Hepatocyte Behavior Within Three-Dimensional Porous Alginate Scaffolds

Rachel Glicklis,1 Lilia Shapiro,1 Riad Agbaria,2 Jose C. Merchuk,1,3 Smadar Cohen1
1 Unit Biotechnology, Faculty of 2 Department of Pharmacology, 3

Engineering Sciences Faculty of Health Sciences Department of Chemical Engineering, Faculty of Engineering Sciences Ben-Gurion University of the Negev, Beer-Sheva, Israel; telephone: +972-7-646-1798; fax: +972-7-647-2915; e-mail: scohen@bgumail.bgu.ac.il

Received 24 March 1999; accepted 12 September 1999

Abstract: A potential approach to facilitate the performance of implanted hepatocytes is to enable their aggregation and re-expression of their differentiated function prior to implantation. Here we examined the behavior of freshly isolated rat adult hepatocytes seeded within a novel three-dimensional (3-D) scaffold based on alginate. The attractive features of this scaffold include a highly porous structure (sponge-like) with interconnecting pores, and pore sizes with diameters of 100150 m. Due to their hydrophilic nature, seeding hepatocytes onto the alginate sponges was efficient. DNA measurements showed that the total cell number within the sponges did not change over 2 weeks, indicating that hepatocytes do not proliferate under these culture conditions. Nearly all seeded cells maintained viability, according to the MTT assay. Within 24 h post-seeding, small clusters of viable cells, were seen scattered within the sponge. More than 90% of the seeded cells participated in the aggregation; the high efficiency is attributed to the non-adherent nature of alginate. The spheroids had smooth boundaries and by day 4 in culture reached an average diameter of 100 m, which is at the same magnitude of the sponge pore size. The cells appeared to synthesize fibronectin which was deposited on the spheroids. No laminin or collagen type IV were detected in the deposit. The 3-D arrangement of hepatocytes within the alginate sponges promoted their functional expression; within a week the cells secreted the maximal albumin secretion rate of 60 g albumin/106 cells/day. Urea secretion rate did not depend on cell aggregation and was similar to that obtained when hepatocytes were cultured on collagen type I coated dishes (100 g/106 cells/day). Our studies show that alginate sponges can provide a conducive environment to facilitate the performance of cultured hepatocytes by enhancing their aggregation. 2000 John Wiley &
Sons, Inc. Biotechnol Bioeng 67: 344353, 2000.

Keywords: alginate scaffolds; hepatocyte aggregation; 3-D hepatocyte culture; tissue engineering

INTRODUCTION Tissue engineering is an emerging scientific approach that attempts to develop biological substitutes from isolated cells

Correspondence to: Smadar Cohen

and three-dimensional (3-D) polymeric scaffolds (Langer and Vacanti, 1993). Because the tissue-engineered constructs contain living cells, they may have the potential for growth and cell self-repair. The living cells may also provide the profile of growth factors and signals required for their integration into the host and wound healing. In this construct, the primary role of the 3-D polymer scaffold is to provide a temporary template with the biomechanical structural characteristics of the native extracellular matrix (ECM) until the cells produce their own. Thus, selecting the material for such a scaffold is critically important for the success of tissue engineering. We are interested in developing 3-D scaffolds that are suitable for hepatic tissue engineering. To this end, the research on 3-D scaffolds for hepatocyte culture and transplantation has been largely confined to designing matrices made of synthetic polymers such as poly(lactide-coglycolide) (PLGA) (Mikos et al., 1993, 1994), or from the natural ECM components, such as the two-layer collagen gels (Dunn et al., 1991) and EHS gels (ECM prepared from the Engelberth-Holm Swarm mouse sarcoma) (Bissell et al., 1990; Moghe et al., 1996). Hepatocytes cultured within the PLGA scaffolds (foams or sponges) retained viability; however, their liver-specific gene expression was limited (Kaufmann et al., 1997). In contrast, hepatocytes seeded within the natural ECM-derived scaffolds maintained their differentiated function for prolonged times (Bader et al., 1992; Dunn et al., 1991). A major limitation of ECM-derived systems, in particular those made from EHS, is their potential antigenicity and tumoregenicity when the material is implanted into a host. Our group has recently developed a 3-D porous scaffold based on the polysaccharide alginate from brown seaweeds (Shapiro and Cohen, 1997). Alginate, the monovalent salt form of alginic acid is a block copolymer composed of 1,4-linked -D-mannuronic (M) and -L-guluronic acid (G) residues in a linear polysaccharide chain. The two monomers exist as chains composed of one unit or the other, referred to as M-blocks or G-blocks, or as chains of the two

