Cytotechnology10: 205-215, 1992. 9 1992KluwerAcademicPublishers.Printedin the Netherlands.
Primary culture of rat hepatocytes entrapped in cylindrical collagen gels: An in vitro system with application to the bioartificial liver Rat hepatocytes cultured in cylindrical collagen gels
Scott L. Nyberg 1,2, Russell A. Shatford 1, William D. Payne ~, Wei-Shou Hu 2 and Frank B. Cerra 1
1Department of Surgery and 2Department of Chemical Engineering and Materials Science University of Minnesota, Minneapolis, MN 55455, USA Received 9 October 1991; accepted in revisedform 24 February 1992
Key words: artificial organ, bioartificial liver, gel entrapment, hepatocyte, metabolism
Abstract
A static culture model employing cylindrical collagen-hepatocyte gels is reported for large scale testing of conditions relevant to the three compartment hollow fiber bioartificial liver. High density hepatocyte cultivation was achieved by cell entrapment within the collagen-hepatocyte gel. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, lidocaine biotransformation, and cholate conjugation. Although hepatocyte viability remained stable through the seven day incubation period, hepatocyte functions were not uniformly preserved. Albumin synthesis remained stable, while representative P-450 and conjugation activities decreased with time. This static culture system will facilitate the development of a hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.
Abbreviations: FDA - fluorescein diacetate; EB - ethidium bromide; MEGX - monoethylglycinexylidide
Introduction
The development of a bioartificial liver requires a technology wherein normal metabolic functions can be maintained by large numbers of liver cells in high density. The use of gel entrapment of hepatocytes is one such technology (Akimoto, 1985; Miura, 1986; Yanagi, 1989; Hu, 1991). Cultivation of mammalian cells on reconstituted collagen has been studied for over 35 years (Ehrmann, 1956). It is well established that collagen (gel or membrane) provides an adequate substratum for cultivation of differentiated mammalian
cells (Elsdale, 1972; Michalopoulos, 1975; Bell, 1979; Anderson, 1990). In the case of liver epithelial cells, collagen provides a matrix for cellcell contact, which is felt to be important for maintenance of organ-specific function (Rubin, 1984; Goulet, 1988). Hepatocytes can be dispersed on collagen gels (Michalopoulos, 1975), sandwiched between collagen gels (Dunn, 1989), or entrapped (suspended) in collagen gels (Akimoto, 1985). Along with preserved liver cell morphology and prolonged tyrosine amino transferase activity, Michalopoulos observed that parenchymal liver cells
206 dispersed on floating collagen gel were capable of gel contraction (Michalopoulos, 1975). Maintenance of cell polarity by sandwiching hepatocytes between two layers of collagen gel was associated with improved viability and prolonged liverspecific function during in vitro cultivation (Dunn, 1989). Likewise, entrapment of rat hepatocytes in collagen gels at high cell density (0.31.0 • 106 cells/ml) was associated with high activities of liver specific functions (Akimoto, 1985). Addition of various growth hormones (Koch, 1990) and extracellular matrix components (Bucher, 1990) at the time of cell entrapment may improve hepatocyte performance and promote hepatocyte regeneration. Besides collagen, other materials used for hepatocyte entrapment include fibrin, calcium alginate, carrageenin, agar, agarose and hydrogel (Akimoto, 1985; Miura, 1986; Yanagi, 1989). Urea synthesis, indole conjugation, phenol conjugation, and short chain fatty acid metabolism were reported by calcium alginate entrapped hepatocytes (Miura, 1986). In recent years, calcium alginate has increased in use for cultivation of hepatocytes (Cai, 1988; Kashani, 1988; Tompkins, 1988), possibly because of its favorable gelling properties and ease of preparation (Akimoto, 1985). Nonetheless, entrapment within type I collagen, calcium alginate, or a similar gelling polymer allows anchorage-dependent cell cultivation at significantly higher density than is possible with standard monolayer culture techniques. Gel entrapment of mammalian cells in a hollow fiber bioreactor was recently described (Scholz, 1991). This bioreactor configuration has been proposed for the development of a three compartment hollow fiber bioartificial liver (Nyberg, 1990; Hu, 1991; Shatford, 1991). The three compartments of this extracorporeal support device consist of the following: (1) an extracapillary space located outside the hollow fibers is perfused with blood from a patient with liver failure; (2) a highly porous collagen gel containing hepatocytes entrapped at high cell density is located within the hollow fiber lumens; mass transfer to and from the intraluminal hepatocytes occurs
through the walls of the porous hollow fibers; (3) an intraluminal channel is also present within the hollow fibers for supply of large molecular weight growth factors to the gel-entrapped hepatocyte~ metabolic products such as conjugated bilirubin and drug metabolites may also be eliminated by the intraluminal channel. Optimization of such a gel-entrapment bioreactor requires an appropriate static culture model for efficient testing of multiple cultivation parameters. In the current study, we report a new static culture technique for cultivation of hepatocytes entrapped in collagen gel (collagen-hepatocyte gels). The new static culture system allowed the study of hepatocytes at high density within intact cylindrical gels, and the study of individual hepatocytes following collagenase digestion of the collagen matrix. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, P-450 biotransformation of lidocaine (phase I activity), and cholic acid conjugation (phase II activity). This technology will facilitate the development of a three compartment hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.
