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Formation of porcine hepatocyte spheroids for use in a bioartificial liver
Cell Transplantation, Vol. 4, No. 3, pp. 259-268, 1995 Copyright 0 1995Elsevier Science Ltd Printed in the USA. All rights reserved 0963.6897/95$9.50 + .OO
Pergamon 0963-6897(94)00058-l
Original Contribution FORMATION OF PORCINE HEPATOCYTE SPHEROIDS FOR USE IN A BIOARTIFICIAL LIVER ARYE
LAZAR, *l MADHUSJDAN
V. PESHWA,* FLORENCE J. Wu,*
FRANK B. CERRA,~
AND WEI-SHOU
Departments of *Chemical Engineering and Materials Science and tsurgery,
?? Abstract - Xenogeneic
CHUNG-MING
CHI,*
HUES
University of Minnesota, Minneapolis, MN 55455-0132
a hepatocyte-entrapment hollow fiber system as a bioartificial liver (BAL) device for interim support of patients in hepatic failure (17,22). This BAL is also being evaluated for use in in vitro studies of liver metabolism (18). By entrapping rat hepatocytes in such a BAL, biochemical functions were shown in an anhepatic rabbit model (15). To support patients in liver failure, large amounts of hepatocyte are required. A D-galactosamine induced liver failure model in canines was developed for further testing of this BAL (23). Logistical considerations of the hepatocyte harvest and bioreactor preparation make pigs a better source of hepatocytes than rats. Therefore, porcine hepatocytes were employed in the dog trials. Preliminary results indicate improved clinical parameters in dogs receiving BAL treatment as compared to the control group which did not receive BAL treatment (unpublished data). In the anticipated human trials or potential clinical use the of BAL, a much higher activity of liver functions will be needed. This requires the scale-up of the BAL used in the dog trials, either by increasing the number of hepatocytes in the BAL, or by enhancing the specific activities of hepatocytes, or by combining the two approaches. It has been reported that the growth viability and function of hepatocytes in culture are strongly affected by cell density and morphology (1,2,13,14). Higher cell density with cell-cell contact was correlated to a higher degree of differentiated functions and cell viability (8,26). Hepatocytes from newborn rats formed spheroids when cultured on a positively charged surface (7) or a surface coated with poly hydroxyethylmethacry-
hepatocytes have recently been used in a bioartificial liver device as a potential short-term extracorporeal support of acute liver failure. Scaling up the system requires large quantities of viable and highly active cells. Hepatocytes grown as spheroids manifest higher metabolic activities for longer time periods as compared to those in monolayer cultures. Use of hepatocyte spheroids for application in a bioartificial liver can possibly alleviate the need of scaling up. Porcine hepatocytes when cultured under stirred conditions, form multicellular spheroids in a defined culture medium. Spheroids were formed 24 h after cell inoculation with an efficiency of 80-90% and a mean diameter of about 135 pm. Scanning electron microscopy revealed numerous microvilli projecting from the entire surface of the spheroids. Transmission electron microscopy revealed differentiated hepatocytes which displayed well-developed cytoplasmic structures separated by bile canaliculus-like structures. The morphological studies show a resemblance between cells in the spheroids and in the liver in vivo. Ureagenesis by spheroids was twice as active and was sustained for a longer culture period than that by hepatocytes cultured as monolayers. Preparation of porcine hepatocyte spheroids in an agitated vessel is simple efficient and reproducible. It will allow for preparation of large quantities of spheroids to be employed in a bioartificial liver device as well as in liver metabolism studies.
0 Keywords - Porcine hepatocytes; Spheroids; Image analysis; Ureagenesis; Bioartificial liver. INTRODUCTION
Liver failure is a major cause of mortality. Except for liver transplantation effective treatments of many forms of liver failure are lacking. In addition, shortages of donor organs have resulted in a critical need of developing an artificial support. We have developed ACCEPTED 9/21/94. ‘Visiting from the Department of Biotechnology, Israel Institute for Biological Research, Ness-Ziona, 70450, Israel.
‘To whom correspondence
259
should
be addressed.
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late (HEMA) (10). Liver specific metabolic activities of hepatocytes which formed into spheroids were reported to be higher than those in monolayer cultures (8). Viability and albumin synthesis were maintained for a longer period. The aggregated cells exhibited a cuboidal shape and conserved cell-cell contacts. It was suggested that isolated liver cells possess and retain part of the information required to form a complex tissue architecture. This information provides an in vivo-like microenvironment that self-generates key factors to maintain viability and differentiated functions (10). Liver cells of both newborn and adult rats can form spheroidal aggregates in hormonally defined media (25). When such hormones and growth factors as dexamethasone, glucagon, insulin and epidermal growth factor (EGF) were added to culture medium, the secretion of albumin and transferrin remained detectable for at least two months. A minimum supplementation of 1 ng/mL EGF and 0.4 pg/mL insulin was found to be required for formation of spheroids (6). These factors enhance hepatocyte attachment during the early phase of culture, a process which is thought to be necessary before aggregation occurs. Adult rat hepatocytes in long-term spheroid culture can metabolize lidocaine to its main metabolite monoethylglycinexylidine (MEGX), up to 14 days in culture, indicating the presence of relatively high levels of cytochrome P-450 enzymes (15,18). Compared to unaggregated hepatocytes or hepatocytes entrapped in collagen gels, the specific MEGX production rate for hepatocyte spheroids was approximately two-fold higher (27). Other investigators reported high albumin and urea production by hepatocyte spheroids (24) for up to 15 days (4,11). The enhanced metabolic activity together with prolonged viability of hepatocyte spheroids make them attractive for use in toxicological and pharmacological studies as well as in bioartificial liver devices. However, the efficiency of spheroid formation is low. On tissue culture dishes only 30-40% of hepatocytes plated initially formed spheroids. The rate at which spheroids were formed was slow (4-6 days). Recently, improved methods for rat spheroid formation involving polyurethane foam reactor (5), and suspension culture methods (20) were reported. Human hepatocytes were also reported to form spheroids in a rotational tissue culture technique (12). However, little is known about spheroid formation by porcine hepatocytes and the resulting liver specific activities. Since our BAL employs porcine hepatocytes, we are interested in developing an efficient method of preparing porcine hepatocyte spheroids and in exploring the feasibility of entrapping spheroids in the BAL. Here we report that porcine
0 Volume 4, Number 3, 1995
hepatocytes do form spheroids when kept in suspension by agitation, and that these spheroids exhibit higher ureagenesis activities than those cultured on surfaces. MATERIALS
AND METHODS
Porcine Hepatocyte Harvest Hepatocytes were harvested from 8 to 10 kg male pigs by a two-step in situ collagenase perfusion technique modified from the original method developed for rat hepatocytes by Seglen (21). The porcine was first anesthetized with ketamine (100 mg/mL): rompun (100 mg/mL), 5 ml:lmL IM to allow for intubation and mechanical ventilation. The porcine was then anesthetized with isoflurane (1.5%) per endotracheal tube and paralyzed with succinylcholine (20 mg IV). The abdomen was entered through a bilateral subcostal chevron incision. The venous vascular supply to and from the liver was completely isolated and looped with ties. The hepatic artery, common bile duct gastrohepatic omentum and phrenic veins were ligated. The portal vein was cannulated with pump tubing and perfusion was initiated at 300 mL/min with oxygenated perfusion solution I (Per I). Per I is a of calciumfree solution with 143 mM sodium chloride, 6.7 mM potassium chloride, 10 mM hydroxyethylpiperazineethanesulfonic acid (HEPES)(Gibco, Grand Island, NY), 1 g/L ethylene glycol-bis-aminoethyl ether (EGTA), at pH 7.4. The suprahepatic and infrahepatic vena cavae were ligated, and a vent was made in the infrahepatic cava to modulate perfusion back pressure. The liver was excised, placed in a large sterile basin and perfused at 300 mL/min with oxygenated perfusion solution II (Per II). Per II consisted of 100 mM HEPES, 67 mM sodium chloride, 6.7 mM potassium chloride, 4.8 mM calcium chloride, 1% (v/v) bovine albumin and 1 g/L collagenase-D (Sigma Chemical Co, St. Louis, MO), pH 7.6. After 20-30 min, upon visual and palpable evidence of liver dissolving, the capsule was broken and the liver substance was raked and irrigated with cold William’s E medium (Gibco) supplemented with 15 mM HEPES, 0.2 U/mL insulin, 2 mM L-glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin. The released cells were filtered through nylon mesh with 100 pm openings and resuspended in fresh William’s E medium. Viability was assessed by trypan blue exclusion.
