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ARTICLES Kinetics of Hepatocyte Spheroid Formation Madhusudan V. Peshwa? Florence J. W U , ~Brian D. Follstad; Frank B. Cerra,*and Wei-ShouHu*tt Department of Chemical Engineering and Materials Science and Department of Surgery, University of Minnesota, Minneapolis, Minnesota 55455
Cultured hepatocytes have been explored for use in a bioartificial liver. Spheroids formed by cultured hepatocytes exhibit enhanced liver-specific functions. The kinetics of spheroid formation, using rat hepatocytes, was studied on positively charged surfaces that were either uncoated or coated with collagen or (hydroxyethy1)methacrylate (HEMA). Optimal spheroid formation was obtained on positively charged (Primaria) surfaces at inoculum densities in the range of (3-9) x lo4 cells/cm2. Cells initially attached and spread out on the surface. Subsequent retraction led to t h e emergence of small clumps of cells attached to the surface, from which spheroids formed and shed off into suspension. The process of spheroid formation took more t h a n 72 h and was accompanied by a decrease in the surface area occupied by attached cells. Optical sectioning of fluorescently stained spheroids using confocal microscopy indicated that most of the cells in the spheroid were viable. Spheroids also maintained a constant albumin synthesis rate for over 7 days in culture. Spheroid formation was evaluated in terms of the changes in spheroid diameter, the surface area covered by attached cells, and the total protein content of the fraction of cells t h a t formed spheroids. The quantitative methodologies developed were used to assess the effect of inoculum cell concentration on spheroid formation and to evaluate the kinetics of spheroid formation on different surfaces both favorable and nonfavorable to spheroid formation.
Introduction The need for an interim liver-assist device as a bridge to transplant for patients in hepatic failure has been welldocumented (1-3 ). Devices designed to provide extracorporeal liver support have already been tested on human patients (4). A hollow fiber bioreactor employing xenogenic hepatocytes entrapped in a three-dimensional collagen matrix within the luminal space has been developed as a bioartificial liver (BAL) (5). Hepatocytespecific functions in the BAL have been demonstrated in vitro (5,6) and in vivo (7, 8). The demonstration of efficacy and application for human therapy requires the BAL to be scaled up. The cultivation of hepatocytes as spheroids or multicellular aggregates with well-defined cellular morphology has been demonstrated to lead to enhanced specific or per cell activity of the hepatocytes (9-12). Thus, the use of hepatocyte spheroids in bioartificial liver devices has been explored (13, 14). It is postulated that using entrapped spheroids in the BAL would lead to improvements in device performance. Hepatocytes form spheroids when cultivated on certain types of surfaces, such as positively charged nonadherent surfaces and surfaces coated with liver-derived proteoglycans or poly((hydroxyethy1)methacrylate) (poly-HEMA) (9-12, 16). Hepatocytes in spheroids reorganize themselves into structures that have morphological and functional similarities to in vivo liver tissue (15,26).The
* Author to whom correspondence is to be addressed. iDepartment
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of Chemical Engineering and Materials Science. Department of Surgery. 8756-7938/94/3010-0460$04.50/0
prolonged maintenance of differentiated functions has been correlated to the maintenance of cell polarity in spheroids; isolated hepatocytes are unable to maintain cell membrane polarity (17), whereas hepatocytes in spheroids retain their ability to repolarize in culture (18). The process of spheroid formation takes 3-4 days, and only a fraction of the cells initially inoculated form spheroids (9,10,12,16). For the use of spheroids in the BAL, the rate and efficiency of spheroid formation need to be improved. To facilitate evaluation of the efficiency and rate of spheroid formation, we have established quantitative methodologies. The effect of inoculum cell concentration on spheroid formation was evaluated. The efficacy of the developed methodologies was demonstrated by quantitatively elucidating the kinetics of spheroid formation on different surface types and comparing the results obtained to qualitative observations reported in the literature. Our results also demonstrate the maintenance of cell viability and liver-specific function in hepatocyte spheroids in prolonged culture. These observations indicate that employing spheroids may lead to possible improvements in the performance of a bioartificial liver (BAL)-assist device.