Materials and methods Cell harvest
Hepatocytes were harvested from 4-6 week old male Sprague-Dawley rats, weighing 200-250 grams by a two-step in situ collagenase perfusion technique modified from the method described by Seglen (Seglen, 1976). Briefly, following intraperitoneal pentobarbital injection (4.5 mg/100 gm body weight) and portal vein cannulation, in vivo perfusion (25 ml/min • 15 min) was performed with a calcium-free hydroxyethylpiperazineethanesulfonic acid (HEPES) buffered solution (143 mM NaC1, 6.7 mM KC1, 10 mM HEPES, 100 mg% ethylene glycol-bis-aminoethyl ether (EGTA), pH 7.4), and then perfused (20 ml/min x 10 min) with a second HEPES buffered solution
207 (67 mM NaC1, 6.7 mM KCI, 4.8 mM CaC12, 100 mM HEPES, 1.0 g% fatty acid free bovine albumin, pH 7.6) containing 0.05% collagenase D (clostridiopeptidase A, Boehringer Mannheim Corp., Indianapolis, IN). The liver capsule was peeled back from all lobes, and the liver was then gently combed to isolate hepatocytes. The hepatocyte pellet was resuspended in harvest medium (Williams E medium (Williams, 1974) supplemented with 5% calf serum, 2 mM L-glutamine, 15 mM HEPES, 1.5 mg/l insulin, 10,000 U/I penicillin G, 100 mg/l streptomycin sulfate) following gauze filtration and three centrifugation (50 x g for 1 minute) washings in harvest medium. Each rat harvest yielded from 3.0-5.0 x 108 hepatocytes with average viability ranging from 85 to 90% by both trypan blue exclusion and fluorescent vital staining (described below).
Hepatocyte entrapment Hepatocytes which had been resuspended in harvest medium were centrifuged (50 x g for 1 minute) to form a soft cell pellet. Supematant was removed and the hepatocytes were resuspended in collagen solution (3 to 1 mixture by volume of type 1 collagen solution (Vitrogen 100, Collagen Corp., Palo Alto, CA) and 4 fold concentrated William's E medium supplemented with 8.0 mM L-glutamine, 40 mg/l insulin, 40,000 U/1 penicillin G, 400 mg/1 streptomycin sulfate, pH 7.5). Hepatocytes were suspended at 0.2-2.0 x 107 cells/ml. The collagen-cell suspension was then inoculated into gas sterilized 0.64 m m l D x 1.20 mm OD silicone tubing (Dow, Coming, MI) with a tuberculin syringe and a 23 gage hypodermic needle. Inoculated tubing was incubated at 37~ for ten minutes to accelerate gel formation.
Cultivation of hepatocytes entrapped in collagen gel Following 10 minutes of incubation, newly formed cylindrical gels were transferred from the silicone tubing into individual wells of a 24 well plate. Gels were removed from the tubing by applying
gentle pressure to one end of the tubing with a tuberculin syringe and a 23 gage hypodermic needle. Each well contained one gel which measured 5 cm in length, 17 ~tl in volume, and contained approximately 75,000 hepatocytes. Twenty-four well plates were maintained at 37~ in a 5% CO e incubator. Serum-free culture medium reported by Lanford (William's E medium supplemented with 100 ng/ml epidermal growth factor, 10 ~tg/ml insulin, 4 ~tg/ml glucagon, 100 ng/ml prolactiri, 1 ~tg/ml somatotropin, 2 ng/ml cholera toxin, 20 ng/ml glycyl-histidyl-lysine, 5 ~tg/ml transferrin, 0.5 mg/ml albumin, 5 ~tg/ml linoleic acid, 10-6 M thyrotropin releasing factor, 10-6M hydrocortisone, 10-7 M selenium, 10-6 M ethanolamine) was used for hepatocyte cultivation (Lanford, 1989). Culture medium (0.5 ml/ well) was changed daily and collected for appropriate functional assays. Samples were stored at -20~ prior to analysis.
