- Email: [email protected]
Galactosylated alginate as a scaffold for hepatocytes entrapment
Biomaterials 23 (2002) 471–479
Galactosylated alginate as a scaffold for hepatocytes entrapment Jun Yanga, Mitsuaki Gotob, Hirohiko Isec, Chong-Su Chod, Toshihiro Akaikea,c,* a
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan b Bio Quest Research Co. Ltd., Tokyo 150-0043, Japan c Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan d School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, South Korea Received 10 September 2000; accepted 26 March 2001
Abstract Galactose moieties were covalently coupled with alginate through ethylenediamine as the spacer for enhancing the interaction of hepatocytes with alginate. Adhesion of hepatocytes onto the galactosylated alginate (GA)-coated polystyrene (PS) surface showed an 18-fold increase as compared with that of the alginate-coated surface and it increased with an increase in the concentration of GA. The morphologies of attached hepatocytes were observed to spread out at the 0.15 wt% GA-coated PS surface while round cells were observed at the 0.5 wt% GA-coated PS surface. Inhibition of hepatocytes attachment onto the galactose-carrying PS-coated surface occurred with the addition of the GA into the hepatocyte suspension, indicating the binding of GA with hepatocytes via the patch of asialoglycoprotein receptors. Primary hepatocytes were entrapped in the GA/Ca2+ capsules (GAC). Higher cell viability and more spheroid formation of hepatocytes were obtained in the GAC than in the alginate/Ca2+ capsules (AC). Moreover, liver functions of the hepatocytes such as albumin secretion and urea synthesis in the GAC were improved in comparison with those in the AC. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Alginate; Synthetic scaffold; Galactose; Hepatocytes; Bioartificial liver; Capsules
1. Introduction In recent years, many attempts to develop a highgrade cell-based hybrid artificial liver (HAL) have been focused on immobilizing primary hepatocytes in scaffolds which can be designed to mimic a physiological microenvironment essential for controlling the growth, differentiation of hepatocytes and supporting reconstruction of the liver in vitro [1–3]. Moreover, with the elucidation of receptor-mediated cell functions and the progress in biomaterial science, developments of synthetic extracellular matrices to replace the natural extracellular matrices (ECMs) are increasingly essential and promising in tissue engineering. Alginate obtained from brown seaweed as a heteropolysaccharide has been extensively used as a gelling *Corresponding author. Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Tel.: +81-45-924-5790; fax:+81-45924-5815. E-mail address: [email protected] (T. Akaike).
agent in many biotechnological and medical applications [4]. It is a block copolymer composed of 1,4-linked b-d mannuronic acid (M) and a-l-guluronic acid (G) residues and the G-block can rapidly be crossed by a divalent cation such as calcium to form hydrogels at room temperature and neutral pH. Therefore, recently, alginate was also used as a three-dimensional synthetic ECM (3D-ECM) for immobilization of cells in tissue engineering [5]. In addition, Lim et al. reported that encapsulation of cells into alginate capsules (AC) could produce a very high-density cell culture system with mechanical support to the cells as well as immunoprotection in the event of implantation [6]. Experimentally, hepatocytes encapsulated in alginate have been used in the treatment of acute liver failure and congenital liver enzyme deficiencies such as bilirubin-diphosphate deficiency of the liver (Gunn rat) [7,8]. However, hepatocytes encapsulated in AC are of low viability and do not maintain the long-term liver functions due to the lack of cell–matrix and cell–cell interactions within AC [9,10]. For an anchorage-sensitive cell such as hepatocyte, cell anchorage and cell aggregation regulate the cell
0142-9612/02/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 1 2 9 - 6
472
J. Yang et al. / Biomaterials 23 (2002) 471–479
functions in vitro and in vivo [11]. Additionally, these are closely correlated with the ECMs [12,13]. Therefore, a major challenge of encapsulated hepatocytes for HAL is to provide an optimum 3D-ECM in capsules, which replace many functions of the native ECMs for adjusting the interaction of hepatocytes with the scaffold and promoting hepatocytes to organize into a threedimensional architecture while enhancing cellular functions in capsules. Recently, many researchers have shown that synthetic ECMs with covalent coupling of cell-specific ligands or extracellular signaling molecules have advantages of both enhancing the interaction of cell with biomaterials and controlling the growth, differentiation and behavior of cells in cultures [14–17]. Rowley et al. reported that alginate modified with RGD-containing cell adhesion ligands promoted the adherence of myoblasts and the expression of a differentiated phenotype [14]. Also, it was reported that galactose moieties were recognized by asialoglycoprotein receptors (ASGP-R) on hepatocyte surfaces and promoted the hepatocytes adhesion [17– 20]. In two-dimensional culture, we previously reported [3,17,20–22] that adult rat hepatocytes attached well on the galactose-carrying PS (poly-N-p-vinylbenzyl-d-lactoneamide, abbreviated as PVLA)-coated surface via the patch of ASGP-R. Furthermore, hepatocytes attached onto PVLA were changed from spreading to round shapes with an increase of PVLA concentration and maintained the differentiated functions with promoting spheroids formation, which is in contrast to the tendency of natural ECM such as collagen and fibronectin [20–22]. In this study, we modified alginate with galactose moieties as ASGP-R ligands to improve the hepatocytes anchorage and the interaction of hepatocytes with alginate, and then enhance the liver functions of encapsulated hepatocytes in three-dimensional culture. Galactosylated alginate (GA) was synthesized utilizing aqueous carbodimide chemistry and the specific interaction between primary mouse hepatocytes and GA was investigated. Also, hepatocytes were entrapped in the GA/Ca2+ capsule (GAC). Furthermore, the liver functions and the spheroids formation of hepatocytes were observed in the GAC. The GA may present an efficient synthetic scaffold and the GAC may offer a promising alternative for the development of a HAL.
2. Materials and methods 2.1. Materials Sodium alginate from macrocystis pyrifera and polyl-lysine (PLL, MW 22,000) was purchased from Sigma (St. Louis, MO). Lactobionic acid was purchased from TCI (Tokyo, Japan). Sulfo-NHS was purchased from
Fluka (Chemika-Biochemika Analytika, Tokyo, Japan). All other chemicals were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) unless otherwise stated. PVLA was synthesized by a similar method previously reported [23]. 2.2. Coupling of galactose moieties with alginate Firstly, primary amine was introduced into lactobionic acid. Briefly, lactobionic lactone prepared by dehydration of lactobionic acid was refluxed with 30fold excess ethylenediamine dissolved in anhydrous dimethyl sulfoxide (DMSO) at 701C for 2 h. The monoamine terminated lactobionic lactone (L-NH2) was precipitated with chloroform and the obtained precipitate was vacuum-dried. Then, 0.8 g of the L-NH2 (2 mmol) was reacted with 1 g of alginate (5 mmol) dissolved in 100 ml of 50 mm TEMED (N0 ,N0 ,N0 -tetramethylethylenediamine, with 0.3 m NaCl, pH 6.5) for 24 h at room temperature using 1-ethyl-(dimethylaminopropyl) carbodiimide (EDC) and a co-reactant Nhydroxy-sulfosuccinimide (sulfo-NHS) as the activation agents. The obtained GA was purified by dialysis against Milli Q for one week and lyophilized. The reaction scheme is shown in Fig. 1. Maltose-modified alginate (MA) was also synthesized by this method. 2.3. Cell preparation Primary hepatocytes were isolated from an ICR mouse (5–7 weeks old, male) (SLC, Shizuoka, Japan) by the modified in situ perfusion method as described previously [24]. The dead parenchyma hepatocytes were removed by density gradient centrifugation in Percoll (Pharmacia, Piscataway, NJ, USA). The viable primary hepatocytes were suspended in William’s E medium (Gibco BRL, New York, USA) that contained antibiotics (50 mg penicillin/ml, 50 mg streptomycin/ml and 100 mg neomycin/ml) and HEPES (18 mm). Only isolated hepatocytes with greater than 90% viability by trypan blue dye exclusion were used. 2.4. Hepatocyte attachment One ml of PVLA (100 mg/ml), GA (0.05, 0.15 or 0.5 wt%) or MA (0.15%) was placed in a 35 mm PS dish (Falcon, 1008) at 371C overnight. The solution was decanted and the surface was rinsed 3 times with William’s E medium. The isolated mouse hepatocytes were cultured in a quantity of 4 104 cells/cm2 at 371C in a humidified air/CO2 incubator (95/5 vol%) for 30 min, and the non-adhered cells were collected by decantation and washing the dish with 1 ml medium. The non-adhered hepatocytes were counted with the Sysmex cell counter. The area of adhered hepatocyte on the coated surfaces was measured by OLYMPUS IX70
J. Yang et al. / Biomaterials 23 (2002) 471–479
473
Fig. 1. Reaction scheme of galactosylated alginate.