Materials and Methods Hepatocyte Culture. Hepatocytes were harvested from 4-6-week-old male Sprague-Dawley rats weighing 200-250 g by a modified two-step in situ collagenase perfusion technique (19). Postharvest hepatocyte viability ranged from 85 to 90% based on trypan blue exclusion. Hepatocytes were cultivated in basal Williams E medium supplemented with 0.2 u n i a insulin (Lilly
0 1994 American Chemical Society and American Institute of Chemical Engineers
Biotechnol. Prog., 1994,Vol. 10, No. 5
Research Laboratories, Indianapolis, IN), 100 u n i t s b penicillin (Gibco Laboratories, Grand Island, NY),100 mg/L streptomycin (Gibco Laboratories), 2 mM L-glutamine (Gibco Laboratories), 15 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonicacid (HEPES) (Gibco Laboratories), 0.1 pM copper (CuSO.&HzO), 3 nM selenium (HZSeOs),50 pM zinc (ZnS04*7HzO),50 ng/mL epidermal growth factor (Sigma Chemical Co., St. Louis, MO), and 50 ng/mL linoleic acid (Sigma Chemical Co.) (9,161. The medium was replenished with fresh medium every 2-3 days. Preparation of the Culture Surface. Three different types of culture plates (60 x 15 mm), Falcon 1008, Falcon 3001, and Falcon Primaria, were used. All three types were used uncoated and coated with either collagen or (hydroxyethy1)methacrylate(HEMA). Surface coating with collagen and HEMA was performed according to the following procedure. A suspension of 3 vol of Vitrogen 100 (Collagen Corporation, Palo Alto, CA) containing 2.9 mg/mL of type I rat tail collagen and 1 vol of 4-foldconcentrated Williams E medium (Gibco Laboratories) was prepared. The final pH was adjusted to approximately 7.2 with sterile 1.0 N NaOH, and 0.5 mL of this collagen-medium suspension was added onto each plate. The plates were swirled to achieve a n even coating and incubated a t room temperature until gelation (-40 min). Plates were then washed twice with phosphate-buffered saline (PBS) and stored hydrated at 4 "C. To prepare HEMA-coated plates, 2.0 mL of a solution containing 2.5% HEMA (Gibco Laboratories) in 95% ethanol was added to each plate. The plates were airdried overnight, washed twice with PBS, and stored a t 4 "C. Quantitative Estimation of Spheroid Formation. Spitemid Diameters. In the process of spheroid formation, cells agglomerate to form clumps, and the aggregate shpe is not well defined. Spheroids were defined as multicellular aggregates that had completely dissociated from the plate surface and were freely floating in suspension. The lengths along two axes, perpendicular to each other, were measured under a n inverted microscope (Olympus, Tokyo) using a l o x ocular lens equipped with a Vernier scale. The average of the two lengths was defined as the spheroid diameter. At least 25 spheroids per plate were measured to obtain representative average diameters and standard deviations. Surface Area Coverage. To estimate the surface area covered by attached cells, 10 fields were examined microscopically by scanning across each plate from top to bottom and then from left to right. The images of each field were recorded using a video camera. The percentage of plate area covered by attached cells was determined by reviewing recorded images on a gridded TV monitor and counting the number of grids occupied by attached cells. Total Protein Content. Culture dishes contained both free-floating spheroids already detached from the surface, as well as cell aggregates and single cells still attached to the surface. Attached cells were freed from the surface by pipeting medium onto the plate surface without removing the collagen or HEMA coating. Samples were obtained by collecting the culture medium from each plate into a 10-mL pipet, allowing the heavier aggregated cells to settle to the bottom one-fourth volume fraction in the pipet, and then separating the culture medium into two fractions: a spheroid fraction and an unaggregated cell fraction. The spheroid fraction was considered to be the aggregated cells that had settled within the first minute into the bottom one-fourth volume fraction of the sampling pipet. The separation of spheroids from cells
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in suspension was confirmed by microscopic observation. The unaggregated fraction consisted of the remaining three-quarters of the sample. Both fractions were sonicated, frozen at -20 "C, and assayed for total protein content at a later date. Total protein was measured by using a bicinchoninic acid (BCA)protein assay kit (Pierce, Rockford, IL). The absorbance at 562 nm, measured on a spectrophotometer (W-160, Shimadzu), was correlated to the total protein content in the sample. Albumin Concentration. Rat albumin concentrations were determined by a competitive enzyme-linked immunoassay (ELISA). Samples were serially diluted, and peroxidase conjugated rabbit antibody against rat albumin (Cappel, Durham, NC) was added to a final concentration of 400 ng/mL. After incubation a t 37 "C for 2 h, 100-pL aliquots of each sample were transferred to a precoated 96-well plate (Nunc, Naperville, IL). Precoated