Viability staining technique Hepatocyte viability was determined with an fluorescein diacetate:ethidium bromide (FDA:EB) stain (Nikolai, 1991). FDA stock solution (5 mg/ml in acetone) was stored at -20~ EB (10 ~tg/ml) in phosphate buffered saline was stored at 4~ The working FDA:EB (5 ~tg/ml):(10 ~tg/ml) solution was prepared fresh before each use by combining 10 ~tl of FDA stock solution and 10 ml of EB stock solution. Viability of individual hepatocytes following gel degradation was determined with the working FDA:EB stain. Following removal of the culture medium, collagen gels were digested with 0.25 ml collagenase solution (clostridiopeptidase A, Sigma #C-6885 type II, 530 U/ml in PBS) to each well. Incubation (25 to 30 minutes, 37~ was allowed for collagen digestion. Gentle pipetting of the partially digested gels improved cell dispersion. Each well then received 0.25 ml FDA:EB working solution and was incubated for 5 minutes at 37~ In most cases, the gel was totally dissolved after this period of incubation. In some older cultures, however, it was difficult to completely separate all hepatocytes; small (5-10 cell)
208 clumps of hepatocytes were noted. Further incubation in collagenase solution did not disperse such hepatocyte aggregates. Hepatocyte viability was quantified with a hemocytometer and epifluorescent microscope configured for FITC (450-490 nm excitation filter, 510 nm barrier filter). Viability was assessed by counting from 100 to 200 cells in several fields. Cells with green staining cytoplasm were scored as viable. Cells with orange staining nuclei were scored as dead. Unstained cells, and cells with both green cytoplasm and orange nuclei, were rarely observed and not scored. Hepatocyte viability distribution was also assessed on intact FDA:EB stained gels by standard epifluorescent microscopy and confocal microscopy (Van Der Voort, 1989). The confocal apparatus was described previously for three-dimensional imaging of isolated islets of Langerhans (Brelje, 1989), and mammalian cells entrapped in macroporous microcarriers (Nikolai, 1991). In order to assess hepatocyte viability by confocal microscopy, intact collagen-hepatocyte gels were stained with working FDA:EB solution, washed in PBS solution, and optically sectioned.
Measurement of gel contraction The rate of gel contraction by the entrapped hepatocytes was measured using disk-shaped collagen gels. This technique was described previously for measuring collagen contraction by fibroblasts (Bell, 1979) and recombinant human cell, 293 (Scholz, 1991). Collagen-cell suspensions were made with hepatocyte concentrations ranging from 0.2 to 1.8 x 107 cells/ml. Collagen gel disks were formed by adding 0.25 ml of collagencell suspension into individual wells (1.6 cm diameter) of a 24 well plate. Plates were incubated at 37~ for 10 minutes to accelerate gel formation. Gels containing entrapped hepatocytes were then freed from the bottom of the plate with a sterile glass spatula. Culture medium (0.5 ml) was added to each well, and the plates were maintained at 37~ in a 5% CO 2 incubator. Gel diameters were measured daily using a ruler technique (Bell, 1979), Gels maintained well formed
disk structure during contraction. The average of the major and minor axis was taken as the diameter.
Insulin concentrations and ureagenesis Insulin levels were determined by a double antibody radioimmunoassay (Ventrex Laboratories, Cat, #72656, Portland, ME). Appropriate dilutions were performed for insulin level determination on a standardized curve. Urea levels were determined by high performance liquid chromatography (Konstantinides, 1987) from culture medium sampled on day 1 and day 2.
Measurement of albumin concentration Rat albumin concentrations were determined by competitive ELISA. Briefly, unknown samples and 10 gg/ml rat albumin standards (Sigma, St. Louis, MO) were serially diluted, and combined with peroxidase bound polyclonal rabbit anti-rat albumin (Organon Teknika, Durham, NC) at a final concentration of 1.29 gg protein/ml. Samples were incubated at 37~ for 2 hours. 100 pl aliquots of each sample were transferred to a precoated maxisorp plate (Nunc, Naperville, IL). Precoating included: (1) addition of 100 I.tl of rat albumin (100 ng/ml in PBS) to each well; (2) two hour incubation at room temperature in a humidified chamber; (3) wash wells three times with 0.05% Tween-20 (Bior-Rad Labs, Richmond, CA) in PBS. Following addition of 100 gl aliquots, precoated plates were returned to the room temperature humidified chamber. After two hours, plates were removed from the humidified chamber and again washed three times with 0.05% Tween-20 in PBS. Each well then received 100 gl (55 mg/ml) of 2,2,Azino-di-[3-ethylbenzthiazoline-6-sulfonate] (Boehringer Mannheim, Indianapolis, IN) in buffer (0.1 M Na Citrate, pH 4.2, with 0.1 gl 30% H2Offl00 gl). The plates were returned to the humidified chamber for 1 hour. Plates were read on an automated plate reader at 405-490 nm. Of note, culture medium contained bovine serum albumin. For this reason, the polyclonal rabbit antibody was preincubated with bo-
209 vine albumin solution (3 gm/dl in 0.05% Tween20 in PBS) in order to block sites cross-reactive with bovine albumin. Serial dilutions of standards and unknown samples were also performed in bovine albumin solution.
Lidocaine biotransformation Lidocaine biotransformation was assessed by the appearance of the N-deethylation intermediate, monoethylglycinexylidide (MEGX). Cylindrical gels were incubated in 0.5 ml culture medium containing 2 mg/1 lidocaine for 60 minutes. MEGX levels were determined by a fluorescence polarization immunoassay (Abbott Laboratories, Chicago, IL) (Oellerich, 1987).
Taurochola~ appearance Measurement of taurocholate appearance in culture medium has been described previously (Boelsterli, 1988). Briefly, hepatocytes were incubated in fresh medium (0.5 mI per well) supplemented with 1.0 mM taurine and 4.22 ~tM [carboxy-14C] cholic acid. Medium was collected after 90 minutes of incubation and stored at -20~ Samples were later thawed and following cholate extraction in ethyl acetate, 14C-taurocholate levels were determined by scintillation counting of residual radioactivity in the aqueous phase.
