Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
BIOMAT., ART. CELLS, ART. ORG., 1 8 ( 4 ) , 5 4 9 - 5 5 4 ( 1 9 9 0 )
CHARACTERIZATION OF IMMOBILIZED HEPATOCYTES AS LIVER SUPPORT Yoshiharu Miura, Toshihiko Akimoto, Noriko Yoshikawa, and Kiyohito Yagi Department of Biochemical Engineering, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565 Japan
ABSTRACT Hepatocytes isolated from rat liver were immobilized wirrhin Ca-alginate. Immobilized hepatocytes could remove ammonia and other toxic substances causing hepatic coma, such as indole, phenol, bilirubin, and short chain fatty acids. Although free hepatocytes lost those activities within 2 days, immobilized hepatocytes maintained those activities for more than 7 days. Immobilized hepatocytes induced tyrosine aminotransferase(TAT) in the presence of dexamethasone and dibutyryl-CAMP and retained the ability to induce TAT for more than 7 days. Biologically active form of coagulation factor 11, prothrombin could be synthesized and secreted into medium by immobilized hepatocytes. Moreover, immobilized hepatocytes produced glucose from lactate, alanine, fructose, and galactose. Like adult rat liver, growth-related function and liver-specific function in immobilized hepatocytes were reciprocally controlled by cell density. There are b o t h d , and p -adrenergic receptors in membrane of liver cells, and the adrenergic action of epinephrine is d-predominant in adult rat liver. Monolayer-cultured hepatocytes can not maintaind adrenergic response. However, immobilized hepatocytes maintained -adrenergic response as shown in vivo. Those characteristics of immobilized and three-dimensionally cultured hepatocytes are regarded almost the same as liver cells in vivo. Furthermore, therapeutic effect of immobilized hepatocytes on the 549
Copyright 0 1990 by Marcel Dekker, Inc.
550
MIURA ET AL.
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
hepatic failure were confirmed in the experiment using hepatocytes damaged with D-galactosamine. Therefore, it is suggested that immobilized hepatocytes could be applied to a hybrid artificial liver support.
INTRODUCTION
Diversity of liver functions makes it difficult to construct an hepatic support, and an effective treatment of acute hepatic failure has not been established. We have been trying to construct hybrid artificial liver. The system should stably possess at least the abilities to detoxify substances causing hepatic coma and to supply plasma proteins. Excised whole liver ( l ) , liver slices(2), and isolated hepatocytes (3) lose liver function very rapidly. It was shown that isolated hepatocytes recovered their function during monolayer culture and survived f o r u p t o s e v e r a l weeks. However, hepatocytes change physiologically as well as morphologically during monolayer culture. Therefore, we tried to immobilize and cultivate hepatocytes three-dimensionally, because the hepatocytes are expected to maintain almost the same physiological state as in vivo. MATERIALS AND METHODS
Cell isolation Hepatocytes were isolated from male SD rats weighing 250-300 g by perfusion of the liver with collagenase, essentially as The isolated cells were suspended at described by Seglen ( 4 ) . 2.0 x l o 6 cells/ml in basal medium, which was Williams medium E(WE) containing 1 0 % fetal bovine serum, 1 pM insulin, 1 0 pM dexamethasone. Immobilization of hepatocytes A mixture of 0 . 5 ml cell suspension and 0.5 m l 2% sodium alginate was dropped into 0 . 1 M CaC12(pH 7.2) through a 23-gauge syringe needle. The resulting globular gels ( 1 .5-2.0 mm in diameter) were washed with the basal medium. Culture conditions Inocula of 1.0 x l o 6 entrapped cells were seeded in 1 ml of basal medium in a Falcon plastic culture dish(3.5 cm in diameter). The same number of isolated cells was cultured as a
IMMOBILIZED HEPATOCYTES AS L l V E R SUPPORT
551
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
monolayer in 1 ml of the basal medium in the dish. These two cultivations were carried out at 37'C under a gas mixture of 5% C02, 45% N2, and 50% 0 2 .