and KEYENCE VQ-7000. The average area was calculated with 100 cells from six experiments on every surface. The cells having an average area larger than 600 mm2/cell were classified as spread cells, while those less than 500 mm2/cell were classified as round cells (the average area of a freshly isolated mouse hepatocyte was 396720 mm2). 2.5. Inhibition of hepatocyte attachment onto PVLA by GA After incubation of hepatocytes in WE medium containing GA (0.15 wt%), alginate (0.15 wt%) or PVLA (100 mg/ml) at 371C for 30 min in an incubator, the incubated hepatocytes were seeded into the PVLAcoated dish and incubated again at 371C for 1 h in a humidified air/CO2 incubator (95/5 vol%). The nonadhesive hepatocytes were collected and counted. As a control, the same experiment was performed with a collagen-coated dish. 2.6. Encapsulation of hepatocytes Isolated hepatocytes were encapsulated in GAC or AC by the method described by Lim et al. [25]. Briefly, isolated hepatocytes were suspended in sodium alginate
(2 wt%) or a mixture of alginate and GA (1 : 1, w/w) at a cell density of 8 105 cells/ml. The cells/alginate (or GA) suspension was dropped into CaCl2 solution (100 mm, pH 7.4) to form alginate gel beads. The alginate beads were coated with PLL (0.05 wt %) and alginate (0.2 wt%) to form an alginate–PLL–alginate (APA) membrane. Then, treatment of coated beads by sodium citrate (50 mm) was carried out to liquefy the alginate (or GA) gel inside the APA membrane, which enhances the permeability of capsules. Next, 1 ml of hepatocyte capsules was cultured with 3 ml William’s E medium supplemented antibiotics (50 mg penicillin/ml, 50 mg streptomycin/ml and 100 mg neomycin/ml), HEPES (18 mm), EGF (20 ng/ml) and insulin (100 nm) in a 60 mm PS dish at 371C in a humidified air/CO2 incubator (95/5 vol%). The medium was changed every day and the collected medium was stored for biochemical assays. 2.7. Measurement of hepatocytes functions The amounts of albumin in the medium were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit anti-mouse albumin serum (Inter-cell Technologies Inc., Hopewell, NJ, USA) and sheep anti-mouse albumin polyclonal
474
J. Yang et al. / Biomaterials 23 (2002) 471–479
antibody (The Binding Site Ltd., Birmingham, England). The secondary antibody was peroxidase-conjugated anti-rabbit IgG (Seikagaku Co., Tokyo, Japan). Orthophenylenediamine (OPD) (Wako Pure Chemical Industries Ltd., Osaka, Japan) solution (0.4 mg/ml OPD and 0.4 ml/ml H2O2 in citrate–phosphate buffer, pH 5.0) was used as a substrate of peroxidase. Absorbency at 492 nm was measured by a microplate reader (MTP-32, Corona Electric Co., Ibaraki, Japan). The urea concentration was quantified with a Wako Urea N B kit (Code No. 279-36201). 2.8. Viability test by Alamer Blue staining of hepatocytes Hepatocytes in capsules were stained with 10% (v/v) Alamer Blue (Iwaki Glass Co., Tokyo, Japan). Alamer Blue stains only viable cells with red by reductive reaction with mitochondria. Alamer Blue staining was done at 371C for 4 h. The stained hepatocytes in GAC were observed with a confocal laser-scanning microscope (Leica, Germany).
3. Results and discussion 3.1. Synthesis of GA Galactose moieties were coupled to alginate according to the reaction scheme shown in Fig. 1. The first step was the introduction of ethylenediamine into lactobionic acid by reaction of lactobionic lactone dehydrated from lactobionic acid with 30-fold excess of ethylenediamine. The purity of the product was checked by thin-layer chromatography and showed no detectable free ethylenediamine in it. The Rf values for L-NH2 and ethylenediamine were 0.51 and 0.23, respectively. The second step was the coupling of the L-NH2 into the alginate using EDC and sulfo-NHS as activation agents. The GA was analyzed with NMR; however, the peaks of galactose residue in the GA were difficult to be assigned due to the overlapping with those of alginate. The content of galactose moieties in the GA was evaluated by element analysis of carbon, hydrogen and nitrogen content and it showed that 2675% of uronic acid in alginate reacted with galactose moieties. 3.2. Adhesion of hepatocytes onto GA-coated surfaces Freshly isolated mouse hepatocytes were cultured on the GA-coated PS dish to check the specific interaction of hepatocytes with GA. Attachment of hepatocytes onto the alginate- or GA-coated PS surface for 30 min at 371C is shown in Fig. 2. The results indicated that 55% of hepatocytes were attached onto 0.15 wt % GA-coated surface while only 3% of hepatocytes were attached onto 0.15 wt% alginate-coated surface. Almost similar
Fig. 2. Attachment of hepatocytes onto alginate (A)-, GA- or MAcoated PS surface for 30 min at 371C in WE medium.
amounts of hepatocytes as alginate were attached onto the MA-coated surface, indicating that the enhancing of attachment of hepatocytes to GA was caused by the galactose residue coupled to alginate. Also, the sugar residues on the GA-coated plate were examined by a direct lectin–enzyme assay. The results showed that the density of galactose residues on GA-coated surface increased as the concentration of GA increased. Furthermore, the cell attachment increased with an increase of GA concentration as shown in Fig. 2, suggesting that the cell binding was dependent on the density of galactose residues on the GA-coated surfaces. The mean cell areas measured by image analysis of microscopy after 4 h were 1279738, 456724, 779718 and 493715 mm2 for PS surface, 0.05, 0.15 and 0.5 wt% GA-coated surfaces, respectively. The cells having an average area larger than 600 mm2/cell were classified as spread cells whereas those less than 500 mm2/cell were classified as round cells. The morphologies of attached hepatocytes after 4 h were round, spread and round on the surface coated with 0.05, 0.15 and 0.5 wt% GA, respectively, as shown in Fig. 3. The results indicated that the adhesion and shapes of hepatocytes were closely related with the concentration of GA. On the 0.05 wt% GA-coated surface, the spherically adhered hepatocytes were observed to be obviously different from the spreading ones on the PS surfaces (Fig. 3A). It is thought that the coating with 0.05 wt% GA was sufficient to inhibit the hydrophobic binding of hepatocytes to the PS surface. However, the galactose residues on the 0.05 wt% GA-coated surface were too sparse to mediate cell spreading. The results of increased cell attachment and decreased spreading of hepatocytes on increasing the concentration of GA from 0.15 to 0.5 wt% might be a sign of regulating the behavior of hepatocytes by the density of galactose residues in the
J. Yang et al. / Biomaterials 23 (2002) 471–479
475
Fig. 3. Phase-contrast micrographs of hepatocytes cultured on PS surface (A), surface coated with 0.15% A (B), 0.05% GA (C), 0.15% GA (D) and 0.5% GA (E). The cells were cultured in WE medium for 4 h.