Statistical test Unpaired student's t-test was used to determine statistical significance between control and study groups. Significance was defined as p < 0.05 between groups. Variables for comparison included percent viability, urea production, albumin production, MEGX production, and taurocholate formation. Average values (+ standard error of the mean [SEM]) are reported. A simple regression test was used to assess the significance of gel contraction and urea production.
Results
Hepatocyte viability in static culture Hepatocyte viability was determined by FDA:EB staining before and after gel digestion with collagenase. In order to determine hepatocyte viability before collagenase digestion, intact cylindrical collagen gels were stained with FDA:EB and examined with standard epifluorescence microscopy (Fig. l a - c ) and with confocal microscopy (Fig. ld). The change in hepatocyte viability from day 0 (shortly after gel entrapment) until day 7 is shown in Fig. l a,b. Cytoplasmic and nuclear detail were better appreciated at high magnification (Fig. lc,d). Confocal microscopy enabled the examination of multiple hepatocytes at high magnification even after being entrapped in gel at a high cell density. In general, static culture hepatocyte viability before and after collagenase digestion correlated well. Average hepatocyte viability after gel degradation in collagenase solution is summarized in Fig. 2. Hepatocyte viability was determined on day 0 (soon after gel formation) and on days 2,4,6,8. Initial harvest viability ranged between 85 and 90% by both trypan blue exclusion and FDA:EB staining. The viability remained above 40% after 8 days in culture. A significant drop was noted between day 0 (76.5 +_ 1.7%) and day 2 (46.8 _+4.0%), however, viability remained stable for the remainder of the cultivation period. Differences between day 2 and day 8 (44.1 + 5.9%) average viability were not statistically significant.
Determination of gel contraction Gel diameters were measured daily in well formed collagen discs. Figure 3 demonstrates the average diameter of 12 gel discs measured daily over a 10 day period. Initial gel diameter was 1.6 cm. The decrease in average gel diameter was highly significant (p < 0.001) over the first week of cultivation and then stabilized at approximately 60% of the initial gel diameter. Gel contraction was not significant after day 7. Control gels,
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211 Fig. 1. (Previous page) Standard epifluorescence micrographs (Fig. la--c) and a confocal micrograph (Fig. ld) of FDA:EB stained hepatocytes entrapped within cylindrical collagen gels. The distribution of viable cells with green cytoplasm (c) and dead cells with orange nuclei (n) within intact gels is demonstrated six hours (Fig. la), thirty hours (Fig. lc,d), and seven days (Fig. lb) after gel entrapment. Examination of gel-entrapped hepatocytes at high magnification (Fig. lc,d) is improved with confocal microscopy (Fig. ld). The confocal micrograph was generated by the superimposition often longitudinal images optically sectioned at 10 lam intervals through an intact gel. Magnifications: Fig. la (x75); Fig. lb (x150); Fig. lc (x600); Figure ld (xl000).
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containing no ceils or containing cells killed by exposure to 50% ethanol for 10 minutes, showed no signs of contraction. Cell density influenced the rate of gel contraction, and, to a lesser extent, the extent of gel contraction (data not shown). Gel contraction by hepatocytes did not appear to be as rapid as gel contraction reported for cultured fibroblasts (Bell, 1979). In summary, Fig. 3 demonstrates that viable hepatocytes were necessary for gel contraction and that gel contraction proceeded gradually over the first week of cultivation.
Insulin profiles Insulin concentrations remained relatively stable in cell-free control gels and in culture medium containing no gel or no hepatocytes. However, in medium from wells containing gel entrapped hepatocytes, less than 10% of the insulin was detected after the first 10 hours of culture, and less than 1% of the insulin was detected after 24 hours. Based on these findings, medium was
Fig. 3. Contraction of collagen gel discs containing entrapped hepatocytes. Control gels (closed circles) contained no cells or ethanol killed cells. Significant contraction was measured over the first week of culture, while contraction was not significant after day 7. Open circles represent the average of t 2 gel discs containing 0.2-1.8 x 107 hepatocytes per ml of gel.
changed in subsequent experiments every 24 hours in order to avoid insulin depletion.
Urea profiles Media from six gels were analyzed for urea concentration after 24 hours and 48 hours. Average urea concentrations (+ SEM) after 24 hours were 755 (+ 19) I.tmol/l, and after 48 hours were 1413 (+ 106) txmol/1. Urea production was significant during the first 48 hours of cultivation (R a = 0.938). No urea was detected in control wells containing only medium or cell-free gels.