plates were prepared by the addition of 100 pL of rat albumin (100 ng/mL in PBS) to each well, subsequent overnight incubation at 4 "C, followed by three washes with 0.05% Tween-20 (Bio-Rad, Richmond, CA) in PBS. After transfer to the precoated plates, the samples were incubated at room temperature for 2 h in a humidified chamber. Subsequently, the plates were again washed thrice with 0.05%Tween-20 in PBS and filled with 100 pL per well of a solution containing 55 mg/mL 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonate~ (Boehringer Mannheim, Indianapolis, IN) in citrate buffer (0.1 M sodium citrate, pH 4.2, with 0.1 pL of 30% hydrogen peroxide per 100 pL). Plates were further incubated a t room temperature in a humidified chamber for 30-45 min and read on a n automated plate-reader. The difference in absorbance at 405 and 490 nm was correlated to the concentration of rat albumin in the sample. Viability Staining. Cells were stained with fluorescein diacetate (FDA) and ethidium bromide (EB) for viability (20). The stained samples were observed using either an epifluorescence microscope (Carl Zeiss Inc., Thornwood, NY)or a confocal microscope. The epifluorescence microscope was equipped with a 35-mm camera, a mercury vapor lamp for W illumination, and a 450490-nm band-pass filter with a 510-nm cutoff filter for fluorescent emission. The confocal microscope system consisted of a n Olympus BH-2 microscope illuminated by a n argon ion laser (wavelength = 488 nm), equipped for epifluorescence, and linked to a n MRCdOO confocal imaging system (Bio-Rad, Boston, MA) (21).
Results and Discussion Three different surface types, hydrophobic uncharged (Falcon 1008), hydrophilic negatively charged (Falcon 3001), and plasma arc treated positively charged (Falcon Primaria) surfaces, were examined. All three surfaces were also used coated with HEMA or collagen. Spheroid formation on these three uncoated and coated plates were qualitatively evaluated. Uncoated Primaria plates appeared to result in optimal spheroid formation. Hepatocytes formed monolayers on all three collagen-coated surfaces and on uncoated and HEMA-coated hydrophobic (Falcon 1008) surfaces. In contrast, hepatocytes formed aggregatedcell clumps on Primaria and Falcon 3001 surfaces coated with HEMA. All further studies discussed in this paper refer to spheroid formation on Primaria surfaces. Hepatocytes inoculated on Primaria surfaces initially attached to the surface and then spread out. Subsequent retraction led to clustering of cells a t certain regions on the culture dish. These clusters matured to form sphe-
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Figure 1. Light micrographs indicating the change in surface area covered by attached cells as a function of culture time: (A) day 1; (B)day 2; (C)day 3; (D) day 7; (E, bottom) light microscopic image of a hepatocyte spheroid.
roids that shed off into the suspension. Figure IA-D shows these qualitative changes observed during the course of spheroid formation. Besides spheroids, some cell clumps and aggregates with undefined morphology were also observed to form. Only those aggregates in which the cells were in close contact with each other and resulted in a well-defined structure with smooth, undulating boundaries, when viewed under an optical microscope, were defined as spheroids (Figure 1E). To examine the effect of inoculum cell density on spheroid formation, hepatocytes were inoculated on Primaria plates a t varying densities in the range of 6.0 x
lo5to 2.4 x lo6 cells/plate (yielding a surface density of 3.1 x lo4to 8.4 x lo4cells/cm2). No spheroid formation was observed a t inoculum cell densities lower than 3.1 x lo4 cells/cm2. At cell densities higher than 8.4 x lo4 cells/cm2,hepatocytes formed multilayers and resulted in aggregatedclumps instead of forming spheroids (data not presented). Thus, there appears to be an optimal range of inoculum cell density for spheroid formation. The diameter of spheroids was measured microscopically. The mean diameter increased during the first 3-5 days of culture to approximately 100 pm and was maintained relatively constant thereafter. At later stages
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of culture, there was a wider distribution for the size of the spheroids, resulting in an increased standard deviation of the mean spheroid diameter. Figure 2 shows the profile or spheroid size a t inoculum cell densities of 6.0 x lo5, 1.2 x lo6, and 2.4 x lo6 cells/plate. Spheroid size a t the three cell densities analyzed increased a t the same rate. The mean spheroid sizes a t the end of the culture duration all fell within a narrow range (98 & 21 pm on day 6). The increase in spheroid diameter coincided with a decrease in the percentage of plate area covered by attached cells as observed under light microscopy. To quantify this observation, the surface area covered by attached rat hepatocytes inoculated a t 6.0 x lo5, 1.2 x lo6,and 2.4 x lo6celldplate was determined as described in Materials and Methods. The initial surface area occupied