Culturing of Hepatocytes as Spheroids The isolated hepatocytes were resuspended in hormonally defined culture medium at a concentration of 0.5-1~10~ cells/ml. The medium was a modification of the serum-free medium of Enat et al. (3) con-
Porcine spheroid formation 0 A. LAZAR ET AL.
taining Williams E basal medium supplemented with 100 units/ml penicillin, 100 pg/mL streptomycin, 0.2 units/ml insulin (Lilly Co., Indianapolis, IN), 1 nmole/mL dexamethasone, 4 ng/mL glucagon, 25 pg/mL EGF, 20 ng/mL liver growth factor, 6.25 pg/mL transferrin, 50 ng/mL linoleic acid, 500 pg/mL albumin, 0.1 pm CuS04 5H20, 3 nM H2Se03, 50 pM ZnS047H2 0, and 15 mM HEPES (Gibco) at pH 7.4. All the medium supplements were from Sigma unless otherwise specified. The cell suspension was placed in 250 mL siliconized spinner flasks and stirred by a magnetic stirrer at 80 rpm in a humidified 5% CO2 incubator at 37°C. Medium was changed 24 h after cell inoculation and every 2-3 days thereafter by stopping agitation, allowing gravity sedimentation of the spheroids at room temperature, followed by aspiration of the spent medium and replacement by fresh medium. Viable cell count was performed by trypan blue exclusion. Spheroid diameter was assessed under an inverted microscope using a 10x ocular lens equipped with a vernier scale. Only particles of above 30 pm in diameter were counted. The average of lengths along two perpendicular axes of the spheroid was defined as the spheroid diameter. Between 80-100 spheroids were evaluated to obtain representative average diameters. Electron Microscopy Spheroids from 5 day-old culture were fixed with 4% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) for 90 min, followed by 2% 0~0~ for 30 min. The cells were dehydrated through increasing concentrations of ethanol. For scanning electron microscopy (SEM), cells were dried in a critical point dryer and coated with gold in a Sputter Coater (EMS 76M, Ernest Fullham, NY). The coated samples were observed under a scanning electron microscope (Hitachi, Japan). For transmission electron microscopy (TEM), after dehydration cells were embedded in Quetol and polymerized. Ultrathin sections were cut with a Reichert Ultracut E ultramicrotome, stained with uranyl acetate and observed under a transmission electron microscope (Hitachi, Japan). Confocal Microscopy Spheroid samples were stained with fluorescein diacetate (FDA) and ethidium bromide (EB) as described previously (16). Samples were examined with an epifluorescent inverted microscope (Olympus BH-2) linked with a confocal imaging system (MRC-500) and an argon ion laser light source (wavelength 488 nm). The confocal system was equipped with appropriate filters and two photomultiplier tubes for dual-wavelength imaging of FDA/EB stained samples. Optical sectioning of spheroids were performed serially at 5.4 pm inter-
261
vals by a computer-driven stepper motor. Hepatocyte viability at each section was assessed by counting the number of FDA-stained viable cells (green) and EBstained dead cells (red). The collected serial images were stacked together to reconstruct a three-dimensional projection of cell viability within the spheroids. Image Analysis of Particle Size Distribution Spheroid samples were placed on a petri dish and scanned using an inverted microscope (Carl Zeiss, Thornwood, NY) equipped with a video camera (Hamamatsu Photonics, Hamamatsu, Japan). A total of 75 microscopic fields were scanned with each sample. The acquired images were stored on an optical disk in a CD-ROM recorder (Matsushita, Osaka, Japan). Image preprocessing was performed on a microcomputer with array processor (Kontron Bildanalyse, Eching, Germany) to obtain the spheroid contours. A data file containing the coordinates of one contour point and the centroid, for each object, was created. Spheroid contours and data files were transferred concurrently to a workstation (Appolo Computer, Chelmsford, MA). The size distribution and estimated concentration of spheroids were obtained using the procedures described elsewhere (27). Urea and Protein Assays Urea levels were determined by the Urea Nitrogen Diagnostic Kit (Sigma). Urea production rates were calculated from urea levels in a 24 h culture supernatant divided by the cell number determined by protein content of the culture. Total protein was assayed by the Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce Chemical Company, Rockford, IL, Product No. 23225), using bovine serum albumin as a standard. RESULTS
Spheroid Formation Freshly isolated porcine hepatocytes aggregate very quickly in culture medium or in isolation buffer when kept in suspension after harvest. Within minutes after isolation from the liver, clumps of 3-10 cells were observed (Fig. la). The aggregates were loosely attached to each other and could be easily separated by repetitive pipetting. Within 24 h after being transferred to spinner flasks, the cells formed small spheroids of 40-70 pm in diameter and a mean diameter of 57.2 pm as determined by microscopic measurements (Fig. 2). Only those cell clumps exhibiting compact morphology and smooth surfaces were considered as spheroids. The clumps with relatively loosely associated cells and lumpy surfaces are referred to as aggregates. Photomicrographs taken at this stage showed a mixture of
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Fig. 1. Photomicrographs of primary porcine hepatocytes, x 167; (A) Immediately after isolation; (B) 24 h in stirred culture; and (C) 6 days in stirred culture.