Albumin production Figure 4 demonstrates stable albumin production by rat hepatocytes in cylindrical collagen gels over an 8 day period. Each data point represents the albumin production by 7.5 • 104 cells over a 24 hour period in 0.5 ml culture medium. From
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compared to no cell controls, however, MEGX production fell steadily over this period. The decrease in MEGX production was not significant from day 4 to day 6, but was significant between earlier successive measurements. Taurocholate appearance
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Primary culture of rat hepatocytes entrapped in cylindrical collagen gels: An in vitro system with application to the bioartificial liver Rat hepatocytes cultured in cylindrical collagen gels
Scott L. Nyberg 1,2, Russell A. Shatford 1, William D. Payne ~, Wei-Shou Hu 2 and Frank B. Cerra 1
1Department of Surgery and 2Department of Chemical Engineering and Materials Science University of Minnesota, Minneapolis, MN 55455, USA Received 9 October 1991; accepted in revisedform 24 February 1992
Key words: artificial organ, bioartificial liver, gel entrapment, hepatocyte, metabolism
Abstract
A static culture model employing cylindrical collagen-hepatocyte gels is reported for large scale testing of conditions relevant to the three compartment hollow fiber bioartificial liver. High density hepatocyte cultivation was achieved by cell entrapment within the collagen-hepatocyte gel. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, lidocaine biotransformation, and cholate conjugation. Although hepatocyte viability remained stable through the seven day incubation period, hepatocyte functions were not uniformly preserved. Albumin synthesis remained stable, while representative P-450 and conjugation activities decreased with time. This static culture system will facilitate the development of a hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.
Abbreviations: FDA - fluorescein diacetate; EB - ethidium bromide; MEGX - monoethylglycinexylidide
Introduction
The development of a bioartificial liver requires a technology wherein normal metabolic functions can be maintained by large numbers of liver cells in high density. The use of gel entrapment of hepatocytes is one such technology (Akimoto, 1985; Miura, 1986; Yanagi, 1989; Hu, 1991). Cultivation of mammalian cells on reconstituted collagen has been studied for over 35 years (Ehrmann, 1956). It is well established that collagen (gel or membrane) provides an adequate substratum for cultivation of differentiated mammalian
cells (Elsdale, 1972; Michalopoulos, 1975; Bell, 1979; Anderson, 1990). In the case of liver epithelial cells, collagen provides a matrix for cellcell contact, which is felt to be important for maintenance of organ-specific function (Rubin, 1984; Goulet, 1988). Hepatocytes can be dispersed on collagen gels (Michalopoulos, 1975), sandwiched between collagen gels (Dunn, 1989), or entrapped (suspended) in collagen gels (Akimoto, 1985). Along with preserved liver cell morphology and prolonged tyrosine amino transferase activity, Michalopoulos observed that parenchymal liver cells
206 dispersed on floating collagen gel were capable of gel contraction (Michalopoulos, 1975). Maintenance of cell polarity by sandwiching hepatocytes between two layers of collagen gel was associated with improved viability and prolonged liverspecific function during in vitro cultivation (Dunn, 1989). Likewise, entrapment of rat hepatocytes in collagen gels at high cell density (0.31.0 • 106 cells/ml) was associated with high activities of liver specific functions (Akimoto, 1985). Addition of various growth hormones (Koch, 1990) and extracellular matrix components (Bucher, 1990) at the time of cell entrapment may improve hepatocyte performance and promote hepatocyte regeneration. Besides collagen, other materials used for hepatocyte entrapment include fibrin, calcium alginate, carrageenin, agar, agarose and hydrogel (Akimoto, 1985; Miura, 1986; Yanagi, 1989). Urea synthesis, indole conjugation, phenol conjugation, and short chain fatty acid metabolism were reported by calcium alginate entrapped hepatocytes (Miura, 1986). In recent years, calcium alginate has increased in use for cultivation of hepatocytes (Cai, 1988; Kashani, 1988; Tompkins, 1988), possibly because of its favorable gelling properties and ease of preparation (Akimoto, 1985). Nonetheless, entrapment within type I collagen, calcium alginate, or a similar gelling polymer allows anchorage-dependent cell cultivation at significantly higher density than is possible with standard monolayer culture techniques. Gel entrapment of mammalian cells in a hollow fiber bioreactor was recently described (Scholz, 1991). This bioreactor configuration has been proposed for the development of a three compartment hollow fiber bioartificial liver (Nyberg, 1990; Hu, 1991; Shatford, 1991). The three compartments of this extracorporeal support device consist of the following: (1) an extracapillary space located outside the hollow fibers is perfused with blood from a patient with liver failure; (2) a highly porous collagen gel containing hepatocytes entrapped at high cell density is located within the hollow fiber lumens; mass transfer to and from the intraluminal hepatocytes occurs
through the walls of the porous hollow fibers; (3) an intraluminal channel is also present within the hollow fibers for supply of large molecular weight growth factors to the gel-entrapped hepatocyte~ metabolic products such as conjugated bilirubin and drug metabolites may also be eliminated by the intraluminal channel. Optimization of such a gel-entrapment bioreactor requires an appropriate static culture model for efficient testing of multiple cultivation parameters. In the current study, we report a new static culture technique for cultivation of hepatocytes entrapped in collagen gel (collagen-hepatocyte gels). The new static culture system allowed the study of hepatocytes at high density within intact cylindrical gels, and the study of individual hepatocytes following collagenase digestion of the collagen matrix. Hepatocyte viability was assessed by vital staining, gel contraction, and insulin utilization. Measures of hepatocyte-specific function included albumin synthesis, ureagenesis, P-450 biotransformation of lidocaine (phase I activity), and cholic acid conjugation (phase II activity). This technology will facilitate the development of a three compartment hollow fiber bioartificial liver which utilizes cylindrical collagen-hepatocyte gels.
