had the maintenance effect. Subcultured HSCs were activated to myofibroblast-like cells (MFBs) and decreased the maintenance effect on hepatocyte function. However, the MFBs were found to resume the ability to maintain the hepatocyte function by cultivation on type I collagen. The coculture of hepatocytes and HSCS/MFB could be applied to the development of bioartificial liver support system and liver regenerative medicine. © 2003 Elsevier B.V. All rights reserved. Keywords: Nonparenchymal liver cells; HGF; Bioartificial liver; Urea synthesis; Albumin secretion
1. Introduction Recent experimental clinical studies of bioartificial liver support (BAL) systems indicate that they are potential new therapeutic approaches for use as liver supports [1]. An effective BAL, which incorporates liver cells, is expected to perform the multiple synthetic, metabolic and detoxifying functions of the liver. The clinical trials in the USA have been carried out using porcine hepatocytes cultured on collagen-coated dextran microcarriers and packed in hollow-fiber-type bioreactors. We call it the first-generation BAL for the treatment of patients with severe hepatitis. For the construction of future-type BAL with high performance, which can be used for wide range of patients, it is necessary to develop effective scaffolds for liver cells and to maintain liver functions for long time in vitro. For the cellular component, most systems employ primary hepato∗ Corresponding author. Tel./fax: +81-6-6879-8195. E-mail address: [email protected] (K. Yagi).
1369-703X/$ – see front matter © 2003 Elsevier B.V. All rights reserved. doi:10.1016/j.bej.2003.07.002
cytes [2]. However, hepatocytes lose functions following isolation within 3–4 days of culture [3]. Therefore, there have been many attempts to maintain and improve liver functions expressed in hepatocytes [4–6]. Many groups have shown that hepatocytes can survive for long periods and maintain specific functions when they are cocultured with other cell types, such as nonparenchymal liver cells (NPCs) [7,8]. We previously reported that formation of multicellular spheroids consisting of hepatocytes and NPC in a hierarchical coculture, in which both cell-types were separated by a collagen layer [9], was very effective for the maintenance of liver functions, such as albumin secretion, urea synthesis and induction of tyrosine aminotransferase. However, NPCs consist of several cell-types, such as Kupffer cells, sinusoidal endothelial cells and stellate cells. It is considered necessary to choose the most effective cell-type as a partner for the maintenance of differentiated functions of the hepatocytes. Since much attention has been focused on the role of hepatic stellate cells (HSCs) for hepatocyte growth and
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S. Higashiyama et al. / Biochemical Engineering Journal 20 (2004) 113–118
functions [10], we chose HSCs for the partner of hepatocytes. HSCs are NPC with a characteristic stellate morphology, which are present in the perisinusoidal space of Disse. HSCs contain variable amounts of lipid droplets mainly containing vitamin A [11]. When there is an injury to the liver, HSCs undergo activation, which also occurs when HSCs are cultured on polystyrene plates. Activation is a characteristic transformation from a quiescent vitamin A-storing cell type to a myofibroblastic-like cell-type (activated stellate cells) eliciting active proliferation, increased extracellular matrix production and enhanced contractility. Activated HSCs also produce various biologically active mediators such as transforming growth factor-␣ and -, interleukin-6 and tumor necrosis factor-␣, but they lose the ability to express hepatocyte growth factor (HGF) found in quiescent HSCs [12–14]. Several studies have recently been carried out to regulate hepatocyte proliferation, when cocultured with HSCs. Uyama et al. reported that hepatocytes proliferation was stimulated in the presence of quiescent HSCs through HGF, extracellular heparan sulfate (HS) and HS proteoglycan [15]. Uemura and Gandhi reported that DNA synthesis of hepatocytes was inhibited in a conditioned medium prepared from endotoxin-activated HSC culture [16]. In this study, we focused on the maintenance of liver-specific functions in hepatocytes, when cocultured with HSCs, particularly in the absence of direct contact between the two types of cell. This coculture system, which incorporates hepatocytes and HSCs, is considered to be important in the development of high-performance BAL. 2. Materials and methods 2.1. Media The basal medium (BM) used consisted of 100 U/ml penicillin G, 100 g/ml streptomycin, 50 ng/ml amphotericin B, and 100 ng/ml aprotinin (Nacalai Tesque, Kyoto, Japan) in William’s medium E (WE, ICN Biochemicals, Costa Mesa, CA, USA). Medium A contained 10% fetal bovine serum (FBS, ICN Biochemicals) in BM. Medium B contained 1 nM insulin (Sigma Chemicals, St. Louis, MO, USA) and 1 nM dexamethasone (Nacalai Tesque, Kyoto, Japan) in Medium A. 2.2. Isolation and culture of hepatocytes and HSCs Hepatocytes were isolated from male Sprague-Dawley rats weighing 150–200 g by perfusing the liver with collagenase (from Clostridium histolyticum Type I; Sigma Chemicals, St. Louis, MO, USA) according to the method of Seglen [17]. HSCs were isolated from male Sprague-Dawley rats weighing 300–400 g by digesting the liver with Pronase-E (Merck Darmstadt, FRG) and collagenase (from C. histolyticum Type I; Wako Pure Chemical Co., Osaka, Japan) as