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monomers in an alternating sequence. The G-block regions can dimerize to form hydrogels in the presence of aqueous divalent cations, such as calcium, barium, and strontium, at room temperature (Grant et al., 1973). Because of the allaqueous method of preparation, alginate matrices are widely investigated for many biotechnological and medical applications. Furthermore, alginate is a biocompatible material (Klo ck et al., 1994; Kulseng et al., 1999), and has been approved by the regulatory authorities as a wound dressing, for dental impression and as a food supplement. In addition to the material properties, the morphology and microstructure of the 3-D scaffold are crucially important as they may determine the nature of the engineered tissue. The alginate scaffolds have been designed to be highly porous (sponge-like), with interconnecting pores, and large pore size (100 m) (Shapiro and Cohen, 1997). These scaffolds should allow the cultivation of a large cell mass, the rearrangement of dispersed cells into a functioning tissue, and following implantation, they may enable a close interaction and metabolite transfer between the transplanted engineered tissue and the host. In the present paper, we characterize the behavior of rat adult hepatocytes within the newly designed alginate sponges, in aim to evaluate the culture conditions needed for these cells to fully express their differentiated function. Several criteria were used to assess their behavior within the alginate sponges such as cell longevity and distribution, cellmatrix interactions, cell aggregation, and hepatospecific function. Our studies demonstrated that hepatocytes maintain their viability when seeded within the alginate sponges but that they do not proliferate. The seeded hepatocytes efficiently aggregated into spheroids with an average diameter of 100 m, matching that of the sponge pore size. It appears that the aggregation is induced by the nonadherent nature of alginate. It took the isolated hepatocytes 7 days to reach their maximal albumin secretion rates, which is the time period needed for spheroid maturation. Urea secretion rates, on the other hand, were not affected by cell aggregation and resembled those from cell monolayers on collagen-coated dishes. MATERIALS AND METHODS Preparation of Alginate Sponges The sponges were prepared from high-G alginate (Protanal LF 120, 65% G, Pronova Biopolymer, Drammen, Norway) by the gelationfreeze technique (Shapiro and Cohen, 1997), which consists of the following steps: (i) preparation of 2% (w/v) sodium alginate stock solutions; (ii) crosslinking the alginate solution by adding an equal volume of calcium gluconate solution (the final concentration of Ca2+ is 0.01 M), while stirring intensively using a homogenizer with a dispenser tool 6G (Heidolph Elektro, Kelheim, Germany) at 31,800 rpm for min; (iii) freezing the cross-linked material, at 18C, overnight; and (iv) lyophilizing the fro-

zen material (Freeze Dry Systems, Labconco Co., Kansas City, MO) at 0.007 mmHg and a temperature of 60C. The resulting sponges were sterilized using an ethylene oxide gas apparatus and stored in laminated bags at room temperature until use. Preparation of Ca-Alginate Films and Collagen Type I Coated Plates Calcium cross-linked alginate films were prepared by spreading 1 mL of 1% (w/v) sterile sodium alginate on a 55 mm bacteriological Falcon Petri dish and then overlying with 1.5% (w/v) calcium gluconate solution for 15 min. Following the complete gelation of the films, they were washed several times with the culture medium, before cell seeding. Collagen Type I coated dishes were prepared from 40 g/mL stock solution of rat-tail collagen (Sigma Chemical Co., St. Louis, MO). Four milliliters of the solution was spread over the surface of a 55 mm tissue culture plate, swirled to achieve an even coating, and the plates were allowed to dry in a sterile laminar hood overnight. The plates were washed with neutral buffered physiological salt solution prior to use. The coating density of collagen was approximately 2 g/cm2. Hepatocyte Isolation and Culture Hepatocytes were harvested from 46 week-old male Sprague-Dawley rats, weighing 180250 g, by the modified 3-step in situ collagenase perfusion procedure of Berry and Friend (1969), and purified by isodensity Percoll (Pharmacia Biotech AB, Uppsala, Sweden) centrifugation (Kreamer et al., 1986). Upon isolation, the hepatocytes were suspended in chemically defined serum-free Williams E medium, supplemented with 10 ng/mL epidermal growth factor (EGF, Sigma), 20 mU/mL bovine insulin (Sigma), 5 nM dexamethasone, 20 mM pyruvate, 0.05 mg/mL ascorbic acid, 100 units/mL penicillin/streptomycin, and 2 mM glutamine. The media and supplements were purchased from Biological Industries, Kibutz Beit HaEmek (Israel), unless otherwise specified. Postharvest hepatocyte viability was above 90% based on a trypan blue exclusion assay. The isolated hepatocytes were seeded at a concentration of 1 106 cells/sponge within a cylindrical sponge (1.5-cm diameter 1.0 cm-height) placed in a well of a 24-well tissue culture plate, filled with 2 mL of medium. The cultures were incubated in a humidified atmosphere of 5% CO2 and 95% air, at 37C, with daily medium changes. In the adhesion studies on alginate films or collagen type I coated dishes, the hepatocytes were seeded at a concentration of 30,000 cells/cm2. After an attachment period of 24 h, the medium was changed to remove unattached cells, after which the cells were incubated in the culture medium with daily medium changes. The adhesion efficiency, i.e., the percent of initial seeded cells that were attached to alginate films or to collagen type I coated dishes, was determined by