Materials and methods Cell harvest
Hepatocytes were harvested from 4-6 week old male Sprague-Dawley rats, weighing 200-250 grams by a two-step in situ collagenase perfusion technique modified from the method described by Seglen (Seglen, 1976). Briefly, following intraperitoneal pentobarbital injection (4.5 mg/100 gm body weight) and portal vein cannulation, in vivo perfusion (25 ml/min • 15 min) was performed with a calcium-free hydroxyethylpiperazineethanesulfonic acid (HEPES) buffered solution (143 mM NaC1, 6.7 mM KC1, 10 mM HEPES, 100 mg% ethylene glycol-bis-aminoethyl ether (EGTA), pH 7.4), and then perfused (20 ml/min x 10 min) with a second HEPES buffered solution
207 (67 mM NaC1, 6.7 mM KCI, 4.8 mM CaC12, 100 mM HEPES, 1.0 g% fatty acid free bovine albumin, pH 7.6) containing 0.05% collagenase D (clostridiopeptidase A, Boehringer Mannheim Corp., Indianapolis, IN). The liver capsule was peeled back from all lobes, and the liver was then gently combed to isolate hepatocytes. The hepatocyte pellet was resuspended in harvest medium (Williams E medium (Williams, 1974) supplemented with 5% calf serum, 2 mM L-glutamine, 15 mM HEPES, 1.5 mg/l insulin, 10,000 U/I penicillin G, 100 mg/l streptomycin sulfate) following gauze filtration and three centrifugation (50 x g for 1 minute) washings in harvest medium. Each rat harvest yielded from 3.0-5.0 x 108 hepatocytes with average viability ranging from 85 to 90% by both trypan blue exclusion and fluorescent vital staining (described below).
Hepatocyte entrapment Hepatocytes which had been resuspended in harvest medium were centrifuged (50 x g for 1 minute) to form a soft cell pellet. Supematant was removed and the hepatocytes were resuspended in collagen solution (3 to 1 mixture by volume of type 1 collagen solution (Vitrogen 100, Collagen Corp., Palo Alto, CA) and 4 fold concentrated William's E medium supplemented with 8.0 mM L-glutamine, 40 mg/l insulin, 40,000 U/1 penicillin G, 400 mg/1 streptomycin sulfate, pH 7.5). Hepatocytes were suspended at 0.2-2.0 x 107 cells/ml. The collagen-cell suspension was then inoculated into gas sterilized 0.64 m m l D x 1.20 mm OD silicone tubing (Dow, Coming, MI) with a tuberculin syringe and a 23 gage hypodermic needle. Inoculated tubing was incubated at 37~ for ten minutes to accelerate gel formation.
Cultivation of hepatocytes entrapped in collagen gel Following 10 minutes of incubation, newly formed cylindrical gels were transferred from the silicone tubing into individual wells of a 24 well plate. Gels were removed from the tubing by applying
gentle pressure to one end of the tubing with a tuberculin syringe and a 23 gage hypodermic needle. Each well contained one gel which measured 5 cm in length, 17 ~tl in volume, and contained approximately 75,000 hepatocytes. Twenty-four well plates were maintained at 37~ in a 5% CO e incubator. Serum-free culture medium reported by Lanford (William's E medium supplemented with 100 ng/ml epidermal growth factor, 10 ~tg/ml insulin, 4 ~tg/ml glucagon, 100 ng/ml prolactiri, 1 ~tg/ml somatotropin, 2 ng/ml cholera toxin, 20 ng/ml glycyl-histidyl-lysine, 5 ~tg/ml transferrin, 0.5 mg/ml albumin, 5 ~tg/ml linoleic acid, 10-6 M thyrotropin releasing factor, 10-6M hydrocortisone, 10-7 M selenium, 10-6 M ethanolamine) was used for hepatocyte cultivation (Lanford, 1989). Culture medium (0.5 ml/ well) was changed daily and collected for appropriate functional assays. Samples were stored at -20~ prior to analysis.