RESULTS AND DISCUSSION Isolated rat hepatocytes were immobilized within various gel matrix and cultivated three-dimensionally. Hepatocytes immobilized within collagen, fibrin, and Ca-alginate showed high Among these activities of ammonia removal and urea synthesis. three materials, Ca-alginate was chosen a s the best entrapping material because it gels under very gentle conditions and the resulting gel has good mechanical intensity, and it is easy to sterilize alginate with an autocrave. Therefore , the hepatocytes entrapped within Ca-alginate were used in the following experiments. Besides ammonia, hepatocytes immobilized within Ca-alginate could remove other toxic substances causing hepatic coma, such as indole, phenol, bilirubin, and short chain fatty acids. Although free hepatocytes lost those activities within 2 days, immobilized hepatocytes maintained those activities for more than 7 days. These results suggest that entrapped cells maintained function of microsomal drug metabolizing system, cytochrome P450. Synthesis and secretion of proteins are indispensable function in hybrid artificial liver. First, we tried to examine whether entrapped hepatocytes can respond to hormon and retain Tyrosine t h e a b i l i t y of d e n o v o p r o t e i n synthesis. aminotransferase(TAT) is one of the liver-specific enzymes and is reported to be induced in the presence of dexamethasonefDEX) and dibutyryl-cAMP(DBcAMP) in monolayer culture(5). Entrapped hepatocytes cultured for 1 , 3 , 5, 7 days were incubated with or without DEX and DBcAMP for 6 hours and homogenized with teflon glass homogenizer. Intracellular TAT activity was measured according to the method described by Granner and Tomkins(6). The addition of hormons increased intracellular TAT activity to 2.5-5 times the control value. Stimulation of TAT synthesis was These observed even in immobilized cells cultured for 7 days. results indicate that immobilized hepatocytes can respond to
MIURA ET AL.
5 52 Table I
Effect of cycloheximide on secretion of prothrombin, cholinesterase, and LCAT in cultured hepatocytes
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
Activities Entrapment culture Monolayer culture CycH(-) ,CycH(+) CycH(-) CycH(+) 2.710.6 Prothrombin (unit/1o6 cells) Cholinesterase 13724 (uunit/1o6 cells) LCAT 0.3 3T0.04 (munit/lO6 cells)
Means
2
SD (n=3), N.D.;
0.7+0.2
5 722 5
N.D.
3.7k1.5 74211
0.4920.11
0
N.D. 0.15+0.06
not detectable
hormones and retain the ability of protein synthesis through 7 Next, we examined the ability of immobilized days of culture. hepatocytes to synthesize and secrete three kinds of plasma proteins, prothrombin, 1ecitin:cholesterol acyltransferase(LCAT), and cholinesterase, because the levels of plasma proteins drop drastically at hepatic failure. Hepatocytes were cultured in basal medium for 2 4 hours and were incubated further for 2 4 hours in the absence or presence of 10 pM cycloheximide.Table 1 summarize the results. Immobilized hepatocytes could synthesize and secrete the three kinds of plasma protein. The addition of cycloheximide inhibited secretions of three kinds of plasma proteins. Therefore, it was confirmed that immobilized hepatocytes did not simply release three preexisting kinds of plasma proteins but synthesize them de novo and excreted them into the medium. In terminal differntiated hepatocytes, two types o f functions, growth-related and liver-specific functions, are reciprocally controlled by cell density(7). We tried to examine whether immobilized hepatocytes maintain a differentiated state as shown in vivo. Induction of serine dehydratase(SDH) by DEX and glucagon was used a s a marker of liver-specific functions. Induction o f glucose-6-phosphate dehydrogenase(G6PDH) by epidermal growth factorfEGF) and insulin was used as a marker of growth-related functions. The induction of SDH was stimulated by increasing cell density. In contrast, the induction of G6PDH
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
IMMOBILIZED HEPATOCYTES AS LIVER SUPPORT
3
553
5 Cell deniity [ X 10 cells/ml gel)
Figure 1. Cell density-dependent control of serine dehydratase (SDHf and glucose-6-phosphate dehydrogenase(G6PDH) induction in hepatocytes immobilized within Ca-alginate. Circles indicate SDH activities induced by DEX and glucagon. Triangles indicate G6PDH activities induced by EGF and insulin.
Figure 2. Effect of adrenerqic agonists on qluconeoqenesis from lactate in cultured hepatocytes. Hepatocytes were seeded at 1 x lo6 cells/ml gel and 1 x lo5 cells/cm2 as entrapment ( A ) and monolayer (B), respectively. CONT, control(n0 hormon); EPI, epinephrine; PRO, prOpranOlOl.
was strongly stimulated by decreasing cell density. Thus, liver-specific functions and growth-related functions were reciprocally controlled by cell density in entrapment culture. These results suggest that immobilized hepatocytes maintain the terminal-differentiated state as in vivo. Gluconeoqenesis from lactate, alanine, fructose, and galactose were observed in immobilized hepatocytes. A s shown in Fig. 2, in monolayer culture, qluconeogenesis from lactate was stimulated by the adrenergic agonist epinephrine. This stimulative effect was antagonized by t h e p -blocker propranolol. It was reported that the effect of epinephrine on gluconeogenesis was d, -adrenergic action in freshly isolated hepatocytes(8). Thus, conversion from an A - to a P -adrenergic action occurred
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
5 54
MIURA ET AL.