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counting the number of attached cells after their removal from the dishes using 0.05% trypsin/EDTA (Biological Industries, Kibutz Beit HaEmek, Israel). Hepatocyte Number and Viability The total cell number was evaluated by quantifying the DNA content of a crude cellular homogenate of the hepatocytes using the fluorescence enhancement of 4 ,6diamidino-2-phenylindole (DAPI) complexed with DNA (Brunk et al., 1979). Samples were lysed by suspension in 1 mL of 1 M NaOH, followed by incubation for 30 min at 70C. After the suspension pH was adjusted to 7.0, 150 L of 100 ng/mL DAPI was added to 850 L of the suspension and the mixture was read at 360-nm excitation and 450-nm emission by a fluorimeter. The cell number in samples was determined by comparison to standard curves, in which DNA content was measured for a range of known cell concentrations. The number of viable cells in 23 representative wells was evaluated by the MTT assay, which measures the ability of mitochondrial dehydrogenase enzymes to convert the soluble yellow MTT salt into insoluble purple formazan salt. The assay was carried out in tissue culture plates containing the cell-seeded sponges. The tetrazolium salt (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma) was prepared as a 5 mg/mL stock solution in phosphate buffered saline (PBS, pH 7.4). The solution was filtered through a 20 m-pore size filter to remove undissolved particles, and kept in the dark at 4C. MTT (100 L) was added to each well, and the plates were incubated for 5 h at 37C. The medium was aspirated from the wells, taking care not to remove the layer of formazan crystals. The dark purple crystals were dissolved in 1 mL of SDS/formamide, and the plates were agitated until complete dissolution of the crystals. The optical density of the formazan solution was read on a microtitre plate spectrophotometer at 550 nm. Blanks consisted of empty sponges treated in a similar way. Absorbance was found to be directly proportional to cell number (data not shown) in accordance with previous studies (Wagner et al., 1997). Estimation of Spheroid Size and Distribution in Sponges Spheroid size and distribution within the sponges at 7 days post-seeding were measured microscopically after staining the cells with fluorescein diacetate (FDA) which stains viable cell cytoplasm green. The sponges were cut horizontally into 200-m sections using a cryostat and the frozen sections were placed on microscope slides. The slides were viewed under an inverted fluorescence microscope (Model IX70, Olympus, Hamburg, Germany), equipped with a 490nm band-pass filter with a 510-nm cutoff filter for fluorescence emission. Spheroid size was estimated by measuring the lengths along two axes, perpendicular to each other, on 35 mm photomicrographs taken of the sponge serial sec-