Viability staining technique Hepatocyte viability was determined with an fluorescein diacetate:ethidium bromide (FDA:EB) stain (Nikolai, 1991). FDA stock solution (5 mg/ml in acetone) was stored at -20~ EB (10 ~tg/ml) in phosphate buffered saline was stored at 4~ The working FDA:EB (5 ~tg/ml):(10 ~tg/ml) solution was prepared fresh before each use by combining 10 ~tl of FDA stock solution and 10 ml of EB stock solution. Viability of individual hepatocytes following gel degradation was determined with the working FDA:EB stain. Following removal of the culture medium, collagen gels were digested with 0.25 ml collagenase solution (clostridiopeptidase A, Sigma #C-6885 type II, 530 U/ml in PBS) to each well. Incubation (25 to 30 minutes, 37~ was allowed for collagen digestion. Gentle pipetting of the partially digested gels improved cell dispersion. Each well then received 0.25 ml FDA:EB working solution and was incubated for 5 minutes at 37~ In most cases, the gel was totally dissolved after this period of incubation. In some older cultures, however, it was difficult to completely separate all hepatocytes; small (5-10 cell)
208 clumps of hepatocytes were noted. Further incubation in collagenase solution did not disperse such hepatocyte aggregates. Hepatocyte viability was quantified with a hemocytometer and epifluorescent microscope configured for FITC (450-490 nm excitation filter, 510 nm barrier filter). Viability was assessed by counting from 100 to 200 cells in several fields. Cells with green staining cytoplasm were scored as viable. Cells with orange staining nuclei were scored as dead. Unstained cells, and cells with both green cytoplasm and orange nuclei, were rarely observed and not scored. Hepatocyte viability distribution was also assessed on intact FDA:EB stained gels by standard epifluorescent microscopy and confocal microscopy (Van Der Voort, 1989). The confocal apparatus was described previously for three-dimensional imaging of isolated islets of Langerhans (Brelje, 1989), and mammalian cells entrapped in macroporous microcarriers (Nikolai, 1991). In order to assess hepatocyte viability by confocal microscopy, intact collagen-hepatocyte gels were stained with working FDA:EB solution, washed in PBS solution, and optically sectioned.
Measurement of gel contraction The rate of gel contraction by the entrapped hepatocytes was measured using disk-shaped collagen gels. This technique was described previously for measuring collagen contraction by fibroblasts (Bell, 1979) and recombinant human cell, 293 (Scholz, 1991). Collagen-cell suspensions were made with hepatocyte concentrations ranging from 0.2 to 1.8 x 107 cells/ml. Collagen gel disks were formed by adding 0.25 ml of collagencell suspension into individual wells (1.6 cm diameter) of a 24 well plate. Plates were incubated at 37~ for 10 minutes to accelerate gel formation. Gels containing entrapped hepatocytes were then freed from the bottom of the plate with a sterile glass spatula. Culture medium (0.5 ml) was added to each well, and the plates were maintained at 37~ in a 5% CO 2 incubator. Gel diameters were measured daily using a ruler technique (Bell, 1979), Gels maintained well formed
disk structure during contraction. The average of the major and minor axis was taken as the diameter.
Insulin concentrations and ureagenesis Insulin levels were determined by a double antibody radioimmunoassay (Ventrex Laboratories, Cat, #72656, Portland, ME). Appropriate dilutions were performed for insulin level determination on a standardized curve. Urea levels were determined by high performance liquid chromatography (Konstantinides, 1987) from culture medium sampled on day 1 and day 2.
Measurement of albumin concentration Rat albumin concentrations were determined by competitive ELISA. Briefly, unknown samples and 10 gg/ml rat albumin standards (Sigma, St. Louis, MO) were serially diluted, and combined with peroxidase bound polyclonal rabbit anti-rat albumin (Organon Teknika, Durham, NC) at a final concentration of 1.29 gg protein/ml. Samples were incubated at 37~ for 2 hours. 100 pl aliquots of each sample were transferred to a precoated maxisorp plate (Nunc, Naperville, IL). Precoating included: (1) addition of 100 I.tl of rat albumin (100 ng/ml in PBS) to each well; (2) two hour incubation at room temperature in a humidified chamber; (3) wash wells three times with 0.05% Tween-20 (Bior-Rad Labs, Richmond, CA) in PBS. Following addition of 100 gl aliquots, precoated plates were returned to the room temperature humidified chamber. After two hours, plates were removed from the humidified chamber and again washed three times with 0.05% Tween-20 in PBS. Each well then received 100 gl (55 mg/ml) of 2,2,Azino-di-[3-ethylbenzthiazoline-6-sulfonate] (Boehringer Mannheim, Indianapolis, IN) in buffer (0.1 M Na Citrate, pH 4.2, with 0.1 gl 30% H2Offl00 gl). The plates were returned to the humidified chamber for 1 hour. Plates were read on an automated plate reader at 405-490 nm. Of note, culture medium contained bovine serum albumin. For this reason, the polyclonal rabbit antibody was preincubated with bo-
209 vine albumin solution (3 gm/dl in 0.05% Tween20 in PBS) in order to block sites cross-reactive with bovine albumin. Serial dilutions of standards and unknown samples were also performed in bovine albumin solution.
Lidocaine biotransformation Lidocaine biotransformation was assessed by the appearance of the N-deethylation intermediate, monoethylglycinexylidide (MEGX). Cylindrical gels were incubated in 0.5 ml culture medium containing 2 mg/1 lidocaine for 60 minutes. MEGX levels were determined by a fluorescence polarization immunoassay (Abbott Laboratories, Chicago, IL) (Oellerich, 1987).
Taurochola~ appearance Measurement of taurocholate appearance in culture medium has been described previously (Boelsterli, 1988). Briefly, hepatocytes were incubated in fresh medium (0.5 mI per well) supplemented with 1.0 mM taurine and 4.22 ~tM [carboxy-14C] cholic acid. Medium was collected after 90 minutes of incubation and stored at -20~ Samples were later thawed and following cholate extraction in ethyl acetate, 14C-taurocholate levels were determined by scintillation counting of residual radioactivity in the aqueous phase.