by attached cells decreased with time for 3-5 days and therefater was held relatively constant (Figure 3a). The surface area occupied by cells varies with inoculum cell density. However, by normalizing the area covered by attached cells to the area initially covered by cells, all three curves were determined to be superimposable (Figure 3b). This indicated that spheroid formation a t the three inoculum densities examined is independent of the inoculum concentration. Thus, the kinetics of spheroid formation is independent of inoculum cell density. The efficiency of spheroid formation, p, was estimated by measuring the total protein content in the spheroid fraction (Psph) and in the unaggregated cell fraction (Pucd. An inherent assumption involved in the above analysis is that the total protein content per hepatocyte is the same irrespective of whether the hepatocyte is in monolayer or spheroid culture. Thus, p is an indicator of the number of inoculated cells that eventually form spheroids and can be expressed as
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can be seen in Figure 4). A total of 40% of inoculated cells formed spheroids after 5 days in culture. Similar efficiencies, between 35 and 45%, were obtained for cultures inoculated a t two of the other inoculum densities analyzed. Albumin synthesis by hepatocytes cultivated as spheroids was measured and compared to hepatocytes that did not form spheroids. The cumulative albumin synthesis profile for spheroid cultures was linear over 7 days. The specific albumin synthesis rate was calculated to be 1.25 pg/cell/h (Figure 5), whereas for hepatocytes that do not form spheroids, the specific albumin synthesis rate is lower than that for spheroids and it also decreases with time over 7 days of culture. The developed methodologies were subsequently used to quantitatively evaluate spheroid formation on collagen-coated and HEMA-coated plates. The results obtained were compared against spheroid formation on uncoated plates. Figure 6 depicts spheroid diameter, the area covered by attached cells, and the albumin synthetic activity of hepatocytes cultivated on uncoated, collagencoated, and HEMA-coated Primaria plates. The inoculum cell density used was 1.5 x lo6 celldplate (or 5.3 x lo4 cells/cm2)in each case. The spheroid diameters on uncoated and HEMA-coated dishses appeared to be similar (Figure 6a). Consistent with these results, the area covered by attached cells on uncoated and HEMAcoated dishes decreased from about 60% on day 1to about 15% on day 4 and was maintained constant thereafter (Figure 6b), whereas in the case of collagen-coated dishes the area covered by attached cells was constant (-40%) over the 10-day cultivation period and no spheroid formation was observed. Hepatocytes seeded on collagen-
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coated dishes did not spread out to the extent that they did on uncoated or HEMA-coated dishes; hence, the same inoculum density on collagen-coated dishes occupied only 45% of the area on day 1versus approximately 55-60% on uncoated dishes. The efficiency of spheroid formation based on total protein content was 35% and 45% for HEMA-coated and uncoated Primaria plates, respectively. The quantitative results obtained using the developed methodologies correlate with qualitative results of spheroid formation observed by us and also reported by other researchers (9,11,16). This indicates the validity of the developed methodologies in quantitatively evaluating spheroid formation kinetics. Although the kinetics and efficiency of spheroid formation appear to be similar on uncoated and HEMA-coated plates, the albumin synthetic activity was lower for spheroids cultivated on HEMA-coated plates compared t o those on uncoated plates (Figure 6c). This was possibly due to the fact that hepatocytes on HEMA-coated surfaces, in addition to forming spheroids, also formed a significant number of cell clumps/aggregates that do not have spheroidlike morphology. The methodology developed to evaluate the kinetics and efficiency of spheroid formation was unable to differentiate between spheroids and nonspheroidal clumps. Hence, in addition to measuring kinetic parameters to evaluate spheroid formation, it was necessary to quantify liver-specific function to ensure optimal spheroid formation. The hepatocyte spheroids were found to maintain the same cell morphology and similar levels of viability and hepatocyte-specific function for over 7 days in culture. Histological sectioning indicated that most spheroids were relatively spherical and were made up of 6-8 cell layers across. Intact nuclei were seen in the cells a t the center of the spheroid, indicating that the cells a t the
Figure 6. Comparison of the hepatocyte spheroid formation process for cells cultivated on uncoated (01,collagen-coated( A ) , and HEMA-coated (0)Primaria dishes inoculated at 1.5 x lo6 celldplate: (a) spheroid diameter as a function of culture duration; (b) area covered by attached cells as a function of culture duration; (cj albumin synthesis.