previously described [18]. These animals were housed in an air-conditioned room at 22± ◦ C before the experiment. Hepatocytes were seeded at a density of 1 × 105 cells/cm2 onto 12-well polystyrene culture plates (Nippon Becton Dickinson, Tokyo, Japan). After 6 h cultivation in Medium B, the cells were cultivated in BM. Isolated HSCs were seeded at a density of 2×105 cells/cm2 onto 12-well polystyrene culture plates or cell culture inserts (12-well format, 3.0 m pore size, PET track-etched membrane, Nippon Becton Dickinson, Tokyo, Japan). 2.3. Coculture of hepatocytes with HSCs Freshly isolated hepatocytes suspended in Medium B were inoculated onto the culture plates in which isolated HSCs had already been cultured for 4 days. After 6 h, the culture medium was replaced with BM. The medium was replaced with a fresh one every 24 h for 2 days of cultivation and culture was continued up to 6 days (mixed coculture (Co-mix)). For the separated coculture (Co-sep), hepatocytes and HSCs were cocultured without any cell-to-cell contact using a cell culture insert. Freshly isolated HSCs were plated on the cell culture insert. After 4 days, hepatocytes were inoculated on the culture plate below the cell culture insert in BM. The medium was replaced with a fresh one every 24 h for 2 days of hepatocyte cultivation and the culture continued up to 6 days. 2.4. Cell count Adherent cells were treated with 0.25% trypsin solution containing 0.02% EGTA in Ca2+ and Mg2+ free phosphate-buffered saline at 37 ◦ C for 5 min, and the viable cell number was determined using a hemocytometer after trypan blue staining. 2.5. Quantification of urea and albumin in the culture medium The amount of urea was determined according to the method of Ormsby [19]. Albumin secretion was measured using ELISA in 96-well maxisoap microtiter plates (Nunc, Rochester, NY, USA). Rat albumin standard solutions (1, 0.5, 0.25, 0.125, 0.0625, 0 m/ml) and culture supernatant with serial dilutions were incubated overnight at 4 ◦ C. After solutions were removed, nonspecific binding was blocked by filling wells to the top with 1% gelatin in PBS for 2 h at room temperature (RT). After a washing with 0.05% Tween 20 in PBS (T-PBS), plates were incubated for 2 h at RT with horseradish peroxidase-conjugated sheep anti-rat albumin antibody (ICN Pharmaceuticals, Aurora, OH, USA). After washing with T-PBS, color was produced by addition of o-phenylenediamine substrate and reaction was stopped with the addition of hydrochloric acid. Absorbance at 492 nm was determined using Microplate Manager III software linked to a Model 550 microplate reader (Bio-Rad Laboratories, Hercules, CA, USA).
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2.6. Labeling of proliferating cell nuclei with BrdU Hepatocytes cultured for 5 days was treated with 40 M BrdU for 24 h. Incorporated BrdU was immunocytochemically evaluated. The number of cells with brown-colored nuclei was counted in three microscopic fields randomly selected in each well. The BrdU labeling index (BrdU L.I.) was calculated as the number of BrdU-positive cells/number of cells in the same area × 100 (%). 3. Results 3.1. Effect of coculture on albumin secretion of hepatocytes Albumin secretion, which represents the liver-specific function, was measured in hepatocytes cocultured with HSCs. After 5 days of cultivation, the amount of albumin secreted into the culture medium for 24 h was measured using an ELISA, as described in Section 2. Fig. 1 shows that the Co-mix, in which hepatocytes were inoculated directly
Fig. 1. Maintenance of albumin secretion in hepatocytes cocultured with hepatic stellate cells. Hepatocyte monoculture (Control), hepatocyte cocultured with hepatic stellate cells without any cell-to-cell contact using a culture insert (Co-sep), hepatocyte cocultured with hepatic stellate cells on the same surface (Co-mix). Albumin secretion was measured using ELISA. Albumin secretion was expressed as the value per unit area after 6 days of cultivation. Values are mean ± S.D. of three experiments. Asterisk indicate values significantly different from values for control (∗ P < 0.05).