(C)
single cells, aggregates and spheroids of various sizes (Fig. lb). Viable cell counts at 24 h after the start of the culture revealed that 80-90% of the viable hepatocytes were integrated into cell clumps. After 48 h in
1
4
2
Days
6
9
13
in Culture
Fig. 2. Spheroid diameter as a function of time in culture. Each column represents a mean of 80-100 microscopic measurements, error bars are standard deviation.
stirred culture, most of the cells appeared as spheroids with a mean diameter of 93 pm . The spheroids continued to grow in size up to an average diameter of 135 pm by day 6 (Fig. 2) forming compact cell structures of uniform size (Fig. lc). Later on, little increase in size was observed. Size distribution of cell clumps was measured by image analysis at days one, two and six. Two distinct cell populations could be seen: the hepatocyte fraction having a diameter range of 7-30 pm, and the spheroid fraction having a diameter of above 30 pm (Fig. 3). Hepatocytes grown in spinner cultures for 24 h show that 80% of the cells were located in the spheroid fraction with a mean diameter of 67 pm and an average of 90 cells per spheroid, determined by dividing the volume of the spheroid by volume of a single cell (Table 1). By the second day in culture, the spheroid diameter increased to an average of 98 pm, with an average of 282 cells per spheroid. This spheroid fraction accounts for 98% of the total population. At the sixth day in culture the spheroids reached a mean diameter of 142 pm containing 849 cells per spheroid. The image analysis based measurements are in agreement with the results obtained through quan-
Porcine spheroid formation 0 A. LAZAR ET AI..
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0.25 0.2 6 s
0.15
: I;:
0.1 0.05 0 1 o* Diameter (Fm)
Fig. 3. Size distribution (0): day 2; (A): day 6.
of porcine hepatocytes
by image analysis at 1, 2, and 6 days after cell inoculation.
titative microscopic analysis of spheroid diameter. It is unclear whether cell growth or coalescence of preformed spheroids is responsible for the increase of spheroid size. However, measurements of the total number of spheroids in the culture between the second and the sixth day revealed a decline in number from 2430 (k 360) to 305 (k 90) spheroids/ml. This suggests that small spheroids coalesce into larger ones. However, increase in cell volume (1) or growth of some mesenchymal cells (8) are also possible causes of such a change in mean diameter. Optical confocal microscopy was used to observe cell morphology and viability. Figure 4a depicts a reconstruction of 25 optical sections each 5.4 pm apart through a representative sample of single cells and
Table 1. Size distribution of hepatocytes and spheroids by image analysis Hepatocyte Fraction
Day 1
2 6
Spheroid Fraction
% of Total
Diameter (pm)
% of Total
Diameter (rm)
Cells/ Spheroid
20 2 1
14.9 + 6.6 15.4 f 6.5 15.4 f 6.5
80 98 99
67 + 30 98 k 59 140 f 90
90* 41 280 k 170 850 k 550
Cell diameter (average + standard deviation) and culture volume were calculated from measured surface area. Cell number in spheroids was calculated from average diameters and distribution frequency.
(0): day 1;
spheroids after 24 h in stirred culture. Only hepatocytes which formed spheroids were maintained viable (green) whereas single cells were dead (red). Figure 4b presents an optical section through the middle of a spheroid after six days of culture. The spheroid surface was smooth and undulating, cell viability as measured by FDA/EB staining indicated good viability with even distribution of dead and viable cells. Electron Microscopy Scanning electron microscopy of spheroids at five days in culture showed that most of them were relatively spherical except for some dumbbell shaped, ones which probably formed by the coalescence of two spheroids (Fig. 5a). At a higher magnification extensive cell-cell contact, numerous microvilli, and small (2-4 pm) holes on the surface of the spheroid can be seen (Fig. 5b). Such holes were frequently found at contact points of three cells (Fig. 5c,d). One may speculate that these pores on the spheroid surface are localized in areas of junction between adjacent cells and are surface openings of differentiated bile canaliculuslike structures. Transmission electron microscopy of hepatocytes in spheroids exhibited extensive cell-cell contact and nuclei of round or oval shape (Fig. 6a). Numerous mitochondria and lipid droplets of various sizes were observed in the cytoplasm of several cells (Fig. 6b). Morphological characteristics predominant in spheroids are junctional complexes such as desmosomes (Fig. 6c), and bile-canaliculus like structures between
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Fig. 4. Confocal micrographs of porcine hepatocytes (FDA) and dead cell nuclei stain red (EB).
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after; (A) 24 h and (B) six days in stirred culture.
hepatocytes (Fig. 6d). Continuous ductular structures of approximately 0.1 pm in diameter were distributed throughout the spheroid and can be seen to open as pores on the spheroid surface. Numerous microvilli protruded into these structures (Fig. 6d insert).
Viable cells stain green
Urea Production Ureagenesis by hepatocytes cultivated as spheroids or as monolayers on tissue culture plates was compared (Fig. 7). Both cultures were inoculated with 5 x lo5 cells/ml, culture medium was changed daily in the
Porcine spheroid formation 0 A. LAZAR ET AL.
265
(A)
Fig. 5. Scanning electron micrographs of porcine hepatocyte spheroids; (A) Spheroid size is relatively uniform. (B) Extensive cell-cell contact. (C) Numerous microvilli and some holes on the surface of a spheroid. (D) High magnification of a spheroid pore.
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(A)
(D) Fig. 6. Transmission electron micrographs of porcine hepatocyte spheroids; (A) Cells within the spheroid exhibit extensive cell-cell contact and differentiated cellular morphology, x3600. (B) Numerous mitochondria (m) and lipid droplets (1) in the cytoplasm of hepatocytes, x3600. (C) Desmosome (d) structures at cell-cell junctions, x 15,300. (D) Golgi (g) apparatus and bile canaliculi (bc)-like structure open as a pore on the spheroid surface, x 10,800. (D insert) Bile duct-like structure lined with microvilli.
first three days and every 3-4 days thereafter. Specific productivity by spheroid culture increased for the first three days to 320 pg/106 cells/day. Thereafter, the production rate continuously decreased probably due
to loss in cell viability. Hepatocytes cultured on tissue culture plates, exhibited lower cell productivity (80160 pg/106 cells/day), and were maintained in culture for shorter time period. During most of the studied
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Porcine spheroid formation 0 A. LAZAR ET AL.