center of the spheroids were viable. Optical confocal microscopy was used to observe cell morphology, cellcell contact, and cell viability. Figure 7a depicts a reconstruction of 25 optical sections through a hepatocyte spheroid. The spheroid surface was smooth and undulating (as was seen in Figure 2), and there was extensive cell-cell contact. The cell viability of hepatocytes cultivated as spheroids was investigated by fluorescent staining using fluorescein diacetate (FDA) and ethidium bromide (EB). FDA stains viable cell cytoplasm green, and EB stains dead cell nuclei red. Figure 7b depicts a reconstructed confocal micrograph of a hepatocyte spheroid after 12 days of culture and indicates good cell viability.
Concluding Remarks Hepatocytes cultivated as spheroids have been reported to better maintain liver-specific functions. The use of hepatocyte spheroids in bioartificial liver-assist devices would be expected t o lead to improvements in device performance. For example, the specific rate of albumin synthesis for hepatocyte spheroids is about "-fold higher than that for freshly harvested hepatocytes entrapped in a highly porous three-dimensional collagen gel (data not shown) of the type used in a bioartificial liver design ( 5 , 6). It is thus envisaged that entrapping hepatocyte spheroids in a collagen matrix within the bioartificial liver would improve the hepatocyte-specific function of the device. In order to be employed for such potential applications, the process of spheroid formation needs to be evaluated quantitatively. Also, spheroid formation
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Figure 7. Confocal micrographs depicting the maintenance of cell-cell contact, cell morphology, and viability for hepatocytes in spheroids: (a)reconstructed projection of 25 optical sections; (b) reconstructed projection of 15 optical sections, indicating good viability for cells in the spheroid.
presently requires 3-4 days, during which greater than one-half of the inoculated cells lose their viability and function. In order t o be used in bioartificial devices, the kinetics of spheroid formation needs to be studied further in order to develop techniques aimed a t improving the efficiency and reducing the time required for spheroid formation. We have developed quantitative methodologies to characterize the process of spheroid formation on uncoated Primaria dishes, and their efficacy was demonstrated by using them to describe and quantify spheroid formation on HEMA-coated and collagen-coated dishes. These techniques should allow the critical evaluation of
different methods of spheroid formation, with the final aim being the ability to engineer the process as desired.
Acknowledgment This work was supported in part by grants from the National Institutes of Health (DK45371-02) and the Whitaker Foundation. F.J.W. was supported by a Biotechnology Training Grant from NIGMS (GM0834701A1).
Literature Cited (1) Langer, R.; Vacanti, J. P. Tissue Engineering. Science 1993, 260,920-926.