Fig. 4. Phase-contrast micrographs of hepatocytes cultured on 0.15 wt% GA-coated PS surface after 1 day (A, D), 3 days (B, E) and 5 days (D, F). 20 ng EGF/ml and 100 nm insulin were supplemented in (D–F).
initial adhesion, which is similar to the tendency reported by Kabayashi et al. [26]. Also, it is thought that the receptors on the hepatocytes were pulled together on the surface containing dense clusters of galactose (0.5 wt% GA), resulting in round cells, whereas the receptors were spread on the surface containing sparse clusters of galactose (0.15 wt% GA), resulting in spread cells [26]. Furthermore, when hepatocytes were cultured on 0.15 wt% GA-coated dish for 5 days, the attached hepatocytes aggregated to form multilayer spheroids on adding EGF (20 ng/ml) and
insulin (100 nm) into the WE medium as shown in Fig. 4. It is suggested that the spheroid formation of hepatocytes depended on the concentration of GA and growth factors such as EGF and insulin. 3.3. Inhibition of hepatocytes attachment onto PVLA by GA The specific interaction of hepatocytes with galactose moieties coupled with GA was further evaluated with checking the inhibition of hepatocyte attachment onto
476
J. Yang et al. / Biomaterials 23 (2002) 471–479
reorganization of ASGP-R with galactose moieties covalently coupled with GA enhanced the interaction of hepatocytes with GA. 3.4. Encapsulation of hepatocytes
Fig. 5. Inhibition of hepatocytes attachment onto PVLA- or collagencoated PS surface by adding PVLA (100 mg/ml), alginate (0.15 wt%) (A), GA (0.15 wt%) or MA (0.15 wt %) into hepatocytes suspension for 1 h at 371C.
PVLA by adding the GA into a hepatocyte suspension (Fig. 5). We observed that the adhesion of hepatocytes onto PVLA showed a greater inhibition by the GA compared with that of the alginate. In contrast, addition of the GA into hepatocytes suspension did not inhibit hepatocytes attachment onto the collagen (Fig. 5). PVLA was previously reported to have highly specific affinity to hepatocytes via the specific recognition of galactose moieties with the ASGP-R on hepatocytes in vitro and in vivo [27,28]. The inhibition of hepatocyte attachment showed the competition between the binding of GA and binding of PVLA to hepatocytes, indicating that the galactose moieties coupled with GA were also recognized by the ASGP-R on hepatocytes. Weigel et al. also reported that rat hepatocytes adhere specifically to galactosyl polyacrylamide gel via a patch of ASGP-R [18]. Therefore, it can be said that hepatocytes attach onto GA via the patch of ASGP-R, and the specific
The cell viability estimated by the MTT method [29] immediately after encapsulating the cells into the GAC was 8177% of the original number of cells. Encapsulation with AC showed the same results. In addition, the GAC observed by microscopy showed almost spherical shapes without roughness or irregularities as same as the shapes of AC. From the observation by phase-contrast microscopy, the hepatocyte structures in the GAC were different from those in the AC after 48 h as shown in Fig. 6. Within GAC, more than 80% of the hepatocytes were aggregated to form multicellular spheroids with diameters enlarged up to above 100 mm, and the spheroids were uniformly distributed in each GAC. The tendency of spheroid formation within the GAC was improved by supplementation with EGF and insulin. This is consistent with the hepatocytes cultured on the GA-coated surface, indicating that EGF and insulin enhanced the mobility of attached hepatocytes in GAC. However, most hepatocytes remained as single cells and only a few cells formed aggregates within the AC. Most of the cells were located in the boundary area around the APA membrane. The compressive moduli of alginate and GA gel beads were 10.9371.99 and 6.0172.53 kPa, respectively, an indication of not much difference of compressive modulus between them. Also, the change of cell distribution in AC and GAC after 48 h as shown in Fig. 6 suggested that the mobility of hepatocytes in AC was almost the same as that in GAC. Therefore, it may be said that the specific interaction of ASGP-R on the hepatocytes with the galactose ligands in GA for anchorage sites is closely related to the spheroid formation in three-dimensional
Fig. 6. Phase-contrast micrographs of encapsulated hepatocytes in AC (A) and GAC (B) cultured in WE medium with EGF and insulin for 2 days.
J. Yang et al. / Biomaterials 23 (2002) 471–479
477
Fig. 8. Albumin secretion (A) and urea synthesis (B) of encapsulated hepatocytes in AC and GAC. The encapsulated hepatocytes were cultured in WE medium with EGF and insulin.
Fig. 7. Confocal micrographs of hepatocyte aggregates in GAC (20 days culture) stained with Alamer Blue; whole image (top) and crosssection images from top to bottom (bottom).
culture instead of differences of physical changes between AC and GAC. It has already been reported that the mobility of intra-capsular hepatocytes is dependent on the viscosity inside microcapsules [30]. Also, it was found that cell adhesion molecules termed as N-Cadherin, A-CAM or N-CAM played an important role in cell interactions of different cell types [31,32]. However, little is known about the exact mechanism of spheroid formation of hepatocytes in 3-D cultures. In order to investigate the viability of hepatocytes in the spheroid, we stained hepatocytes cultured in the GAC for 20 days with Alamer Blue and observed the inner part of hepatocytes aggregates using confocal microscopy as shown in Fig. 7. The internal hepatocytes of the spheroid could be stained with Alamer Blue, indicating that the hepatocytes were alive in the spheroid formed in the GAC. Also, more cell viability within the GAC was obtained than that of the AC. It
was reported that the spheroid formation of hepatocytes was a necessary requirement for cell viability and improvement of cell differentiated functions [33]. A comparison of albumin secretion and urea synthesis of the encapsulated hepatocytes between the AC and the GAC is shown in Fig. 8. The results indicated that the albumin secretion and urea synthesis of the AC rapidly decreased with culture time whereas those of the GAC slowly decreased and maintained higher levels than those of the AC. Additionally, as controls, MA and alginate treated with WSC and sulfo-NHS without galactose were also synthesized by the same method. The results showed that much adhesion of hepatocytes, inhibition of hepatocytes attachment to PVLA, the promoted spheroids formation and enhanced cellular functions in capsules were not found. This suggested that the hepatocytes behaviors were enhanced by incorporated galactose moieties with alginate, but not simply by modifying with another sugar. Therefore, it may be regarded that the galactose moieties in the GA provided anchorage sites for hepatocytes in GAC and improved the formation of hepatocyte spheroids, and then enhanced the liver functions of encapsulated hepatocytes. Clayton et al. reported that tissue-specific
478
J. Yang et al. / Biomaterials 23 (2002) 471–479
transcription was maintained at a high rate when hepatocytes were cultured as slices of tissue in which cell contact and cell architecture were maintained [34]. Wu et al. also reported that hepatocyte spheroids maintained higher P450 activity than monolayer on collagen and demonstrated that the higher P450 activity within spheroids was associated with their ability to maintain a greater degree of differentiation compared to the monolayer [35]. The biological tissue organization or three-dimensional structure of hepatocyte spheroids is considered to be very important in maintaining cell stability and liver-specific functions. In this study, we demonstrated that the highly differentiated functions could be expressed and structural organizations could be achieved by culturing hepatocytes in the GAC. The GAC may offer a promising alternative for the development of a high-grade cellbased biohybrid artificial liver. Furthermore, these results suggest that the specific interaction of hepatocytes with galactose moieties coupled with the GA plays an important role in maintaining the liver functions and promoting the structural organization of hepatocytes in the 3-D culture. Therefore, it may be expected that the GAC will be a promising scaffold for 3-D culture of hepatocytes, which can be used in cell biological studies of liver functions in vitro as well as in HAL.