tions. The average of the two lengths was defined as the spheroid diameter. At least 15 spheroids per section were measured to obtain representative average diameters and standard deviations. Percent cell occupation at a given distance from sponge surface was calculated from the 35 mm photomicrographs. Cell outlines were traced from the photographs and then scanned and analyzed with NIH Image 1.49 software, determining spheroid area for a given area. Spheroid Histology To identify the components of the deposit on hepatocyte spheroids, at 7 days post seeding, thin-sections of hepatocyte-seeded alginate sponges were analyzed by an indirect immunofluorescent assay using sheep anti-human fibronectin (Serotec Ltd., Oxford, England), rabbit anti-mouse laminin (ICN Biomedicals, Inc. Aurora, OH), and rabbit antihuman collagen type IV (ICN Biomedicals). The samples were frozen in liquid nitrogen and then processed for cryostat sectioning. Frozen sections (12-m thick) were placed on microscope slides and air-dried. They were overlaid with 0.3 mL of the specific antibody diluted at an appropriate concentration and kept for 1 h at 37C. After washing, the slides were incubated for 1 h in the presence of the appropriate FITC-conjugated anti-immunoglobulin antibody diluted 1:200, rinsed, and viewed under an inverted fluorescence microscope, equipped with a 490-nm band-pass filter with a 510-nm cutoff filter for fluorescence emission. For positive control, Matrigel (Collaborative Biomedical Products, Bedford, MA) coated dishes were used. The Matrigel was adsorbed from a cold buffered solution (20 mg/mL) onto 35-mm bacteriological polystyrene Petri dishes. The dishes were coated with 2 mL of the Matrigel solution and incubated for 90 min at room temperature. After aspiration and washing twice in PBS, the dishes were used in the assay. Spheroid Morphology Hepatocyte morphology within the alginate sponges was followed by scanning electron microscopy (SEM). The cellseeded sponges were washed extensively with PBS, and then fixed in 2.5% (w/v) glutaraldehyde in PBS. After washing with PBS buffer three times, the sponges were dehydrated in a graded series of waterethanol solutions and critical-point dried. Thin sections of the cell-seeded sponges were gold-sputtered (100 ), and examined by SEM (Model JSM 35 CF, Jeol, Japan) at 25 kV electron beam radiation. Hepatocyte Function Urea levels were determined by the Urea Nitrogen Diagnostic Kit (Sigma) that utilizes an urease/Berthelot, quantitative, colorimetric method (Chaney and Marback, 1962). Into test tubes were added 100 L of sample followed by 0.3 mL urease solution. After gentle mixing, the tubes were allowed to stand at room temperature for 1520 min while

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urea hydrolyzed to ammonia. To each test tube was then added 0.6 mL of phenol nitroprusside solution, followed by 0.6 mL of alkaline hypochlorite solution, followed by 1.5 mL of deionized water, with gentle vortexing after each addition. After approximately 30 min at room temperature, upon color development, 200 L from each test tube was pipetted into 96-well plates. Sample concentration were determined using a urea nitrogen standard curve. Urea secretion rates were calculated from urea levels in a 24-h culture supernatant divided by the number of viable cells determined by the MTT assay. The rate of albumin secretion from hepatocytes was quantified by a sandwich enzyme-linked immunosorbent assay (ELISA), using antibodies specific to rats (OrganonTeknika Corporation/Cappel, Durham, NC) (Schwerer et al., 1987). Briefly, 96-well plates (Immunolon II) were coated with 100 L of rabbit anti-rat albumin in PBS buffer, overnight at 4C. The plates were washed three times with PBS containing 0.05% (v/v) Tween 20 (PBS/Tween), and then blocked by adding 100 L of 1% (w/v) gelatin in PBS, to reduce non specific binding. After incubating for 1 h at 37C, the plates were washed three times with PBS/Tween. Samples from cell media were diluted as needed, and 100 L of the appropriate diluted medium was added to the wells. After 1 h incubation at 37C, the wells were washed as described above, followed by the addition of 100 L of peroxidase-conjugated sheep anti-rat albumin in PBS. After incubation for 1 h at 37C, the wells were washed and 100 L of the substrate, 2,2-azinodi-(3-ethylbenzthiazoline sulfonate) (1 mg/mL in 77 mM sodium phosphate/61 mM citrate buffer, pH 4.0/0.01% (v/v) H2O2), was added into each well and incubated for 1 h at 37C. Color change was monitored at 405 nm against a reference at 490 nm, using an ELISA microplate reader. Pure rat albumin (OrganonTeknika-Cappel) was used for establishing the standard curve.

Figure 1. Scanning electron micrograph of a cross-section of the alginate sponge used in the study (100 m).

Hepatocyte Seeding within Alginate Sponges and Longevity Hepatocytes were seeded onto dry alginate sponges, at a cell density of 1 106 cells/sponge, by dropping the cell suspension on top of the sponge. The hydrophilicity of the alginate material allowed a rapid wetting of the sponge and entry of the liquid into the air-filled porous structure of the dry sponges. Because of the large pore diameter, the physical obstruction of liquid flow by the torturous pore pathway was minimal, allowing the transport of cells into the sponge pores. Cell viability assay performed on the sponges, one day after cell seeding, revealed that the extent of MTT conversion into formazan crystals by the entrapped hepatocytes corresponded to (0.91) 106 viable cells per sponge, indicating that nearly all seeded cells were efficiently entrapped within the sponges. The alginate sponges were capable of retaining the hepatocytes in culture and no significant cell leakage from the devices was observed for over 2 weeks. The total number of cells per sponge, as determined by quantifying the DNA content of the hepatocytes, did not significantly change throughout the culture (Fig. 2). These findings indicate that adult rat hepatocytes do not proliferate in vitro within the alginate sponges. According to the MTT viability assay, the cells maintained their initial metabolic activity for over a week in culture; the extent of MTT conversion 1 week post-seeding corresponded to (0.91) 106 viable cells per sponge (Fig. 2). The correlation between the total cell number as determined by quantifying the DNA content of the hepatocytes, and viable cell number as assessed by MTT, indicate that the hepatocytes maintain their viability and metabolic rate when cultured in alginate sponges. Hepatocyte Spheroid Formation Within Alginate Sponges Because of the relative transparency of the alginate matrix, cells seeded within the sponges could be observed by a