_
Owl”““““““““” 0
8
4
12
16
20
Days in Culture Fig. 7. Urea production by hepatocytes cultivated as monolayer cultures on tissue culture dishes, and as spheroids culture. Error bars are standard deviations. (0): spheroids; (0): monolayer cultures.
period,
spheroids
productivity of hepatocytes cultured as was twice as high as monolayer cultures.
the
DISCUSSION
Many potential applications of primary hepatocytes cultured as spheroids including hepatocyte transplantation, drug metabolism and toxicological studies, could benefit from having a large number of hepatocytes which are metabolically active for an extended time period. Recently rat hepatocyte spheroids were used in bioreactors as potential bioartificial liver systems. The reported systems include culture in polyurethane foam matrix in a packed-bed reactor (5) culture in a tubular reactor packed with glass beads (11) encapsulation in calcium-alginate in a spouted bed chamber (5,9) inoculation of collagen-entrapped spheroids in the extracapillary space of a hollow fiber bioreactor (19). The entrapment of hepatocyte spheroids in a gel matrix within hollow fibers in a perfused bioreactor (28) has also currently been investigated in our laboratory. Morphological evaluation revealed the maintenance of differentiated cellular structures for hepatocytes in spheroids. One feature which stands out is the formation of an intricate continuous bile canalicular-like network within the spheroids. The extensive microvilli and junctional complex lining the canalicular channels may indicate maintenance of polarized hepatocyte morphology. The presence of vesicular bodies in these bilecanalicular like structures may possibly indicate the presence of an active secretory pathway for hepato-
in stirred
cytes in spheroids. These channels are envisioned to serve as open structures which facilitate diffusion of nutrients into and waste products out of the interior of the spheroids. Maintaining the cell-cell communication for hepatocytes within the spheroid appears beneficial for preservation of differentiated functions Culturing of hepatocytes as spheroids may be preferable to single-cell hepatocyte culture, as spheroids are metabolically more active and can be maintained in culture for longer time periods. Other liver functions, such as albumin production and drug metabolism capacity are now being studied to determine fully the metabolic activities of the spheroids. Because of the simplicity, the high reproducibility and the easy scaleup potential, this system may be used in an extracorporeal BAL device for support of patients in hepatic failure, and as an in vitro system for liver drug metabolism studies. Acknowledgments- We wish to acknowledge the technical assistance of Kristine Groehler, Daidre Olson, Gien-Liang Jim Hou. This work was partially supported by the Whitaker Foundation and Cellex Biosciences, Inc. REFERENCES 1. Asano, K.; Koide, N.; Tsuji, T. Ultrastructure of multicellular spheroids formed in the primary culture of adult rat hepatocytes. J. Clin. Electron Microsc. 22:243-252; 1989. 2. Ben-Ze’ev, A.; Robinson, G.S.; Bucher, N.L.R.; Farmer, S.R. Cell-cell and cell-matrix interaction differentially
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0 Volume 4, Number 3, 1995 14. Nakumura, T.; Yoshimoto, K.; Nakayama, Y.; Tomita, Y.; Ichihara, A. Reciprocal modulation of growth and differentiated functions of mature rat hepatocytes in primary culture by cell-cell contact and cell membranes. Proc. Natl. Acad. Sci. 80:7229-7233; 1983. 15. Nyberg, S.L.; Mann, H.J.; Remmel, R.P.; Hu, W.-S.; Cerra, F.B. Pharmacokinetic analysis verifies P450 function during in vitro and in vivo application of a bioartificial liver. ASAIO J. 39:M252-M256; 1993. 16. Nyberg, S.L.; Shatford, R.A.; Payne, W.D.; Hu, W.-S,; Cerra, F.B. Staining with fluorescein diacetate correlates with hepatocyte function. Biotechnic and Histochemistry 68:56-63; 1993. 17. Nyberg, S.L.; Shatford, R.A.; Peshwa, M.V.; White, J.G.; Cerra, F.B.; Hu, W.-S. Evaluation of a hepatocyteentrapment hollow fiber bioreactor: a potential bioartificial liver. Biotechnol. Bioeng. 41: 194-203; 1992. 18. Nyberg, S.L.; Shirabe, K.; Peshwa, M.V.; Sielaff, T.D.; Crotty, P.L.; Mann, H.J.; Remmel, R.P.; Payne, W.D.; Hu, W.-S.; Cerra, F.B. Extracorporeal application of a gel-entrapment, bioartificial liver: demonstration of drug metabolism and other biochemical functions. Cell Transplant. 21:441-452; 1993. 19. Sakai, Y.; Suzuki, M. A hollow fiber type bioartificial liver using hepatocyte spheroids entrapped in collagen gel. Extended Abstract, Japanese Association of Animal Cell Technology Annual Meeting. Nagoya, Japan; 1993. 20. Sakai, Y.; Suzuki, M. Stable immobilization and functional expression under conditions of actual clinical uses of a bioartificial liver of hepatocyte spheroids rapidly formed by suspension culture. Jpn. J. Artif. Organs 22: 164-170; 1993. 21. Seglen, P.O. Preparation of isolated rat liver cells. Meth. Cell. Biol. 13:29-38; 1976. 22. Shatford, R.A.; Nyberg, S.L.; Meier, S.J.; White, J.G.; Payne, W.D.; Hu, W.-S.; Cerra, F.B. Hepatocyte function in a hollow fiber bioreactor: a potential bioartificial liver. J. Surgical Res. 53:549-557; 1992. 23. 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 dog model of acute fulminant hepatitis: Surgical Forum 49:61-63; 1993. 24. Takabatake, H.; Koide, N.; Tsuji, T. Encapsulated multicellular spheroids of rat hepatocytes produce albumin and urea in a spouted bed circulating culture system. Artif. Organs 15:474-480; 1991. 25. Tong, J.Z.; Bernarn, 0.; Alvarez, F. Long-term culture of rat liver cell spheroids in hormonally defined media. Exp. Cell Res. 189:87-92; 1990. 26. Tong, J.Z.; De Lagausie, P.; Furlan, V.; Cresteil, T.; Bernard, 0.; Alvarez, F. Long-term culture of adult rat hepatocyte spheroids. Exp. Cell Res. 200:326-332; 1992. 27. Vits, H.; Chi, C.-M.; Staba, E.J.; Cooke,T.J.; Hu, W.S. Characterization of patterns in plant somatic embryo cultures: the morphology and development of embryos. AIChE J. 40:1728-1740; 1994. 28. Wu, F.J.; Peshwa, M.V.; Cerra, F.B.; Hu, W.-S. Entrapment of hepatocyte spheroids in a hollow fiber bioreactor as a potential bioartificial liver. Tissue Eng. (in press).