4. Conclusion Galactose moieties as the ASGP-R ligands were coupled with alginate. Much more hepatocytes were attached onto GA than alginate and the morphologies of attached hepatocytes were correlated with the concentration of GA. Inhibition of hepatocytes attachment onto PVLA occurred with the addition of GA into the cell suspension. The hepatocytes in the GAC were observed to have, spheroids formation and improved liver functions than that in the AC. The GA may offer a promising scaffold for hepatocytes entrapment and transplantation.
References [1] Glicklis R, Shapiro L, Agbaria R, Merchuk JC, Cohen S. Hepatocyte behavior within three-dimensional porous alginate scaffolds. Biotechnol Bioengng 2000;67(3):344–53. [2] Gutsche AT, Lo H, Zurlo J, Yager J, Leong KW. Engineering of a sugar-derivatized porous network for hepatocyte culture. Biomaterials 1996;17:387–93. [3] Akaike T, Tobe S, Kobayashi A, Goto M, Kobayashi K. Design of hepatocyte-specific extracellular matrices for hybrid artificial liver. Gastroenterol Jpn 1993;28:45–52. [4] Skjaek-Break G. Alginates: biosynthess and structure-function relationships relevant to biomedical and biotechnological applications. Biochem Soc Trans 1992;20(1):27–33.
[5] Smidsrod O, Skjak-Braek G. Alginate as immobilization matrix for cells. TIBTECH 1990;8:71–8. [6] Lim F, Moss RD. Microencapsulation of living cells and tissues. J Pharm Sci 1981;70(4):351–4. [7] Dixit V, Arthur M, Gitnick G. A morphological and functional evaluation of transplanted isolated encapsulated hepatocytes following long-term transplantation in Gunn rats. Biomater Artif Cells Immob Biotech 1993;21(2):119–33. [8] Fremond B, Malandain C, Guyomard C, Chesne C, Guillouzo A, Campion JP. Correction of bilirubin conjugation in the Gunn rat using hepatocytes immobilized in alginate gel beads as an extracorporeal bioartificial liver. Cell Transplant 1993;2(6):453–60. [9] Yagi K, Tsuda K, Serada M, Yamada C, Kondoh A, Miura Y. Rapid formation of multicellular spheroids of adult rat hepatocytes by rotation culture and their immobilization within calcium alginate. Artif Organs 1993;17(11):929–34. [10] Smentana K. Cell biology of hydrogels. Biomaterials 1993; 14:1046–50. [11] Irwin M, Arias MD, editor-in chief. The liver: biology and pathobiology, 3rd ed. New York: Raven press, 1994. [12] Moghe PV, Berthiaume F, Ezzell RM, Toner M, Tompkins RG, Yarmush ML. Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function. Biomaterials 1996;17:373–85. [13] Mooney D, Hansen L, Vacanti J, Langer R, Farmer S, Inghber S, Ingber D. Switching from differentiation to growth in hepatocytes: control by extracellular matrix. J Cell Physiol 1992;151: 497–505. [14] Rowley JA, Madlabayan G, Mooney DJ. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 1999;20:45–53. [15] Lopina ST, Wu G, Merrill EW, Cima LG. Hepatocyte culture on carbohydrate-modified star polyethylene oxide hydrogels. Biomaterials 1996;17:559–69. [16] Matsuda T, Moghaddam MJ. Molecular design of three-dimensional artificial extracellular matrix: photosensitive polymers containing cell adhesive peptide. J Polym Sci Polym Chem 1993;31:1589–97. [17] Lee YC, Lee RT. Neoglycoconjugates: preparation and application. San Diego: Academic Press, 1994. [18] Weigel PH. Rat hepatocytes bind to synthetic galactoside surfaces via a patch of asialoglycoprotein receptors. J Cell Biol 1980;87(3 Pt 1):855–61. [19] Ashwell G, Harford A. Carbohydrate-specific receptors of the liver. Annu Rev Biochem 1982;51:531–54. [20] Kobayashi A, Akaike T, Kobayashi K, Sumitomo H. Enhanced division and survival efficiency of liver cells in culture dishes coated with a lactose carrying styrene homopolymer. Macromol Chem Rapid Commun 1986;7:645–50. [21] Tobe S, Takei Y, Kobayashi K, Akaike T. Receptor-mediated formation of multilayer aggregates of primary rat hepatocytes on lactose-substituted polystyrene. Biochem Biophys Res Commun 1992;184:225–30. [22] Tobe S, Takei Y, Kobayashi K, Akaike T. Effect of supplemented EGF on multiplayer aggregation in rat hepatocyte primary culture. Commun Appl Cell Biol 1990;8:16–22. [23] Kobatashi K, Sumitomo H, Ina Y. Synthesis and functions of polystyrene derivative having pendant oligosaccharides. Polym J 1985;17:567–75. [24] Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 1976;13:23–83. [25] Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 1980;210:908–10. [26] Kobayashi A, Goto M, Sekine T, Masumoto A, Yamamoto N, Kobayashi K, Akaike T. Regulation of differentiation and proliferation of rat hepatocytes by lactose-carrying polystyrene. Artif Organs 1992;16(6):564–7.
J. Yang et al. / Biomaterials 23 (2002) 471–479 [27] Goto M, Kobayashi A, kobayashi K, Saito K, Akaike T. Model study of hepatocyte targeting polymeric supper molecular assembly as drug carrier. Chem Lett 1992;123–6. [28] Goto M, Yura H, Chang CW, Kobayashi A, shinoda T, Maeda A, Kojima S, Kobayashi K, Akaike T. Lactose-carrying polystyrene as a drug carrying: investigation of body distributions to parenchyma liver cells using 125I-labelled lactose-carrying polystyrene. J Control Rel 1994;28:223–33. [29] Sun JH, Wang LSH, Waring MA, Wang AM, Woodman KK, Sheil AGR. Simple and reliable methods to assess hepatocyte viability in bioartificial liver support system matrices. Artif Organs 1997;21(5):408–13. [30] Ito Y, Chang TMS. In vitro study of multicellular hepatocyte spheroids formed in microcapsules. Artif Organs 1992;16(4): 422–7.
479
[31] Rouiller DG, Cirulli V, Halban PA. Differences in aggregation properties and levels of the neural cell adhesion molecule (NCAM) between islet types. Exp Cell Res 1990;191:305–12. [32] Knudsen KA, Myers L, McElwee SA. A role for the Ca2+dependent adhesion molecule, N-Cadherin in myoblast interaction during myogenesis. Exp Cell Res 1990;188:175–84. [33] Surapaneni S, Pryor T, Klein MD, Matthew HWT. Rapid hepatocyte spheroid formation: optimization and long-term function in perfused microcapsules. ASAIO J 1997;43(5):M848–53. [34] Clayton DF, Harrelson AL, Darnell Jr JE. Dependence of liverspecific transcription on tissue organization. Mol Cell Biol 1985;5:2623–32. [35] Wu FJ, Friend JR, Remmel RP, Cerra FB, Hu WS. Enhance cytochrome P450 IA1 activity of self-assembled rat hepatocyte spheroids. Cell Transplant 1999;8(3):233–46.