RESULTS

Morphology of Alginate Sponges The alginate sponges in the present study displayed a highly porous structure with interconnected pores and pore sizes ranging between 100150 m in diameter as judged by SEM analysis (Fig. 1). The alginate sponges were easily wetted by the aqueous medium. After equilibration in the chemically defined Williams E medium, the hydrated alginate sponges appeared to be transparent, and displayed a soft consistency. Once wet, the alginate sponges lost part of their mechanical strength (Shapiro and Cohen, 1997). Yet, under in vitro cell culture conditions the sponges were sufficiently stable to hold the cells for more than a month, were easily handled and did not contract as some collagen-based materials do (Nehrer et al., 1997).

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Figure 2. Hepatocyte number and viability within alginate sponges by the () MTT assay and () DNA measurements. Data were converted to cell number/sponge, using the appropriate calibration curves.

phase contrast microscopy throughout the culture. The cells were followed microscopically after staining them with FDA, which stains viable cell cytoplasm green. The photomicrograph in Fig. 3 reveals that within 24 h after seeding, most of the seeded cells in the alginate sponges are arranged in small viable aggregates which stained green. The few cells that had not participated in the aggregate formation were judged as nonviable upon staining with trypan blue (data not shown). SEM pictures of sponge cross-sections at different times after cell seeding reveal that the hepatocytes aggregated into increasingly larger cell clumps that eventually formed into tightly packed spheroid structures. The formation process is depicted in Fig. 4, starting with dispersed cells (Fig. 4A) that aggregated into doublets and triplets within the first 24 h after inoculation. Larger aggregates of over 10 cells per aggregate appeared in the culture by day 2, reaching an average size of 100 m in diameter at day 4 (Fig. 4B).

Figure 4. Scanning electron micrographs of hepatocytes within the alginate sponges at different times post seeding: (A) 1 day; (B) 4 days; (C) 7 days.

Figure 3. Inverted phase fluoromicrographs of hepatocytes within the alginate sponges 1-day post seeding. The cells, stained with FDA, are arranged in small viable aggregates which stained green (magnification 100).

Within 7 days in culture, formation of spheroids, defined in this study as cells aggregates that have assembled into tightly packed spherical structures with smooth boundaries, was observed (Fig. 4C). The spheroids exhibited a morphology which was distinctly different from the initial cell clumps seen on day 4, in which cells were seen to be loosely

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connected and irregularly shaped. The spheroids have a maximal average size of 100 m, which is at the same order of magnitude as the sponge average pore size. Spheroid Size Distribution Within Sponges Spheroids with a narrow particle size distribution, in the range of 85110 m in diameter, could be detected in serial horizontal thin sections (thickness of 200 m) of the alginate sponges, up to a distance of 6.4 mm from the sponge surface (the spongess height is 10 mm) (Fig. 5A). Subsequently, the particle size decreased sharply, and at a distance of 6.75 mm from the sponge surface the particle size was only 20 m in diameter. No spheroids were found in the lower third part of the sponge (Fig. 5B). The reduction in spheroid size at the sponge bottom correlated with the decrease in the percent cell occupation in that region, from an average 14% at the upper and center parts of the sponge to 4% and below at the lower part. This may be explained