Fig. 2. Effect of mixed coculture on DNA synthesis of hepatocytes. BrdU labeling index (BrdU L.I.) of hepatocyte monoculture (Control) and hepatocytes cocultured with hepatic stellate cells on the same surface (Co-mix) after 6 days of cultivation. Values are mean ± S.D. of three experiments. Asterisks indicate values significantly different from values for control (∗∗ P < 0.01).
onto the preformed HSC layer, had significantly lower albumin secretion after 5 days of cultivation. Since hepatocytes in the Co-mix might enter into S-phase resulting in the decrease of differentiated function, DNA synthesis activity was examined by BrdU uptake assay (Fig. 2). A significant number of hepatocytes showed BrdU uptake in Co-mix, as expected. On the contrary, the Co-sep, in which hepatocytes and HSCs affected each other through the membrane having an average pore size of 3 m, had a significantly higher albumin secretion than the control, as shown in Fig. 1. These results indicate that the Co-sep would have an advantage over the Co-mix, when HSCs are used for the BAL system. Hepatocytes and HSCs were thus cultured in the Co-sep in the following experiments. 3.2. Effect of separated coculture on hepatocytes viability and function Hepatocytes cultured in vitro caused apoptotic or necrotic cell death and were detached from the surface of the culture substrate. The number of viable hepatocytes was counted by a trypan blue exclusion test after 6 days of cultivation (Fig. 3A). The initial density of hepatocytes was about 1.0 × 105 cells/cm2 at the start of the cultivation.
Fig. 3. Maintenance of urea synthesis in hepatocytes cocultured with hepatic stellate cells. Hepatocyte monoculture (Control), hepatocytes cocultured with hepatic stellate cells without any cell-to-cell contact using a culture insert (Co-sep). (A) After 6 days of cultivation, viable cells were counted using the trypan blue exclusion test. (B) Urea synthesis was expressed as the value per area after 6 days of cultivation. Values are mean ± S.D. of three experiments. Asterisks indicate values significantly different from values for control (∗∗ P < 0.01).
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Fig. 4. Phase-contrast micrographs of quiescent HSCs and subcultured MFBs. (A) HSC (magnification of 400×). (B) MFB (magnification of 100×).
Approximately 30% of hepatocytes remained viable in both the monoculture and the Co-sep. HSCs did not affect viability of hepatocytes at 6 days of culture. Urea synthesis, which represents a liver-specific function, was then measured in both the monoculture and the Co-sep. After 6 days of cultivation, hepatocytes in the Co-sep had a significantly higher urea synthesis activity than the control, as shown in Fig. 3B. These results indicate that HSCs secreted certain factor(s) that maintain liver functions, such as albumin secretion and urea synthesis, in the Co-sep. 3.3. Effect of separated coculture with MFB on hepatocytes viability and function Considering the use of HSCs for BAL, the proliferation process is necessary to obtain a sufficient number of cells. However, HSCs are known to be activated by subculture using ordinary polystyrene culture plates and to exhibit myofibroblast-like phenotypes. After the passage in the presence of fetal bovine serum the morphology of HSC changed from the round shape to fibroblastic (Fig. 4). We next examined whether cultured myofibroblast-like cells (MFB) could be an alternative source of HSCs. The viability of hepatocytes adhering to the culture plate was examined by a trypan blue exclusion test (Fig. 5A). Hepatocytes were cocultured
with MFB without cell-to-cell contact using a culture insert (MFB Co-sep). There was a significantly lower viability than the control at 6 days of cultivation. In contrast, when hepatocytes were cocultured with MFB without cell-to-cell contact using a BIOCOAT® fibrillar collagen insert that has a deposition of fibrillar rat tail collagen type I on surface of a 1 m PET membrane (MFB collagen Co-sep), the number of viable hepatocytes was equal to that in the monoculture control at 6 days of cultivation. Urea synthesis was measured in hepatocytes cocultured with MFB (Fig. 5B). After 6 days, hepatocytes cocultured with MFB inoculated on an ordinary surface showed no significant difference in urea synthesis activity from the monoculture control. However, hepatocytes cocultured with MFB on collagen showed a significantly higher activity than the control and MFB Co-sep at 6 days of cultivation.