Galactosylated alginate as a scaffold for hepatocytes entrapment Jun Yanga, Mitsuaki Gotob, Hirohiko Isec, Chong-Su Chod, Toshihiro Akaikea,c,* a
Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan b Bio Quest Research Co. Ltd., Tokyo 150-0043, Japan c Institute of Organ Transplants, Reconstructive Medicine and Tissue Engineering, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto 390-8621, Japan d School of Agricultural Biotechnology, Seoul National University, Suwon 441-744, South Korea Received 10 September 2000; accepted 26 March 2001
Abstract Galactose moieties were covalently coupled with alginate through ethylenediamine as the spacer for enhancing the interaction of hepatocytes with alginate. Adhesion of hepatocytes onto the galactosylated alginate (GA)-coated polystyrene (PS) surface showed an 18-fold increase as compared with that of the alginate-coated surface and it increased with an increase in the concentration of GA. The morphologies of attached hepatocytes were observed to spread out at the 0.15 wt% GA-coated PS surface while round cells were observed at the 0.5 wt% GA-coated PS surface. Inhibition of hepatocytes attachment onto the galactose-carrying PS-coated surface occurred with the addition of the GA into the hepatocyte suspension, indicating the binding of GA with hepatocytes via the patch of asialoglycoprotein receptors. Primary hepatocytes were entrapped in the GA/Ca2+ capsules (GAC). Higher cell viability and more spheroid formation of hepatocytes were obtained in the GAC than in the alginate/Ca2+ capsules (AC). Moreover, liver functions of the hepatocytes such as albumin secretion and urea synthesis in the GAC were improved in comparison with those in the AC. r 2001 Elsevier Science Ltd. All rights reserved. Keywords: Alginate; Synthetic scaffold; Galactose; Hepatocytes; Bioartificial liver; Capsules
1. Introduction In recent years, many attempts to develop a highgrade cell-based hybrid artificial liver (HAL) have been focused on immobilizing primary hepatocytes in scaffolds which can be designed to mimic a physiological microenvironment essential for controlling the growth, differentiation of hepatocytes and supporting reconstruction of the liver in vitro [1–3]. Moreover, with the elucidation of receptor-mediated cell functions and the progress in biomaterial science, developments of synthetic extracellular matrices to replace the natural extracellular matrices (ECMs) are increasingly essential and promising in tissue engineering. Alginate obtained from brown seaweed as a heteropolysaccharide has been extensively used as a gelling *Corresponding author. Department of Biomolecular Engineering, Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, Japan. Tel.: +81-45-924-5790; fax:+81-45924-5815. E-mail address: [email protected] (T. Akaike).
agent in many biotechnological and medical applications [4]. It is a block copolymer composed of 1,4-linked b-d mannuronic acid (M) and a-l-guluronic acid (G) residues and the G-block can rapidly be crossed by a divalent cation such as calcium to form hydrogels at room temperature and neutral pH. Therefore, recently, alginate was also used as a three-dimensional synthetic ECM (3D-ECM) for immobilization of cells in tissue engineering [5]. In addition, Lim et al. reported that encapsulation of cells into alginate capsules (AC) could produce a very high-density cell culture system with mechanical support to the cells as well as immunoprotection in the event of implantation [6]. Experimentally, hepatocytes encapsulated in alginate have been used in the treatment of acute liver failure and congenital liver enzyme deficiencies such as bilirubin-diphosphate deficiency of the liver (Gunn rat) [7,8]. However, hepatocytes encapsulated in AC are of low viability and do not maintain the long-term liver functions due to the lack of cell–matrix and cell–cell interactions within AC [9,10]. For an anchorage-sensitive cell such as hepatocyte, cell anchorage and cell aggregation regulate the cell
0142-9612/02/$ - see front matter r 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 1 4 2 - 9 6 1 2 ( 0 1 ) 0 0 1 2 9 - 6
472
J. Yang et al. / Biomaterials 23 (2002) 471–479
functions in vitro and in vivo [11]. Additionally, these are closely correlated with the ECMs [12,13]. Therefore, a major challenge of encapsulated hepatocytes for HAL is to provide an optimum 3D-ECM in capsules, which replace many functions of the native ECMs for adjusting the interaction of hepatocytes with the scaffold and promoting hepatocytes to organize into a threedimensional architecture while enhancing cellular functions in capsules. Recently, many researchers have shown that synthetic ECMs with covalent coupling of cell-specific ligands or extracellular signaling molecules have advantages of both enhancing the interaction of cell with biomaterials and controlling the growth, differentiation and behavior of cells in cultures [14–17]. Rowley et al. reported that alginate modified with RGD-containing cell adhesion ligands promoted the adherence of myoblasts and the expression of a differentiated phenotype [14]. Also, it was reported that galactose moieties were recognized by asialoglycoprotein receptors (ASGP-R) on hepatocyte surfaces and promoted the hepatocytes adhesion [17– 20]. In two-dimensional culture, we previously reported [3,17,20–22] that adult rat hepatocytes attached well on the galactose-carrying PS (poly-N-p-vinylbenzyl-d-lactoneamide, abbreviated as PVLA)-coated surface via the patch of ASGP-R. Furthermore, hepatocytes attached onto PVLA were changed from spreading to round shapes with an increase of PVLA concentration and maintained the differentiated functions with promoting spheroids formation, which is in contrast to the tendency of natural ECM such as collagen and fibronectin [20–22]. In this study, we modified alginate with galactose moieties as ASGP-R ligands to improve the hepatocytes anchorage and the interaction of hepatocytes with alginate, and then enhance the liver functions of encapsulated hepatocytes in three-dimensional culture. Galactosylated alginate (GA) was synthesized utilizing aqueous carbodimide chemistry and the specific interaction between primary mouse hepatocytes and GA was investigated. Also, hepatocytes were entrapped in the GA/Ca2+ capsule (GAC). Furthermore, the liver functions and the spheroids formation of hepatocytes were observed in the GAC. The GA may present an efficient synthetic scaffold and the GAC may offer a promising alternative for the development of a HAL.
2. Materials and methods 2.1. Materials Sodium alginate from macrocystis pyrifera and polyl-lysine (PLL, MW 22,000) was purchased from Sigma (St. Louis, MO). Lactobionic acid was purchased from TCI (Tokyo, Japan). Sulfo-NHS was purchased from
Fluka (Chemika-Biochemika Analytika, Tokyo, Japan). All other chemicals were purchased from Wako Pure Chemical Industries Ltd. (Osaka, Japan) unless otherwise stated. PVLA was synthesized by a similar method previously reported [23]. 2.2. Coupling of galactose moieties with alginate Firstly, primary amine was introduced into lactobionic acid. Briefly, lactobionic lactone prepared by dehydration of lactobionic acid was refluxed with 30fold excess ethylenediamine dissolved in anhydrous dimethyl sulfoxide (DMSO) at 701C for 2 h. The monoamine terminated lactobionic lactone (L-NH2) was precipitated with chloroform and the obtained precipitate was vacuum-dried. Then, 0.8 g of the L-NH2 (2 mmol) was reacted with 1 g of alginate (5 mmol) dissolved in 100 ml of 50 mm TEMED (N0 ,N0 ,N0 -tetramethylethylenediamine, with 0.3 m NaCl, pH 6.5) for 24 h at room temperature using 1-ethyl-(dimethylaminopropyl) carbodiimide (EDC) and a co-reactant Nhydroxy-sulfosuccinimide (sulfo-NHS) as the activation agents. The obtained GA was purified by dialysis against Milli Q for one week and lyophilized. The reaction scheme is shown in Fig. 1. Maltose-modified alginate (MA) was also synthesized by this method. 2.3. Cell preparation Primary hepatocytes were isolated from an ICR mouse (5–7 weeks old, male) (SLC, Shizuoka, Japan) by the modified in situ perfusion method as described previously [24]. The dead parenchyma hepatocytes were removed by density gradient centrifugation in Percoll (Pharmacia, Piscataway, NJ, USA). The viable primary hepatocytes were suspended in William’s E medium (Gibco BRL, New York, USA) that contained antibiotics (50 mg penicillin/ml, 50 mg streptomycin/ml and 100 mg neomycin/ml) and HEPES (18 mm). Only isolated hepatocytes with greater than 90% viability by trypan blue dye exclusion were used. 2.4. Hepatocyte attachment One ml of PVLA (100 mg/ml), GA (0.05, 0.15 or 0.5 wt%) or MA (0.15%) was placed in a 35 mm PS dish (Falcon, 1008) at 371C overnight. The solution was decanted and the surface was rinsed 3 times with William’s E medium. The isolated mouse hepatocytes were cultured in a quantity of 4 104 cells/cm2 at 371C in a humidified air/CO2 incubator (95/5 vol%) for 30 min, and the non-adhered cells were collected by decantation and washing the dish with 1 ml medium. The non-adhered hepatocytes were counted with the Sysmex cell counter. The area of adhered hepatocyte on the coated surfaces was measured by OLYMPUS IX70
J. Yang et al. / Biomaterials 23 (2002) 471–479
473
Fig. 1. Reaction scheme of galactosylated alginate.