by the gravity method of cell seeding which involves dropping the cell suspension on top of the sponge. It might be also that cell concentration per sponge is insufficient to fill the entire void volume of the sponge. Hepatocyte Culture on Alginate Films To further understand the phenomenon of cell aggregation within alginate sponges, we examined whether the alginate material is adhesive to primary hepatocytes from rats. The studies were performed on thin films of calcium crosslinked alginate hydrogels (hydrated) that were characterized with a relatively smooth surface and minimal groovings. Alginate sponges were not suitable for these studies because of the extensive microgrooving created by their highly porous surface structure. The cells were seeded on the alginate films, at a cell density of 30,000/cm2, and their behavior was compared to cells seeded on control collagen-coated dishes. The adhesion efficiency of hepatocytes, 24 h postseeding, was minimal on alginate films and ranged from 7080% on collagen-coated dishes. When cultured on the alginatecovered films, most of the cells were recovered from the supernatants within 24 h after seeding, and they were nonviable. In some instances, cells were observed to concentrate in an aggregate form within microgrooves on the surface of the film and they were viable, as judged by the trypan blue exclusion assay (Fig. 6A). This indicates that the reduced cell viability when cultured on alginate films is not due to material toxicity, but probably because the cells have minimal interactions with the alginate matrix. On the collagen-coated dishes, on the other hand, the attached cells began to spread out and formed networks, or webs, of connected cells in a monolayer (Fig. 6B). After a week, the cells gradually began to lose their adhesivity for collagen, began to contract and eventually were detached from the substrate. Biomatrix Deposition on Spheroids Immunohistochemistry of the hepatocyte spheroids formed within the alginate sponges at day 7 postseeding revealed that they were positively stained for fibronectin, but not for laminin or collagen type IV (Fig. 7). It appears that the fibronectin is deposited on the surface of the spheroids and between the individual cells that constitute the spheroids. Control empty sponges, or cell-seeded sponges reacted only with the second FITC-conjugated antibody, were not stained. Expression of Liver-Specific Functions Albumin and urea secretion rates were evaluated as two major representative liver-specific functions. Hepatocytes seeded within the alginate sponges exhibited a timedependent increase in the rate of albumin secretion during the first week (Fig. 8A). It is suggested that this dynamic behavior reflects the aggregation of hepatocytes into tightly-

Figure 5. Hepatocyte spheroid distribution within alginate sponges, 7 days post seeding. (A) Spheroid size distribution in serial cross-sections of the sponge. (B) Percent cell occupation. The sponges were horizontally cut into 200- sections using a cryostat and stained with FDA. Spheroid size is the average of lengths along two axes, perpendicular to each other.

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Figure 6. Inverted phase photomicrographs of 1-day old hepatocytes on (A) Ca-alginate films and (B) collagen type I coated dish. Most cells seeded on the Ca-alginate films were found in the spent medium, some were concentrated as aggregates within microgrooves on the surface of the films (magnification 100).

packed spheroids during this time period. The albumin secretion rates were maximal during the second week of hepatocyte culture within the sponges (approximately 60 g albumin/106 cells/day). Throughout the culture, albumin secretion rates from hepatocytes seeded within the alginate sponges were considerably higher than from cells cultured

Figure 8. Comparison of liver-specific functions between hepatocytes cultured within alginate sponges () and on collagen type I coated dishes (). Rat hepatocytes were seeded onto the alginate sponge at a concentration of 1 106 cells/sponge, and on collagen-coated dish at a concentration of 3 104 cells/cm2. The culture medium was changed daily (1.5 mL/day). Secretion rates for albumin (A) and urea (B) were normalized to the number of viable cells (based on MTT assay) per dish. Each data point shown is the average of at least 10 replicate dishes.

on the standard collagen type I coated dishes and for longer times. In the later cultures, albumin secretion decreased nearly to zero within 5 days in culture (Fig. 8A). In contrast to albumin secretion, the urea secretion rate by hepatocytes did not depend on the type of culture or the initial cell density (Fig. 8B). Urea secretion rates from hepatocytes seeded within the alginate sponges were similar to the levels obtained from cells cultured on collagen type I coated dishes (100 g urea/106 cells/day) and they remained relatively constant throughout the 2-week culture. DISCUSSION The optimal scaffolding for tissue engineering remains a key issue in the development of successful biological substitutes. In the present paper, we investigated the suitability of a novel 3-D alginate scaffold for hepatic tissue engineering. The scaffold is characterized with a highly porous structure (sponge-like), with an average pore size 100150

Figure 7. Fibronectin deposition on hepatocyte spheroids formed within the alginate sponges, 7 days postseeding.