Pergamon 0963-6897(94)00058-l
Original Contribution FORMATION OF PORCINE HEPATOCYTE SPHEROIDS FOR USE IN A BIOARTIFICIAL LIVER ARYE
LAZAR, *l MADHUSJDAN
V. PESHWA,* FLORENCE J. Wu,*
FRANK B. CERRA,~
AND WEI-SHOU
Departments of *Chemical Engineering and Materials Science and tsurgery,
?? Abstract - Xenogeneic
CHUNG-MING
CHI,*
HUES
University of Minnesota, Minneapolis, MN 55455-0132
a hepatocyte-entrapment hollow fiber system as a bioartificial liver (BAL) device for interim support of patients in hepatic failure (17,22). This BAL is also being evaluated for use in in vitro studies of liver metabolism (18). By entrapping rat hepatocytes in such a BAL, biochemical functions were shown in an anhepatic rabbit model (15). To support patients in liver failure, large amounts of hepatocyte are required. A D-galactosamine induced liver failure model in canines was developed for further testing of this BAL (23). Logistical considerations of the hepatocyte harvest and bioreactor preparation make pigs a better source of hepatocytes than rats. Therefore, porcine hepatocytes were employed in the dog trials. Preliminary results indicate improved clinical parameters in dogs receiving BAL treatment as compared to the control group which did not receive BAL treatment (unpublished data). In the anticipated human trials or potential clinical use the of BAL, a much higher activity of liver functions will be needed. This requires the scale-up of the BAL used in the dog trials, either by increasing the number of hepatocytes in the BAL, or by enhancing the specific activities of hepatocytes, or by combining the two approaches. It has been reported that the growth viability and function of hepatocytes in culture are strongly affected by cell density and morphology (1,2,13,14). Higher cell density with cell-cell contact was correlated to a higher degree of differentiated functions and cell viability (8,26). Hepatocytes from newborn rats formed spheroids when cultured on a positively charged surface (7) or a surface coated with poly hydroxyethylmethacry-
hepatocytes have recently been used in a bioartificial liver device as a potential short-term extracorporeal support of acute liver failure. Scaling up the system requires large quantities of viable and highly active cells. Hepatocytes grown as spheroids manifest higher metabolic activities for longer time periods as compared to those in monolayer cultures. Use of hepatocyte spheroids for application in a bioartificial liver can possibly alleviate the need of scaling up. Porcine hepatocytes when cultured under stirred conditions, form multicellular spheroids in a defined culture medium. Spheroids were formed 24 h after cell inoculation with an efficiency of 80-90% and a mean diameter of about 135 pm. Scanning electron microscopy revealed numerous microvilli projecting from the entire surface of the spheroids. Transmission electron microscopy revealed differentiated hepatocytes which displayed well-developed cytoplasmic structures separated by bile canaliculus-like structures. The morphological studies show a resemblance between cells in the spheroids and in the liver in vivo. Ureagenesis by spheroids was twice as active and was sustained for a longer culture period than that by hepatocytes cultured as monolayers. Preparation of porcine hepatocyte spheroids in an agitated vessel is simple efficient and reproducible. It will allow for preparation of large quantities of spheroids to be employed in a bioartificial liver device as well as in liver metabolism studies.
0 Keywords - Porcine hepatocytes; Spheroids; Image analysis; Ureagenesis; Bioartificial liver. INTRODUCTION
Liver failure is a major cause of mortality. Except for liver transplantation effective treatments of many forms of liver failure are lacking. In addition, shortages of donor organs have resulted in a critical need of developing an artificial support. We have developed ACCEPTED 9/21/94. ‘Visiting from the Department of Biotechnology, Israel Institute for Biological Research, Ness-Ziona, 70450, Israel.
‘To whom correspondence
259
should
be addressed.
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late (HEMA) (10). Liver specific metabolic activities of hepatocytes which formed into spheroids were reported to be higher than those in monolayer cultures (8). Viability and albumin synthesis were maintained for a longer period. The aggregated cells exhibited a cuboidal shape and conserved cell-cell contacts. It was suggested that isolated liver cells possess and retain part of the information required to form a complex tissue architecture. This information provides an in vivo-like microenvironment that self-generates key factors to maintain viability and differentiated functions (10). Liver cells of both newborn and adult rats can form spheroidal aggregates in hormonally defined media (25). When such hormones and growth factors as dexamethasone, glucagon, insulin and epidermal growth factor (EGF) were added to culture medium, the secretion of albumin and transferrin remained detectable for at least two months. A minimum supplementation of 1 ng/mL EGF and 0.4 pg/mL insulin was found to be required for formation of spheroids (6). These factors enhance hepatocyte attachment during the early phase of culture, a process which is thought to be necessary before aggregation occurs. Adult rat hepatocytes in long-term spheroid culture can metabolize lidocaine to its main metabolite monoethylglycinexylidine (MEGX), up to 14 days in culture, indicating the presence of relatively high levels of cytochrome P-450 enzymes (15,18). Compared to unaggregated hepatocytes or hepatocytes entrapped in collagen gels, the specific MEGX production rate for hepatocyte spheroids was approximately two-fold higher (27). Other investigators reported high albumin and urea production by hepatocyte spheroids (24) for up to 15 days (4,11). The enhanced metabolic activity together with prolonged viability of hepatocyte spheroids make them attractive for use in toxicological and pharmacological studies as well as in bioartificial liver devices. However, the efficiency of spheroid formation is low. On tissue culture dishes only 30-40% of hepatocytes plated initially formed spheroids. The rate at which spheroids were formed was slow (4-6 days). Recently, improved methods for rat spheroid formation involving polyurethane foam reactor (5), and suspension culture methods (20) were reported. Human hepatocytes were also reported to form spheroids in a rotational tissue culture technique (12). However, little is known about spheroid formation by porcine hepatocytes and the resulting liver specific activities. Since our BAL employs porcine hepatocytes, we are interested in developing an efficient method of preparing porcine hepatocyte spheroids and in exploring the feasibility of entrapping spheroids in the BAL. Here we report that porcine
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hepatocytes do form spheroids when kept in suspension by agitation, and that these spheroids exhibit higher ureagenesis activities than those cultured on surfaces. MATERIALS
AND METHODS
Porcine Hepatocyte Harvest Hepatocytes were harvested from 8 to 10 kg male pigs by a two-step in situ collagenase perfusion technique modified from the original method developed for rat hepatocytes by Seglen (21). The porcine was first anesthetized with ketamine (100 mg/mL): rompun (100 mg/mL), 5 ml:lmL IM to allow for intubation and mechanical ventilation. The porcine was then anesthetized with isoflurane (1.5%) per endotracheal tube and paralyzed with succinylcholine (20 mg IV). The abdomen was entered through a bilateral subcostal chevron incision. The venous vascular supply to and from the liver was completely isolated and looped with ties. The hepatic artery, common bile duct gastrohepatic omentum and phrenic veins were ligated. The portal vein was cannulated with pump tubing and perfusion was initiated at 300 mL/min with oxygenated perfusion solution I (Per I). Per I is a of calciumfree solution with 143 mM sodium chloride, 6.7 mM potassium chloride, 10 mM hydroxyethylpiperazineethanesulfonic acid (HEPES)(Gibco, Grand Island, NY), 1 g/L ethylene glycol-bis-aminoethyl ether (EGTA), at pH 7.4. The suprahepatic and infrahepatic vena cavae were ligated, and a vent was made in the infrahepatic cava to modulate perfusion back pressure. The liver was excised, placed in a large sterile basin and perfused at 300 mL/min with oxygenated perfusion solution II (Per II). Per II consisted of 100 mM HEPES, 67 mM sodium chloride, 6.7 mM potassium chloride, 4.8 mM calcium chloride, 1% (v/v) bovine albumin and 1 g/L collagenase-D (Sigma Chemical Co, St. Louis, MO), pH 7.6. After 20-30 min, upon visual and palpable evidence of liver dissolving, the capsule was broken and the liver substance was raked and irrigated with cold William’s E medium (Gibco) supplemented with 15 mM HEPES, 0.2 U/mL insulin, 2 mM L-glutamine, 100 U/mL penicillin and 100 pg/mL streptomycin. The released cells were filtered through nylon mesh with 100 pm openings and resuspended in fresh William’s E medium. Viability was assessed by trypan blue exclusion.