and KEYENCE VQ-7000. The average area was calculated with 100 cells from six experiments on every surface. The cells having an average area larger than 600 mm2/cell were classified as spread cells, while those less than 500 mm2/cell were classified as round cells (the average area of a freshly isolated mouse hepatocyte was 396720 mm2). 2.5. Inhibition of hepatocyte attachment onto PVLA by GA After incubation of hepatocytes in WE medium containing GA (0.15 wt%), alginate (0.15 wt%) or PVLA (100 mg/ml) at 371C for 30 min in an incubator, the incubated hepatocytes were seeded into the PVLAcoated dish and incubated again at 371C for 1 h in a humidified air/CO2 incubator (95/5 vol%). The nonadhesive hepatocytes were collected and counted. As a control, the same experiment was performed with a collagen-coated dish. 2.6. Encapsulation of hepatocytes Isolated hepatocytes were encapsulated in GAC or AC by the method described by Lim et al. [25]. Briefly, isolated hepatocytes were suspended in sodium alginate
(2 wt%) or a mixture of alginate and GA (1 : 1, w/w) at a cell density of 8 105 cells/ml. The cells/alginate (or GA) suspension was dropped into CaCl2 solution (100 mm, pH 7.4) to form alginate gel beads. The alginate beads were coated with PLL (0.05 wt %) and alginate (0.2 wt%) to form an alginate–PLL–alginate (APA) membrane. Then, treatment of coated beads by sodium citrate (50 mm) was carried out to liquefy the alginate (or GA) gel inside the APA membrane, which enhances the permeability of capsules. Next, 1 ml of hepatocyte capsules was cultured with 3 ml William’s E medium supplemented antibiotics (50 mg penicillin/ml, 50 mg streptomycin/ml and 100 mg neomycin/ml), HEPES (18 mm), EGF (20 ng/ml) and insulin (100 nm) in a 60 mm PS dish at 371C in a humidified air/CO2 incubator (95/5 vol%). The medium was changed every day and the collected medium was stored for biochemical assays. 2.7. Measurement of hepatocytes functions The amounts of albumin in the medium were determined by a sandwich enzyme-linked immunosorbent assay (ELISA) using rabbit anti-mouse albumin serum (Inter-cell Technologies Inc., Hopewell, NJ, USA) and sheep anti-mouse albumin polyclonal
474
J. Yang et al. / Biomaterials 23 (2002) 471–479
antibody (The Binding Site Ltd., Birmingham, England). The secondary antibody was peroxidase-conjugated anti-rabbit IgG (Seikagaku Co., Tokyo, Japan). Orthophenylenediamine (OPD) (Wako Pure Chemical Industries Ltd., Osaka, Japan) solution (0.4 mg/ml OPD and 0.4 ml/ml H2O2 in citrate–phosphate buffer, pH 5.0) was used as a substrate of peroxidase. Absorbency at 492 nm was measured by a microplate reader (MTP-32, Corona Electric Co., Ibaraki, Japan). The urea concentration was quantified with a Wako Urea N B kit (Code No. 279-36201). 2.8. Viability test by Alamer Blue staining of hepatocytes Hepatocytes in capsules were stained with 10% (v/v) Alamer Blue (Iwaki Glass Co., Tokyo, Japan). Alamer Blue stains only viable cells with red by reductive reaction with mitochondria. Alamer Blue staining was done at 371C for 4 h. The stained hepatocytes in GAC were observed with a confocal laser-scanning microscope (Leica, Germany).
3. Results and discussion 3.1. Synthesis of GA Galactose moieties were coupled to alginate according to the reaction scheme shown in Fig. 1. The first step was the introduction of ethylenediamine into lactobionic acid by reaction of lactobionic lactone dehydrated from lactobionic acid with 30-fold excess of ethylenediamine. The purity of the product was checked by thin-layer chromatography and showed no detectable free ethylenediamine in it. The Rf values for L-NH2 and ethylenediamine were 0.51 and 0.23, respectively. The second step was the coupling of the L-NH2 into the alginate using EDC and sulfo-NHS as activation agents. The GA was analyzed with NMR; however, the peaks of galactose residue in the GA were difficult to be assigned due to the overlapping with those of alginate. The content of galactose moieties in the GA was evaluated by element analysis of carbon, hydrogen and nitrogen content and it showed that 2675% of uronic acid in alginate reacted with galactose moieties. 3.2. Adhesion of hepatocytes onto GA-coated surfaces Freshly isolated mouse hepatocytes were cultured on the GA-coated PS dish to check the specific interaction of hepatocytes with GA. Attachment of hepatocytes onto the alginate- or GA-coated PS surface for 30 min at 371C is shown in Fig. 2. The results indicated that 55% of hepatocytes were attached onto 0.15 wt % GA-coated surface while only 3% of hepatocytes were attached onto 0.15 wt% alginate-coated surface. Almost similar
Fig. 2. Attachment of hepatocytes onto alginate (A)-, GA- or MAcoated PS surface for 30 min at 371C in WE medium.
amounts of hepatocytes as alginate were attached onto the MA-coated surface, indicating that the enhancing of attachment of hepatocytes to GA was caused by the galactose residue coupled to alginate. Also, the sugar residues on the GA-coated plate were examined by a direct lectin–enzyme assay. The results showed that the density of galactose residues on GA-coated surface increased as the concentration of GA increased. Furthermore, the cell attachment increased with an increase of GA concentration as shown in Fig. 2, suggesting that the cell binding was dependent on the density of galactose residues on the GA-coated surfaces. The mean cell areas measured by image analysis of microscopy after 4 h were 1279738, 456724, 779718 and 493715 mm2 for PS surface, 0.05, 0.15 and 0.5 wt% GA-coated surfaces, respectively. The cells having an average area larger than 600 mm2/cell were classified as spread cells whereas those less than 500 mm2/cell were classified as round cells. The morphologies of attached hepatocytes after 4 h were round, spread and round on the surface coated with 0.05, 0.15 and 0.5 wt% GA, respectively, as shown in Fig. 3. The results indicated that the adhesion and shapes of hepatocytes were closely related with the concentration of GA. On the 0.05 wt% GA-coated surface, the spherically adhered hepatocytes were observed to be obviously different from the spreading ones on the PS surfaces (Fig. 3A). It is thought that the coating with 0.05 wt% GA was sufficient to inhibit the hydrophobic binding of hepatocytes to the PS surface. However, the galactose residues on the 0.05 wt% GA-coated surface were too sparse to mediate cell spreading. The results of increased cell attachment and decreased spreading of hepatocytes on increasing the concentration of GA from 0.15 to 0.5 wt% might be a sign of regulating the behavior of hepatocytes by the density of galactose residues in the
J. Yang et al. / Biomaterials 23 (2002) 471–479
475
Fig. 3. Phase-contrast micrographs of hepatocytes cultured on PS surface (A), surface coated with 0.15% A (B), 0.05% GA (C), 0.15% GA (D) and 0.5% GA (E). The cells were cultured in WE medium for 4 h.