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m and interlinked pores (Shapiro and Cohen, 1997). Such a structure is attractive for engineering tissues from isolated cells in vitro, as it can provide a sufficient space for seeding a large cell mass and for the reorganization of the cells into tissue-like structures. Seeding hepatocytes onto the alginate sponges was efficient due to the hydrophilic nature of the alginate polymer and the rapid wetting of the matrix by the culture medium. Consequently, the cell transport into the matrix pores was maximized, ensuring that the most of the device is uniformly cell-rich. The alginate matrix resisted hepatocyte adhesion and spreading. This can be explained by the highly hydrated anionic surface of the alginate material, which reaches over 99% hydration in weight. A surface mainly composed of water may offer a negligible number of surface points where proteins can adsorb. Consequently, hepatocytes do not attach to the alginate films as such interactions are largely mediated via receptors to the adsorbed proteins. In contrast to the 2-D alginate film cultures, hepatocytes were efficiently retained within the 3-D alginate sponges probably due to the tortuous path presented to the cells by the porous scaffold. The alginate sponges were sufficiently stable under the culture conditions to retain the cells for the duration of the study. The inability of the cells to attach to the alginate matrix was not associated with death or impairment of cellular functions of the hepatocytes, instead they immediately clustered into aggregates within the sponge pores, preferring cellcell interactions. More than 90% of the seeded hepatocytes participated in spheroid formation. The few cells which did not aggregate lost their viability. The formation rate of spheroids within the 3-D alginate sponges was significantly more rapid than that of spheroids formed from adherent matrices such as collagen matrices or positively charged polystyrene plates. In the latter, aggregation took 35 days and only 3540% of plated cells formed spheroids (Koide et al., 1990; Peshwa et al., 1994). The greater efficiency of spheroid formation within the alginate sponges should lead to a maximal exploitation of the limited supply of primary hepatocytes. This is particularly important if we consider that no cell proliferation was observed in the spheroid in culture, despite the use of a favorable medium supplemented with growth factors. This phenomenon has been reported by other research groups and was attributed to contact inhibition of growth due to the high cell density in the spheroids (Yuasa et al., 1993). The greater efficiency and rapidity of hepatocyte spheroid formation within the alginate sponges is attributed not only to the non-adherent nature and chemistry of the alginate matrix but also to the macroporous structure of the sponge. The large pore size of the alginate sponges, in the range of 100150 m in diameter, enabled the reorganization and clustering of the cells within the pores. In conventional calcium alginate gels, isolated rat hepatocytes did not form multicellular aggregates (Yagi et al., 1993). The hepatocytes lost the abilities of albumin secretion and TAT (tyrosine aminotransferse) induction within a week. The

conventional gels are characterized with pore sizes that are largely determined by the size of the encapsulated material, and in the case of dispersed hepatocytes, the range of pore size is 1020 m in diameter. Thus, it is possible that the Ca-alginate gel interferes to the formation of hepatocyte spheroids. Other works have also shown the advantage of using macroporous matrices. Lin et al (1995) showed successful long-term maintenance of serum albumin secretion and bile acid formation (around 12 days in culture) in the gelfoam culture systems which was ascribed to the retention of morphology and the formation of spheroids (Lin et al., 1995). The size distribution of hepatocyte spheroids formed within the alginate sponges was relatively narrow, approaching that of the sponge pore size (100 m in diameter). It appears that the pore size of the sponge can dictate the size of the formed spheroids. Whether this phenomenon will have an impact on the performance of the engineered hepatic tissue has yet to be unraveled. The important issue is that spheroids of this size may not suffer nutrient transfer limitations. The results of the MTT viability assay indicate that indeed the cells in the 100-m spheroids formed within the alginate sponges maintained high viability throughout the culture time. Other investigators have also shown, on the basis of experiments and calculations of oxygen diffusion in spheroids, that oxygen may be a limiting factor only if the diameter of the spheroids exceeds 106.2 m and that below this value the spheroids retain high cell viability (Ijima et al., 1998). Our ability to control the size of hepatocyte spheroids to 100 m in diameter, using the alginate sponges, thus ensures that all formed spheroids are highly viable. The formed spheroids were homogeneously distributed in the upper two-third of the sponge, and none were found in the bottom, at 7 days post seeding. The seeding method by gravitation and the relatively low seeding density of the hepatocytes, i.e., 1 million cells per sponge, may both account for that. It appears that most of the sponge void volume is not occupied by the hepatocytes: the percent cell occupation in most of the sponge cross-sections ranged between 8% and 14%. For a sponge of this volume (1.75 cm3) and porosity of 90% (Shapiro and Cohen, 1997), the maximal cell loading can reach 3.8 108 cells/sponge. If we neglect possible mass transfer limitations in highly cellseeded sponges, the above number means that around 6 hepatocyte-seeded sponges of this small size will be needed to be total supportive in humans (the number of transplanted hepatocytes for an adult of 70 kg should be 2 109) (Asonuma et al., 1992). Further studies to estimate the actual maximal cell capacity of alginate sponges will be needed to substantiate their advantage as cell carriers. As yet, the detailed mechanism of spheroid formation within the alginate sponges is not fully understood. In recent years, several possibilities have been suggested to explain the process of self-assembly, including the traditional cell migration, cell-to-cell contacts that are initiated by active membrane protrusions (Powers and Griffith-Cima, 1996), and cell movement toward the neighboring cells due to ma-