Culturing of Hepatocytes as Spheroids The isolated hepatocytes were resuspended in hormonally defined culture medium at a concentration of 0.5-1~10~ cells/ml. The medium was a modification of the serum-free medium of Enat et al. (3) con-
Porcine spheroid formation 0 A. LAZAR ET AL.
taining Williams E basal medium supplemented with 100 units/ml penicillin, 100 pg/mL streptomycin, 0.2 units/ml insulin (Lilly Co., Indianapolis, IN), 1 nmole/mL dexamethasone, 4 ng/mL glucagon, 25 pg/mL EGF, 20 ng/mL liver growth factor, 6.25 pg/mL transferrin, 50 ng/mL linoleic acid, 500 pg/mL albumin, 0.1 pm CuS04 5H20, 3 nM H2Se03, 50 pM ZnS047H2 0, and 15 mM HEPES (Gibco) at pH 7.4. All the medium supplements were from Sigma unless otherwise specified. The cell suspension was placed in 250 mL siliconized spinner flasks and stirred by a magnetic stirrer at 80 rpm in a humidified 5% CO2 incubator at 37°C. Medium was changed 24 h after cell inoculation and every 2-3 days thereafter by stopping agitation, allowing gravity sedimentation of the spheroids at room temperature, followed by aspiration of the spent medium and replacement by fresh medium. Viable cell count was performed by trypan blue exclusion. Spheroid diameter was assessed under an inverted microscope using a 10x ocular lens equipped with a vernier scale. Only particles of above 30 pm in diameter were counted. The average of lengths along two perpendicular axes of the spheroid was defined as the spheroid diameter. Between 80-100 spheroids were evaluated to obtain representative average diameters. Electron Microscopy Spheroids from 5 day-old culture were fixed with 4% glutaraldehyde in 0.2 M sodium cacodylate buffer (pH 7.2) for 90 min, followed by 2% 0~0~ for 30 min. The cells were dehydrated through increasing concentrations of ethanol. For scanning electron microscopy (SEM), cells were dried in a critical point dryer and coated with gold in a Sputter Coater (EMS 76M, Ernest Fullham, NY). The coated samples were observed under a scanning electron microscope (Hitachi, Japan). For transmission electron microscopy (TEM), after dehydration cells were embedded in Quetol and polymerized. Ultrathin sections were cut with a Reichert Ultracut E ultramicrotome, stained with uranyl acetate and observed under a transmission electron microscope (Hitachi, Japan). Confocal Microscopy Spheroid samples were stained with fluorescein diacetate (FDA) and ethidium bromide (EB) as described previously (16). Samples were examined with an epifluorescent inverted microscope (Olympus BH-2) linked with a confocal imaging system (MRC-500) and an argon ion laser light source (wavelength 488 nm). The confocal system was equipped with appropriate filters and two photomultiplier tubes for dual-wavelength imaging of FDA/EB stained samples. Optical sectioning of spheroids were performed serially at 5.4 pm inter-
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vals by a computer-driven stepper motor. Hepatocyte viability at each section was assessed by counting the number of FDA-stained viable cells (green) and EBstained dead cells (red). The collected serial images were stacked together to reconstruct a three-dimensional projection of cell viability within the spheroids. Image Analysis of Particle Size Distribution Spheroid samples were placed on a petri dish and scanned using an inverted microscope (Carl Zeiss, Thornwood, NY) equipped with a video camera (Hamamatsu Photonics, Hamamatsu, Japan). A total of 75 microscopic fields were scanned with each sample. The acquired images were stored on an optical disk in a CD-ROM recorder (Matsushita, Osaka, Japan). Image preprocessing was performed on a microcomputer with array processor (Kontron Bildanalyse, Eching, Germany) to obtain the spheroid contours. A data file containing the coordinates of one contour point and the centroid, for each object, was created. Spheroid contours and data files were transferred concurrently to a workstation (Appolo Computer, Chelmsford, MA). The size distribution and estimated concentration of spheroids were obtained using the procedures described elsewhere (27). Urea and Protein Assays Urea levels were determined by the Urea Nitrogen Diagnostic Kit (Sigma). Urea production rates were calculated from urea levels in a 24 h culture supernatant divided by the cell number determined by protein content of the culture. Total protein was assayed by the Bicinchoninic Acid (BCA) Protein Assay Kit (Pierce Chemical Company, Rockford, IL, Product No. 23225), using bovine serum albumin as a standard. RESULTS
Spheroid Formation Freshly isolated porcine hepatocytes aggregate very quickly in culture medium or in isolation buffer when kept in suspension after harvest. Within minutes after isolation from the liver, clumps of 3-10 cells were observed (Fig. la). The aggregates were loosely attached to each other and could be easily separated by repetitive pipetting. Within 24 h after being transferred to spinner flasks, the cells formed small spheroids of 40-70 pm in diameter and a mean diameter of 57.2 pm as determined by microscopic measurements (Fig. 2). Only those cell clumps exhibiting compact morphology and smooth surfaces were considered as spheroids. The clumps with relatively loosely associated cells and lumpy surfaces are referred to as aggregates. Photomicrographs taken at this stage showed a mixture of
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Fig. 1. Photomicrographs of primary porcine hepatocytes, x 167; (A) Immediately after isolation; (B) 24 h in stirred culture; and (C) 6 days in stirred culture.