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466 (2) Chang, T. M. S. Experimental artificial liver support with emphasis on fulminant hepatic failure: concepts and review. Semin. Liver Dis. 1986,6 , 543-545. (3) Kimoto, S. The artificial liver experiments and clinical application. ASAIO Trans. 1959,5,102-110. (4) Rozga, J.; Holzman, M. D.; Rob, M. S.;Griffin, D. W.; Neuzil, D. F.; Giorgio, T. D.; Moscioni, A. D.; Demetriou, A. A. Development of a hybrid bioartificial liver. Ann. Surg. 1993, 217, 502-509. 15) Nyberg, S. L.; Shatford, S. L.; Peshwa, M. V.; White, J. G.; Cerra, F. B.; Hu, W.-S. Evaluation of a hepatocyte-entrapment hollow fiber bioreactor: A potential bioartificial liver. Biotechnol. Bioeng. 1993,41,194-203. (6) Peshwa, M. V.; Nyberg, S. L.; Cerra, F. B.; Hu, W.-S. Distribution of viability and function of gel entrapped hepatocytes, American Institute of Chemical Engineers (AIChE) Annual Meeting, November 1-6, 1992, Miami Beach, FL. ( 7 ) Nyberg, S. L.; Shirabe, K.; Peshwa, M. V.; Crotty, P.; Payne, W. D.; Hu, W.-S.; Cerra, F. B. Support of anhepatic liver failure by gel-entrapped hepatocytes within a hollow fiber bioartificial liver. Cell Transplant. 1993,in press. ( 8 ) Sielaff, T. D.; Nyberg, S. L.; Amiot, B.; Hu, M. Y.; Peshwa, M. V.; Wu, F. J.; Hu, W.-S.; Cerra, F. B. Application of a bioartificial liver (BAL) in a new model of acute fulminant hepatitis. Surg. Forum. 1993,in press. (91 Koide, N.; Sakaguchi, K.; Koide, Y.; Asano, K.; Kawaguchi, M.; Matsushima, H.; Takenami, T.; Shinji, T.; Mori, M.; Tsuji, T. Formation of multicellular spheroids composed of adult rat hepatocytes in dishes with positively charged surfaces and other nonadherent environments. Exp. Cell Res. 1990,186, 227-235. (10) Tong, J . Z.; Bernard, 0.;Alvarez, F. Long-term culture of rat liver cell spheroids in hormonally defined media. Exp. Cell Res. 1990,189,87-92. (11) Takezawa, T.; Yamazaki, M.; Mori, Y.; Yonaha, T.; Yoshizato, K. Morphological and immuno-cytochemical characterization of a hetero-spheroid composed of fibroblasts and hepatocytes. J . Cell Sci. 1989,101, 495-501. (12) Landry, J.; Bernier, D.; Ouellet, C.; Goyette, R.; Marceau, N. Spheroidal aggregate culture of rat liver cells: Histotypic reorganization, biomatrix deposition, and maintenance of functional activities. J . Cell Biol. 1985,101, 914-923.
(13) Li, A. P.; Barker, G.; Beck, D.; Colburn, S.; Monsell, R.; Pellegrin, C. Culturing of primary hepatocytes as entrapped aggregates in a packed bed bioreactor: a potential bioartificial liver. I n Vitro Cell Dev. Biol. 1993,29A,249-254. (14) Matsushita, T.; Taniguchi, Y.; Ijima, H.; Funatsu, K. Development of a hybrid artificial liver using hepatocyte spheroidsPUF packed-bed. Published abstract, Japanese Association for Animal Cell Technology Annual Meeting, November 13-15, 1991. (15) Asano, K.; Koide, N.; Tsuji, T. Ultrastructure of multicellular spheroids formed in the primary culture of adult r a t hepatocytes. J . Clin. Electron Microsc. 1989,22,243-252. (16) Koide, N.; Shinji, T.; Tanabe, T.; Asano, K.; Kawaguchi, M.; Sakaguchi, K.; Koide, Y.; Mori, M.; Tsuji, T. Continued high albumin production by multicellular spheroids of adult rat hepatocytes formed in the presence of liver-derived proteoglycans. Biochem. Biophys. Res. Commun. 1989,161, 385-391. (17) McMillan, P. N.; Hixson, D. C.; Hevey, K. A.; Naik, S.; Jauregui, H. 0. Hepatocyte cell surface polarity as demonstrated by lectin binding. J . Histochem. Cytochem. 1988,36, 1561-1571. (18) Stamatoglou, S. C.; Hughes, R. C. Dynamic interactions of hepatocytes with fibronectin substrata: temporal and spatial changes in the distribution of adhesive contacts fibronectin receptors, and actin filaments. Exp. Cell Res. 1992,198,179-182. (19) Seglen, P. 0. Preparation of isolated r a t liver cells. Methods Cell Biol. 1976,13,29-83. (20) Nikolai, T. J.; Peshwa, M. V.; Goetghebeur, S.; Hu, W.-S. Improved microscopic observation of mammalian cells on microcarriers by fluorescent staining. Cytotechnology 1991, 5, 141-146. (21) Peshwa, M. V.; Kyung, Y.-S.; McClure, D. B.; Hu, W . 4 . Cultivation of mammalian cells as aggregates in bioreactorsEffect of calcium concentration on spatial distribution of viability. Biotechnol. Bioeng. 1993,41,179-187. Accepted April 19, 1994.@ @
Abstract published in Advance ACS Abstracts, June 1, 1994.