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trix reorganization and displacement (Coger et al., 1997). The later theory best explains what occurs when cells are seeded at a sparse-density, which is apparently the situation in our system. However, as the hepatocytes within the alginate sponges have minimal interactions with the matrix and in fact they do not adhere to the sponge, it is not yet clear whether and how the matrix movement can exert forces on the cells. Another possibility to consider is the involvement of ECM components in the process of cell aggregation and compaction. We found that the hepatocytes within the alginate sponges synthesize fibronectin that is deposited on the surface of the spheroids and between the cells which constitute the particle. Considering the evidence which suggest the presence of a specific receptor for fibronectin on rat hepatocytes (Johansson and Hook, 1984), it is likely that fibronectin helps in the hepatocyte aggregation. Landry et al., when investigating spheroids formed on non adherent plates, found in addition to fibronectin also laminin and collagen (Landry et al., 1985). We did not detect laminin or collagen type IV in the deposits. The absence of the later components is no surprise as they are mainly produced by Ito cells in the native liver (Rojkind and Greenwel, 1994), and our cultures were pure hepatocytes. We believe that fibronectin secretion by the hepatocytes within alginate sponges is part of the cell effort to recreate its own matrix within the alginate sponges, and by doing so to establish sufficient cellmatrix interactions to maintain survival and differentiation. The efficient formation of hepatocyte spheroids and fibronectin deposition are likely to be responsible for the maintenance of hepatocyte function within alginate sponges. Albumin secretion was used as a marker for protein synthesis because it requires liver-specific gene expression and intact translational and secretory pathways in the cells (Bissell et al., 1990). The specific albumin secretion rates increased with culture time and the progression of hepatocyte aggregation within the alginate sponges, until reaching the maximal level of 60 g albumin/106 cells/day at the second week. This value falls within the range of that observed in the liver in vivo (Peter and Peter, 1972). On collagen-coated dishes, the hepatocyte monolayers stopped secreting albumin within a week. Our results suggest that the maintenance of albumin secretion by the hepatocytes in culture is due to spheroid formation. In contrast to albumin secretion, the specific urea secretion rate was not affected by the aggregation state of the hepatocytes. Hepatocytes grown as monolayers on collagen-coated dishes secreted urea at a similar rate to that secreted by the spheroids formed within alginate sponges. These results may suggest that there may be certain cell activities which do not depend on cell aggregation and spheroid formation. The specific albumin and urea secretion rates were similar to values obtained with hepatocytes which were first attached to gelatin-coated polystyrene microcarriers followed by embedding them within the sandwich collagen gels (Stockmann et al., 1997), or when the cells were embedded directly into the sandwich collagen systems (Dunn

et al., 1991; Moghe et al., 1996). However, the sandwich culture system in its current form yields relatively low cell concentrations per matrix volume, and the need to lay down the collagen layers into a flat-plate geometry is cumbersome. In contrast, the 3-D alginate sponges appear to be suitable for high-density cell cultures using a simple seeding method. In summary, this study identifies a potential 3-D scaffold for engineering hepatic tissue. The macroporous structure and non-adherent nature of the alginate sponges enhanced hepatocyte aggregation and facilitated their performance, i.e., albumin secretion. It took the freshly isolated hepatocytes as much as a week to fully re-express their differentiated functions within the alginate sponges. During this time the sponges were able to foster a long-term viability and protection of the cells. This tissue culture system may provide a new model system to study hepatocyte behavior in three dimensions, in vitro, and be applied in the study of xenobiotic drug metabolism. Furthermore, because the alginate sponges are composed of a biocompatible material (Klo ck et al., 1994; Lanza et al., 1995), they are potentially attractive cell carriers intended for implantation. Hepatic tissue engineered within these sponges can find clinical applications in replacing a damaged liver.
The authors thank Ms. Sharon Zmora for preparing the samples for SEM analysis. The research is supported by The Israeli Ministry of Science (Program for Creative Ideas), The Harry Stern Applied Research Grants, and The Institute for Applied BioSciences-Center for Polysaccharide Research at Ben-Gurion University.

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