(C)
single cells, aggregates and spheroids of various sizes (Fig. lb). Viable cell counts at 24 h after the start of the culture revealed that 80-90% of the viable hepatocytes were integrated into cell clumps. After 48 h in
1
4
2
Days
6
9
13
in Culture
Fig. 2. Spheroid diameter as a function of time in culture. Each column represents a mean of 80-100 microscopic measurements, error bars are standard deviation.
stirred culture, most of the cells appeared as spheroids with a mean diameter of 93 pm . The spheroids continued to grow in size up to an average diameter of 135 pm by day 6 (Fig. 2) forming compact cell structures of uniform size (Fig. lc). Later on, little increase in size was observed. Size distribution of cell clumps was measured by image analysis at days one, two and six. Two distinct cell populations could be seen: the hepatocyte fraction having a diameter range of 7-30 pm, and the spheroid fraction having a diameter of above 30 pm (Fig. 3). Hepatocytes grown in spinner cultures for 24 h show that 80% of the cells were located in the spheroid fraction with a mean diameter of 67 pm and an average of 90 cells per spheroid, determined by dividing the volume of the spheroid by volume of a single cell (Table 1). By the second day in culture, the spheroid diameter increased to an average of 98 pm, with an average of 282 cells per spheroid. This spheroid fraction accounts for 98% of the total population. At the sixth day in culture the spheroids reached a mean diameter of 142 pm containing 849 cells per spheroid. The image analysis based measurements are in agreement with the results obtained through quan-
Porcine spheroid formation 0 A. LAZAR ET AI..
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0.25 0.2 6 s
0.15
: I;:
0.1 0.05 0 1 o* Diameter (Fm)
Fig. 3. Size distribution (0): day 2; (A): day 6.
of porcine hepatocytes
by image analysis at 1, 2, and 6 days after cell inoculation.
titative microscopic analysis of spheroid diameter. It is unclear whether cell growth or coalescence of preformed spheroids is responsible for the increase of spheroid size. However, measurements of the total number of spheroids in the culture between the second and the sixth day revealed a decline in number from 2430 (k 360) to 305 (k 90) spheroids/ml. This suggests that small spheroids coalesce into larger ones. However, increase in cell volume (1) or growth of some mesenchymal cells (8) are also possible causes of such a change in mean diameter. Optical confocal microscopy was used to observe cell morphology and viability. Figure 4a depicts a reconstruction of 25 optical sections each 5.4 pm apart through a representative sample of single cells and
Table 1. Size distribution of hepatocytes and spheroids by image analysis Hepatocyte Fraction
Day 1
2 6
Spheroid Fraction
% of Total
Diameter (pm)
% of Total
Diameter (rm)
Cells/ Spheroid
20 2 1
14.9 + 6.6 15.4 f 6.5 15.4 f 6.5
80 98 99
67 + 30 98 k 59 140 f 90
90* 41 280 k 170 850 k 550
Cell diameter (average + standard deviation) and culture volume were calculated from measured surface area. Cell number in spheroids was calculated from average diameters and distribution frequency.
(0): day 1;
spheroids after 24 h in stirred culture. Only hepatocytes which formed spheroids were maintained viable (green) whereas single cells were dead (red). Figure 4b presents an optical section through the middle of a spheroid after six days of culture. The spheroid surface was smooth and undulating, cell viability as measured by FDA/EB staining indicated good viability with even distribution of dead and viable cells. Electron Microscopy Scanning electron microscopy of spheroids at five days in culture showed that most of them were relatively spherical except for some dumbbell shaped, ones which probably formed by the coalescence of two spheroids (Fig. 5a). At a higher magnification extensive cell-cell contact, numerous microvilli, and small (2-4 pm) holes on the surface of the spheroid can be seen (Fig. 5b). Such holes were frequently found at contact points of three cells (Fig. 5c,d). One may speculate that these pores on the spheroid surface are localized in areas of junction between adjacent cells and are surface openings of differentiated bile canaliculuslike structures. Transmission electron microscopy of hepatocytes in spheroids exhibited extensive cell-cell contact and nuclei of round or oval shape (Fig. 6a). Numerous mitochondria and lipid droplets of various sizes were observed in the cytoplasm of several cells (Fig. 6b). Morphological characteristics predominant in spheroids are junctional complexes such as desmosomes (Fig. 6c), and bile-canaliculus like structures between
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Fig. 4. Confocal micrographs of porcine hepatocytes (FDA) and dead cell nuclei stain red (EB).
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after; (A) 24 h and (B) six days in stirred culture.
hepatocytes (Fig. 6d). Continuous ductular structures of approximately 0.1 pm in diameter were distributed throughout the spheroid and can be seen to open as pores on the spheroid surface. Numerous microvilli protruded into these structures (Fig. 6d insert).
Viable cells stain green
Urea Production Ureagenesis by hepatocytes cultivated as spheroids or as monolayers on tissue culture plates was compared (Fig. 7). Both cultures were inoculated with 5 x lo5 cells/ml, culture medium was changed daily in the
Porcine spheroid formation 0 A. LAZAR ET AL.
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(A)
Fig. 5. Scanning electron micrographs of porcine hepatocyte spheroids; (A) Spheroid size is relatively uniform. (B) Extensive cell-cell contact. (C) Numerous microvilli and some holes on the surface of a spheroid. (D) High magnification of a spheroid pore.
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(A)
(D) Fig. 6. Transmission electron micrographs of porcine hepatocyte spheroids; (A) Cells within the spheroid exhibit extensive cell-cell contact and differentiated cellular morphology, x3600. (B) Numerous mitochondria (m) and lipid droplets (1) in the cytoplasm of hepatocytes, x3600. (C) Desmosome (d) structures at cell-cell junctions, x 15,300. (D) Golgi (g) apparatus and bile canaliculi (bc)-like structure open as a pore on the spheroid surface, x 10,800. (D insert) Bile duct-like structure lined with microvilli.
first three days and every 3-4 days thereafter. Specific productivity by spheroid culture increased for the first three days to 320 pg/106 cells/day. Thereafter, the production rate continuously decreased probably due
to loss in cell viability. Hepatocytes cultured on tissue culture plates, exhibited lower cell productivity (80160 pg/106 cells/day), and were maintained in culture for shorter time period. During most of the studied
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Porcine spheroid formation 0 A. LAZAR ET AL.