Statistical test Unpaired student's t-test was used to determine statistical significance between control and study groups. Significance was defined as p < 0.05 between groups. Variables for comparison included percent viability, urea production, albumin production, MEGX production, and taurocholate formation. Average values (+ standard error of the mean [SEM]) are reported. A simple regression test was used to assess the significance of gel contraction and urea production.
Results
Hepatocyte viability in static culture Hepatocyte viability was determined by FDA:EB staining before and after gel digestion with collagenase. In order to determine hepatocyte viability before collagenase digestion, intact cylindrical collagen gels were stained with FDA:EB and examined with standard epifluorescence microscopy (Fig. l a - c ) and with confocal microscopy (Fig. ld). The change in hepatocyte viability from day 0 (shortly after gel entrapment) until day 7 is shown in Fig. l a,b. Cytoplasmic and nuclear detail were better appreciated at high magnification (Fig. lc,d). Confocal microscopy enabled the examination of multiple hepatocytes at high magnification even after being entrapped in gel at a high cell density. In general, static culture hepatocyte viability before and after collagenase digestion correlated well. Average hepatocyte viability after gel degradation in collagenase solution is summarized in Fig. 2. Hepatocyte viability was determined on day 0 (soon after gel formation) and on days 2,4,6,8. Initial harvest viability ranged between 85 and 90% by both trypan blue exclusion and FDA:EB staining. The viability remained above 40% after 8 days in culture. A significant drop was noted between day 0 (76.5 +_ 1.7%) and day 2 (46.8 _+4.0%), however, viability remained stable for the remainder of the cultivation period. Differences between day 2 and day 8 (44.1 + 5.9%) average viability were not statistically significant.
Determination of gel contraction Gel diameters were measured daily in well formed collagen discs. Figure 3 demonstrates the average diameter of 12 gel discs measured daily over a 10 day period. Initial gel diameter was 1.6 cm. The decrease in average gel diameter was highly significant (p < 0.001) over the first week of cultivation and then stabilized at approximately 60% of the initial gel diameter. Gel contraction was not significant after day 7. Control gels,
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211 Fig. 1. (Previous page) Standard epifluorescence micrographs (Fig. la--c) and a confocal micrograph (Fig. ld) of FDA:EB stained hepatocytes entrapped within cylindrical collagen gels. The distribution of viable cells with green cytoplasm (c) and dead cells with orange nuclei (n) within intact gels is demonstrated six hours (Fig. la), thirty hours (Fig. lc,d), and seven days (Fig. lb) after gel entrapment. Examination of gel-entrapped hepatocytes at high magnification (Fig. lc,d) is improved with confocal microscopy (Fig. ld). The confocal micrograph was generated by the superimposition often longitudinal images optically sectioned at 10 lam intervals through an intact gel. Magnifications: Fig. la (x75); Fig. lb (x150); Fig. lc (x600); Figure ld (xl000).
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containing no ceils or containing cells killed by exposure to 50% ethanol for 10 minutes, showed no signs of contraction. Cell density influenced the rate of gel contraction, and, to a lesser extent, the extent of gel contraction (data not shown). Gel contraction by hepatocytes did not appear to be as rapid as gel contraction reported for cultured fibroblasts (Bell, 1979). In summary, Fig. 3 demonstrates that viable hepatocytes were necessary for gel contraction and that gel contraction proceeded gradually over the first week of cultivation.
Insulin profiles Insulin concentrations remained relatively stable in cell-free control gels and in culture medium containing no gel or no hepatocytes. However, in medium from wells containing gel entrapped hepatocytes, less than 10% of the insulin was detected after the first 10 hours of culture, and less than 1% of the insulin was detected after 24 hours. Based on these findings, medium was
Fig. 3. Contraction of collagen gel discs containing entrapped hepatocytes. Control gels (closed circles) contained no cells or ethanol killed cells. Significant contraction was measured over the first week of culture, while contraction was not significant after day 7. Open circles represent the average of t 2 gel discs containing 0.2-1.8 x 107 hepatocytes per ml of gel.
changed in subsequent experiments every 24 hours in order to avoid insulin depletion.
Urea profiles Media from six gels were analyzed for urea concentration after 24 hours and 48 hours. Average urea concentrations (+ SEM) after 24 hours were 755 (+ 19) I.tmol/l, and after 48 hours were 1413 (+ 106) txmol/1. Urea production was significant during the first 48 hours of cultivation (R a = 0.938). No urea was detected in control wells containing only medium or cell-free gels.
Albumin production Figure 4 demonstrates stable albumin production by rat hepatocytes in cylindrical collagen gels over an 8 day period. Each data point represents the albumin production by 7.5 • 104 cells over a 24 hour period in 0.5 ml culture medium. From
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compared to no cell controls, however, MEGX production fell steadily over this period. The decrease in MEGX production was not significant from day 4 to day 6, but was significant between earlier successive measurements. Taurocholate appearance
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