4. Discussion Hepatocytes in the liver stably maintain viability and their functions by interacting with various kinds of NPC. In this study we showed that HSCs isolated from NPC fraction had the potential to maintain liver-specific functions in hepatocytes. The soluble factor(s) secreted from HSCs were
Fig. 5. Maintenance of urea synthesis in hepatocytes cocultured with MFBs. Hepatocyte monoculture (Control), hepatocytes were cocultured with MFB without cell-to-cell contact using a culture insert (MFB Co-sep), hepatocytes were cocultured with MFBs without cell-to-cell contact using a BIOCOAT® fibrillar collagen insert that has a deposition of fibrillar rat tail collagen type I on surface of membrane (MFB collagen Co-sep). (A) After 6 days of cultivation, viable cells were counted using the trypan blue exclusion test. (B) Urea synthesis was expressed as the value per area after 6 days of cultivation. Values are mean ± S.D. of three experiments. Asterisks indicate values significantly different from values for control (∗ P < 0.05).
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responsible for the maintenance effect because the direct contact between hepatocytes and HSCs did not exist in the Co-sep. The conditioned medium prepared from HSC monoculture also had the maintenance effect on urea synthesis activity of hepatocytes (data not shown). We are now analyzing the effective factor(s) in a conditioned medium of the Co-sep by 2D polyacrylamide gel electrophoresis. Several spots were detected only in the Co-sep sample, but not in monoculture sample in our present study. The sequence analysis of the proteins is now underway. Although the effectiveness of NPC was reported by many researchers [7,8], we solely used HSCs as the partner of hepatocytes, thereby the number of HSCs could be increased in a limited space for the BAL system. We found that hepatocytes in the Co-mix had significantly lower albumin secretion and higher DNA synthesis activities. Hepatocytes in the Co-mix might adhere firmly to the extracellular matrix, such as collagens, which are synthesized by HSCs [10]. The quiescent hepatocytes seem to proceed from G0 to G1 phase of the cell cycle on the collagen layer. The priming step is also mediated by cytokines, such as TNF-␣ and IL-6, which are known to be secreted from HSCs [14]. The initiated hepatocytes might then enter into the S phase by the action of HGF secreted from HSCs. The contact between hepatocytes and HSCs might thus play a negative role in the differentiated functions and a positive role in cell proliferation. We can use Co-mix and Co-sep properly depending on the purpose of liver tissue engineering. The Co-mix and Co-sep could be applied to liver regenerative medicine and the future-type BAL, respectively. To realize the clinical use of BAL for the coculture system, a large amount of HSCs is required. Although subcultured HSCs (MFBs) showed decreased ability to maintain hepatocyte function, we found that MFB, when cultured on collagen type I, recovered the effectiveness on hepatocyte function in the Co-sep. HSCs cultured on a polystyrene surface show a fibroblastic-like flattened shape without process. On the contrary, it was reported that the shape of HSCs cultured on collagen gel markedly changed similarly to in vivo shape with mutipolar processes [20]. MFB could be an alternative source to primary and quiescent HSCs under optimum culture conditions. The analysis of effective factor(s) present in the conditioned medium from MFB-Co-sep by 2D polyacrylamide gel electrophoresis is also necessary to clarify the mechanism underlying the maintenance effect. In our previous study, we carried out the coculture of hepatocytes with Kupffer cells or sinusoidal endothelial cells. Kupffer cells had the maintenance effect on hepatocyte function [9]. Since Kupffer cells do not proliferate in vitro so much, HSCs/MFBs seems to be the best partner for the hepatocytes among NPCs. In this study HSCs were shown to have two important effects on hepatocytes. Depending on the configuration of coculture, HSCs could promote the proliferation or maintain the differentiated functions. HSCs are thus considered to
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