Fig. 4. Phase-contrast micrographs of hepatocytes cultured on 0.15 wt% GA-coated PS surface after 1 day (A, D), 3 days (B, E) and 5 days (D, F). 20 ng EGF/ml and 100 nm insulin were supplemented in (D–F).
initial adhesion, which is similar to the tendency reported by Kabayashi et al. [26]. Also, it is thought that the receptors on the hepatocytes were pulled together on the surface containing dense clusters of galactose (0.5 wt% GA), resulting in round cells, whereas the receptors were spread on the surface containing sparse clusters of galactose (0.15 wt% GA), resulting in spread cells [26]. Furthermore, when hepatocytes were cultured on 0.15 wt% GA-coated dish for 5 days, the attached hepatocytes aggregated to form multilayer spheroids on adding EGF (20 ng/ml) and
insulin (100 nm) into the WE medium as shown in Fig. 4. It is suggested that the spheroid formation of hepatocytes depended on the concentration of GA and growth factors such as EGF and insulin. 3.3. Inhibition of hepatocytes attachment onto PVLA by GA The specific interaction of hepatocytes with galactose moieties coupled with GA was further evaluated with checking the inhibition of hepatocyte attachment onto
476
J. Yang et al. / Biomaterials 23 (2002) 471–479
reorganization of ASGP-R with galactose moieties covalently coupled with GA enhanced the interaction of hepatocytes with GA. 3.4. Encapsulation of hepatocytes
Fig. 5. Inhibition of hepatocytes attachment onto PVLA- or collagencoated PS surface by adding PVLA (100 mg/ml), alginate (0.15 wt%) (A), GA (0.15 wt%) or MA (0.15 wt %) into hepatocytes suspension for 1 h at 371C.
PVLA by adding the GA into a hepatocyte suspension (Fig. 5). We observed that the adhesion of hepatocytes onto PVLA showed a greater inhibition by the GA compared with that of the alginate. In contrast, addition of the GA into hepatocytes suspension did not inhibit hepatocytes attachment onto the collagen (Fig. 5). PVLA was previously reported to have highly specific affinity to hepatocytes via the specific recognition of galactose moieties with the ASGP-R on hepatocytes in vitro and in vivo [27,28]. The inhibition of hepatocyte attachment showed the competition between the binding of GA and binding of PVLA to hepatocytes, indicating that the galactose moieties coupled with GA were also recognized by the ASGP-R on hepatocytes. Weigel et al. also reported that rat hepatocytes adhere specifically to galactosyl polyacrylamide gel via a patch of ASGP-R [18]. Therefore, it can be said that hepatocytes attach onto GA via the patch of ASGP-R, and the specific
The cell viability estimated by the MTT method [29] immediately after encapsulating the cells into the GAC was 8177% of the original number of cells. Encapsulation with AC showed the same results. In addition, the GAC observed by microscopy showed almost spherical shapes without roughness or irregularities as same as the shapes of AC. From the observation by phase-contrast microscopy, the hepatocyte structures in the GAC were different from those in the AC after 48 h as shown in Fig. 6. Within GAC, more than 80% of the hepatocytes were aggregated to form multicellular spheroids with diameters enlarged up to above 100 mm, and the spheroids were uniformly distributed in each GAC. The tendency of spheroid formation within the GAC was improved by supplementation with EGF and insulin. This is consistent with the hepatocytes cultured on the GA-coated surface, indicating that EGF and insulin enhanced the mobility of attached hepatocytes in GAC. However, most hepatocytes remained as single cells and only a few cells formed aggregates within the AC. Most of the cells were located in the boundary area around the APA membrane. The compressive moduli of alginate and GA gel beads were 10.9371.99 and 6.0172.53 kPa, respectively, an indication of not much difference of compressive modulus between them. Also, the change of cell distribution in AC and GAC after 48 h as shown in Fig. 6 suggested that the mobility of hepatocytes in AC was almost the same as that in GAC. Therefore, it may be said that the specific interaction of ASGP-R on the hepatocytes with the galactose ligands in GA for anchorage sites is closely related to the spheroid formation in three-dimensional
Fig. 6. Phase-contrast micrographs of encapsulated hepatocytes in AC (A) and GAC (B) cultured in WE medium with EGF and insulin for 2 days.
J. Yang et al. / Biomaterials 23 (2002) 471–479
477
Fig. 8. Albumin secretion (A) and urea synthesis (B) of encapsulated hepatocytes in AC and GAC. The encapsulated hepatocytes were cultured in WE medium with EGF and insulin.
Fig. 7. Confocal micrographs of hepatocyte aggregates in GAC (20 days culture) stained with Alamer Blue; whole image (top) and crosssection images from top to bottom (bottom).
culture instead of differences of physical changes between AC and GAC. It has already been reported that the mobility of intra-capsular hepatocytes is dependent on the viscosity inside microcapsules [30]. Also, it was found that cell adhesion molecules termed as N-Cadherin, A-CAM or N-CAM played an important role in cell interactions of different cell types [31,32]. However, little is known about the exact mechanism of spheroid formation of hepatocytes in 3-D cultures. In order to investigate the viability of hepatocytes in the spheroid, we stained hepatocytes cultured in the GAC for 20 days with Alamer Blue and observed the inner part of hepatocytes aggregates using confocal microscopy as shown in Fig. 7. The internal hepatocytes of the spheroid could be stained with Alamer Blue, indicating that the hepatocytes were alive in the spheroid formed in the GAC. Also, more cell viability within the GAC was obtained than that of the AC. It
was reported that the spheroid formation of hepatocytes was a necessary requirement for cell viability and improvement of cell differentiated functions [33]. A comparison of albumin secretion and urea synthesis of the encapsulated hepatocytes between the AC and the GAC is shown in Fig. 8. The results indicated that the albumin secretion and urea synthesis of the AC rapidly decreased with culture time whereas those of the GAC slowly decreased and maintained higher levels than those of the AC. Additionally, as controls, MA and alginate treated with WSC and sulfo-NHS without galactose were also synthesized by the same method. The results showed that much adhesion of hepatocytes, inhibition of hepatocytes attachment to PVLA, the promoted spheroids formation and enhanced cellular functions in capsules were not found. This suggested that the hepatocytes behaviors were enhanced by incorporated galactose moieties with alginate, but not simply by modifying with another sugar. Therefore, it may be regarded that the galactose moieties in the GA provided anchorage sites for hepatocytes in GAC and improved the formation of hepatocyte spheroids, and then enhanced the liver functions of encapsulated hepatocytes. Clayton et al. reported that tissue-specific
478
J. Yang et al. / Biomaterials 23 (2002) 471–479
transcription was maintained at a high rate when hepatocytes were cultured as slices of tissue in which cell contact and cell architecture were maintained [34]. Wu et al. also reported that hepatocyte spheroids maintained higher P450 activity than monolayer on collagen and demonstrated that the higher P450 activity within spheroids was associated with their ability to maintain a greater degree of differentiation compared to the monolayer [35]. The biological tissue organization or three-dimensional structure of hepatocyte spheroids is considered to be very important in maintaining cell stability and liver-specific functions. In this study, we demonstrated that the highly differentiated functions could be expressed and structural organizations could be achieved by culturing hepatocytes in the GAC. The GAC may offer a promising alternative for the development of a high-grade cellbased biohybrid artificial liver. Furthermore, these results suggest that the specific interaction of hepatocytes with galactose moieties coupled with the GA plays an important role in maintaining the liver functions and promoting the structural organization of hepatocytes in the 3-D culture. Therefore, it may be expected that the GAC will be a promising scaffold for 3-D culture of hepatocytes, which can be used in cell biological studies of liver functions in vitro as well as in HAL.