_
Owl”““““““““” 0
8
4
12
16
20
Days in Culture Fig. 7. Urea production by hepatocytes cultivated as monolayer cultures on tissue culture dishes, and as spheroids culture. Error bars are standard deviations. (0): spheroids; (0): monolayer cultures.
period,
spheroids
productivity of hepatocytes cultured as was twice as high as monolayer cultures.
the
DISCUSSION
Many potential applications of primary hepatocytes cultured as spheroids including hepatocyte transplantation, drug metabolism and toxicological studies, could benefit from having a large number of hepatocytes which are metabolically active for an extended time period. Recently rat hepatocyte spheroids were used in bioreactors as potential bioartificial liver systems. The reported systems include culture in polyurethane foam matrix in a packed-bed reactor (5) culture in a tubular reactor packed with glass beads (11) encapsulation in calcium-alginate in a spouted bed chamber (5,9) inoculation of collagen-entrapped spheroids in the extracapillary space of a hollow fiber bioreactor (19). The entrapment of hepatocyte spheroids in a gel matrix within hollow fibers in a perfused bioreactor (28) has also currently been investigated in our laboratory. Morphological evaluation revealed the maintenance of differentiated cellular structures for hepatocytes in spheroids. One feature which stands out is the formation of an intricate continuous bile canalicular-like network within the spheroids. The extensive microvilli and junctional complex lining the canalicular channels may indicate maintenance of polarized hepatocyte morphology. The presence of vesicular bodies in these bilecanalicular like structures may possibly indicate the presence of an active secretory pathway for hepato-
in stirred
cytes in spheroids. These channels are envisioned to serve as open structures which facilitate diffusion of nutrients into and waste products out of the interior of the spheroids. Maintaining the cell-cell communication for hepatocytes within the spheroid appears beneficial for preservation of differentiated functions Culturing of hepatocytes as spheroids may be preferable to single-cell hepatocyte culture, as spheroids are metabolically more active and can be maintained in culture for longer time periods. Other liver functions, such as albumin production and drug metabolism capacity are now being studied to determine fully the metabolic activities of the spheroids. Because of the simplicity, the high reproducibility and the easy scaleup potential, this system may be used in an extracorporeal BAL device for support of patients in hepatic failure, and as an in vitro system for liver drug metabolism studies. Acknowledgments- We wish to acknowledge the technical assistance of Kristine Groehler, Daidre Olson, Gien-Liang Jim Hou. This work was partially supported by the Whitaker Foundation and Cellex Biosciences, Inc. REFERENCES 1. Asano, K.; Koide, N.; Tsuji, T. Ultrastructure of multicellular spheroids formed in the primary culture of adult rat hepatocytes. J. Clin. Electron Microsc. 22:243-252; 1989. 2. Ben-Ze’ev, A.; Robinson, G.S.; Bucher, N.L.R.; Farmer, S.R. Cell-cell and cell-matrix interaction differentially
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0 Volume 4, Number 3, 1995 14. Nakumura, T.; Yoshimoto, K.; Nakayama, Y.; Tomita, Y.; Ichihara, A. Reciprocal modulation of growth and differentiated functions of mature rat hepatocytes in primary culture by cell-cell contact and cell membranes. Proc. Natl. Acad. Sci. 80:7229-7233; 1983. 15. Nyberg, S.L.; Mann, H.J.; Remmel, R.P.; Hu, W.-S.; Cerra, F.B. Pharmacokinetic analysis verifies P450 function during in vitro and in vivo application of a bioartificial liver. ASAIO J. 39:M252-M256; 1993. 16. Nyberg, S.L.; Shatford, R.A.; Payne, W.D.; Hu, W.-S,; Cerra, F.B. Staining with fluorescein diacetate correlates with hepatocyte function. Biotechnic and Histochemistry 68:56-63; 1993. 17. Nyberg, S.L.; Shatford, R.A.; Peshwa, M.V.; White, J.G.; Cerra, F.B.; Hu, W.-S. Evaluation of a hepatocyteentrapment hollow fiber bioreactor: a potential bioartificial liver. Biotechnol. Bioeng. 41: 194-203; 1992. 18. Nyberg, S.L.; Shirabe, K.; Peshwa, M.V.; Sielaff, T.D.; Crotty, P.L.; Mann, H.J.; Remmel, R.P.; Payne, W.D.; Hu, W.-S.; Cerra, F.B. Extracorporeal application of a gel-entrapment, bioartificial liver: demonstration of drug metabolism and other biochemical functions. Cell Transplant. 21:441-452; 1993. 19. Sakai, Y.; Suzuki, M. A hollow fiber type bioartificial liver using hepatocyte spheroids entrapped in collagen gel. Extended Abstract, Japanese Association of Animal Cell Technology Annual Meeting. Nagoya, Japan; 1993. 20. Sakai, Y.; Suzuki, M. Stable immobilization and functional expression under conditions of actual clinical uses of a bioartificial liver of hepatocyte spheroids rapidly formed by suspension culture. Jpn. J. Artif. Organs 22: 164-170; 1993. 21. Seglen, P.O. Preparation of isolated rat liver cells. Meth. Cell. Biol. 13:29-38; 1976. 22. Shatford, R.A.; Nyberg, S.L.; Meier, S.J.; White, J.G.; Payne, W.D.; Hu, W.-S.; Cerra, F.B. Hepatocyte function in a hollow fiber bioreactor: a potential bioartificial liver. J. Surgical Res. 53:549-557; 1992. 23. 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 dog model of acute fulminant hepatitis: Surgical Forum 49:61-63; 1993. 24. Takabatake, H.; Koide, N.; Tsuji, T. Encapsulated multicellular spheroids of rat hepatocytes produce albumin and urea in a spouted bed circulating culture system. Artif. Organs 15:474-480; 1991. 25. Tong, J.Z.; Bernarn, 0.; Alvarez, F. Long-term culture of rat liver cell spheroids in hormonally defined media. Exp. Cell Res. 189:87-92; 1990. 26. Tong, J.Z.; De Lagausie, P.; Furlan, V.; Cresteil, T.; Bernard, 0.; Alvarez, F. Long-term culture of adult rat hepatocyte spheroids. Exp. Cell Res. 200:326-332; 1992. 27. Vits, H.; Chi, C.-M.; Staba, E.J.; Cooke,T.J.; Hu, W.S. Characterization of patterns in plant somatic embryo cultures: the morphology and development of embryos. AIChE J. 40:1728-1740; 1994. 28. Wu, F.J.; Peshwa, M.V.; Cerra, F.B.; Hu, W.-S. Entrapment of hepatocyte spheroids in a hollow fiber bioreactor as a potential bioartificial liver. Tissue Eng. (in press).