4. Conclusion Galactose moieties as the ASGP-R ligands were coupled with alginate. Much more hepatocytes were attached onto GA than alginate and the morphologies of attached hepatocytes were correlated with the concentration of GA. Inhibition of hepatocytes attachment onto PVLA occurred with the addition of GA into the cell suspension. The hepatocytes in the GAC were observed to have, spheroids formation and improved liver functions than that in the AC. The GA may offer a promising scaffold for hepatocytes entrapment and transplantation.
References [1] Glicklis R, Shapiro L, Agbaria R, Merchuk JC, Cohen S. Hepatocyte behavior within three-dimensional porous alginate scaffolds. Biotechnol Bioengng 2000;67(3):344–53. [2] Gutsche AT, Lo H, Zurlo J, Yager J, Leong KW. Engineering of a sugar-derivatized porous network for hepatocyte culture. Biomaterials 1996;17:387–93. [3] Akaike T, Tobe S, Kobayashi A, Goto M, Kobayashi K. Design of hepatocyte-specific extracellular matrices for hybrid artificial liver. Gastroenterol Jpn 1993;28:45–52. [4] Skjaek-Break G. Alginates: biosynthess and structure-function relationships relevant to biomedical and biotechnological applications. Biochem Soc Trans 1992;20(1):27–33.
[5] Smidsrod O, Skjak-Braek G. Alginate as immobilization matrix for cells. TIBTECH 1990;8:71–8. [6] Lim F, Moss RD. Microencapsulation of living cells and tissues. J Pharm Sci 1981;70(4):351–4. [7] Dixit V, Arthur M, Gitnick G. A morphological and functional evaluation of transplanted isolated encapsulated hepatocytes following long-term transplantation in Gunn rats. Biomater Artif Cells Immob Biotech 1993;21(2):119–33. [8] Fremond B, Malandain C, Guyomard C, Chesne C, Guillouzo A, Campion JP. Correction of bilirubin conjugation in the Gunn rat using hepatocytes immobilized in alginate gel beads as an extracorporeal bioartificial liver. Cell Transplant 1993;2(6):453–60. [9] Yagi K, Tsuda K, Serada M, Yamada C, Kondoh A, Miura Y. Rapid formation of multicellular spheroids of adult rat hepatocytes by rotation culture and their immobilization within calcium alginate. Artif Organs 1993;17(11):929–34. [10] Smentana K. Cell biology of hydrogels. Biomaterials 1993; 14:1046–50. [11] Irwin M, Arias MD, editor-in chief. The liver: biology and pathobiology, 3rd ed. New York: Raven press, 1994. [12] Moghe PV, Berthiaume F, Ezzell RM, Toner M, Tompkins RG, Yarmush ML. Culture matrix configuration and composition in the maintenance of hepatocyte polarity and function. Biomaterials 1996;17:373–85. [13] Mooney D, Hansen L, Vacanti J, Langer R, Farmer S, Inghber S, Ingber D. Switching from differentiation to growth in hepatocytes: control by extracellular matrix. J Cell Physiol 1992;151: 497–505. [14] Rowley JA, Madlabayan G, Mooney DJ. Alginate hydrogels as synthetic extracellular matrix materials. Biomaterials 1999;20:45–53. [15] Lopina ST, Wu G, Merrill EW, Cima LG. Hepatocyte culture on carbohydrate-modified star polyethylene oxide hydrogels. Biomaterials 1996;17:559–69. [16] Matsuda T, Moghaddam MJ. Molecular design of three-dimensional artificial extracellular matrix: photosensitive polymers containing cell adhesive peptide. J Polym Sci Polym Chem 1993;31:1589–97. [17] Lee YC, Lee RT. Neoglycoconjugates: preparation and application. San Diego: Academic Press, 1994. [18] Weigel PH. Rat hepatocytes bind to synthetic galactoside surfaces via a patch of asialoglycoprotein receptors. J Cell Biol 1980;87(3 Pt 1):855–61. [19] Ashwell G, Harford A. Carbohydrate-specific receptors of the liver. Annu Rev Biochem 1982;51:531–54. [20] Kobayashi A, Akaike T, Kobayashi K, Sumitomo H. Enhanced division and survival efficiency of liver cells in culture dishes coated with a lactose carrying styrene homopolymer. Macromol Chem Rapid Commun 1986;7:645–50. [21] Tobe S, Takei Y, Kobayashi K, Akaike T. Receptor-mediated formation of multilayer aggregates of primary rat hepatocytes on lactose-substituted polystyrene. Biochem Biophys Res Commun 1992;184:225–30. [22] Tobe S, Takei Y, Kobayashi K, Akaike T. Effect of supplemented EGF on multiplayer aggregation in rat hepatocyte primary culture. Commun Appl Cell Biol 1990;8:16–22. [23] Kobatashi K, Sumitomo H, Ina Y. Synthesis and functions of polystyrene derivative having pendant oligosaccharides. Polym J 1985;17:567–75. [24] Seglen PO. Preparation of isolated rat liver cells. Methods Cell Biol 1976;13:23–83. [25] Lim F, Sun AM. Microencapsulated islets as bioartificial endocrine pancreas. Science 1980;210:908–10. [26] Kobayashi A, Goto M, Sekine T, Masumoto A, Yamamoto N, Kobayashi K, Akaike T. Regulation of differentiation and proliferation of rat hepatocytes by lactose-carrying polystyrene. Artif Organs 1992;16(6):564–7.
J. Yang et al. / Biomaterials 23 (2002) 471–479 [27] Goto M, Kobayashi A, kobayashi K, Saito K, Akaike T. Model study of hepatocyte targeting polymeric supper molecular assembly as drug carrier. Chem Lett 1992;123–6. [28] Goto M, Yura H, Chang CW, Kobayashi A, shinoda T, Maeda A, Kojima S, Kobayashi K, Akaike T. Lactose-carrying polystyrene as a drug carrying: investigation of body distributions to parenchyma liver cells using 125I-labelled lactose-carrying polystyrene. J Control Rel 1994;28:223–33. [29] Sun JH, Wang LSH, Waring MA, Wang AM, Woodman KK, Sheil AGR. Simple and reliable methods to assess hepatocyte viability in bioartificial liver support system matrices. Artif Organs 1997;21(5):408–13. [30] Ito Y, Chang TMS. In vitro study of multicellular hepatocyte spheroids formed in microcapsules. Artif Organs 1992;16(4): 422–7.
479
[31] Rouiller DG, Cirulli V, Halban PA. Differences in aggregation properties and levels of the neural cell adhesion molecule (NCAM) between islet types. Exp Cell Res 1990;191:305–12. [32] Knudsen KA, Myers L, McElwee SA. A role for the Ca2+dependent adhesion molecule, N-Cadherin in myoblast interaction during myogenesis. Exp Cell Res 1990;188:175–84. [33] Surapaneni S, Pryor T, Klein MD, Matthew HWT. Rapid hepatocyte spheroid formation: optimization and long-term function in perfused microcapsules. ASAIO J 1997;43(5):M848–53. [34] Clayton DF, Harrelson AL, Darnell Jr JE. Dependence of liverspecific transcription on tissue organization. Mol Cell Biol 1985;5:2623–32. [35] Wu FJ, Friend JR, Remmel RP, Cerra FB, Hu WS. Enhance cytochrome P450 IA1 activity of self-assembled rat hepatocyte spheroids. Cell Transplant 1999;8(3):233–46.