during monolayer culture. Also, gluconeogenesis in immobilized hepatocytes was stimulated by epinephrine. But this stimulated effect was not antagonized by A-blocker. These results indicate that hepatocytes immobilized and three-dimensionally cultured were able to maintain&-adrenergic response as shown in vivo. Therapeutic effect of immobilized hepatocytes on the hepatic failure was investigated in vitro by using monolayer cultured hepatocytes damaged with D-galactosamine. When immobilized hepatocytes were co-cultured with damaged monolayer-cultured hepatocytes, immobilized hepatocytes showed stimulative effect on TAT induction in damaged hepatocytes. These characteristics of hepatocytes immobilized within Caalginate and three-dimensionally cultured are regarded as significant when immobilized hepatocytes are applied to a hybrid artificial liver support.
REFERENCES 1. 2. 3.
J. J. Otto, J. C. Pender, J. H. Cleary, D. M. Sensenig, and C. S. Welch. Surgery, 4 3 , 3 0 1 - 3 0 9 ( 1 9 5 8 ) H. Sakamoto, I. Koshino, N. Yoshikawa, Y. Tsuji, Y. Nakajima, Y. Shinada, M. Matsusita, T. Mizutani, Y. Kasai. Jpn. J. Artif. Organs, 7 , 1 0 7 8 - 1 0 8 1 ( 1 9 7 8 ) B. Eiseman, L. Norton, and Kralios. Surg. Gynecol. Obstet., 142,
4. 5.
6.
21-28
(1976)
P. 0. Seglen. Methods Cell Biol., 1 3 , 2 9 - 8 3 ( 1 9 7 6 ) T. Nakamura, C. Noda, and A . Ichihara. Biochem. Biophys. Res. Commun. 99, 7 7 5 - 7 8 0 ( 1 9 8 1 ) U. K. Granner and G. M. Tomkins. Methods Enzymol. 1 7 , 633-637
(1970)
7.
T. Nakamura, K. Yoshimoto, Y. Nakayama, Y. Tomita, and A . Ichihara. Proc. Natl. Acad. Sci. USA, 8 0 , 7 2 2 9 - 7 2 3 3
8.
J. A. Gracia-Sainz and S . M. T. Hernandez-Stmayor. Proc. Natl. Acad. Sci. USA, 82, 6 7 2 7 - 6 7 3 0 ( 1 9 8 5 )
(1983)
BIOMAT., ART. CELLS, ART. ORG., 1 8 ( 4 ) , 5 4 9 - 5 5 4 ( 1 9 9 0 )
CHARACTERIZATION OF IMMOBILIZED HEPATOCYTES AS LIVER SUPPORT Yoshiharu Miura, Toshihiko Akimoto, Noriko Yoshikawa, and Kiyohito Yagi Department of Biochemical Engineering, Faculty of Pharmaceutical Sciences, Osaka University, Suita, Osaka 565 Japan
ABSTRACT Hepatocytes isolated from rat liver were immobilized wirrhin Ca-alginate. Immobilized hepatocytes could remove ammonia and other toxic substances causing hepatic coma, such as indole, phenol, bilirubin, and short chain fatty acids. Although free hepatocytes lost those activities within 2 days, immobilized hepatocytes maintained those activities for more than 7 days. Immobilized hepatocytes induced tyrosine aminotransferase(TAT) in the presence of dexamethasone and dibutyryl-CAMP and retained the ability to induce TAT for more than 7 days. Biologically active form of coagulation factor 11, prothrombin could be synthesized and secreted into medium by immobilized hepatocytes. Moreover, immobilized hepatocytes produced glucose from lactate, alanine, fructose, and galactose. Like adult rat liver, growth-related function and liver-specific function in immobilized hepatocytes were reciprocally controlled by cell density. There are b o t h d , and p -adrenergic receptors in membrane of liver cells, and the adrenergic action of epinephrine is d-predominant in adult rat liver. Monolayer-cultured hepatocytes can not maintaind adrenergic response. However, immobilized hepatocytes maintained -adrenergic response as shown in vivo. Those characteristics of immobilized and three-dimensionally cultured hepatocytes are regarded almost the same as liver cells in vivo. Furthermore, therapeutic effect of immobilized hepatocytes on the 549
Copyright 0 1990 by Marcel Dekker, Inc.
550
MIURA ET AL.
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
hepatic failure were confirmed in the experiment using hepatocytes damaged with D-galactosamine. Therefore, it is suggested that immobilized hepatocytes could be applied to a hybrid artificial liver support.
INTRODUCTION
Diversity of liver functions makes it difficult to construct an hepatic support, and an effective treatment of acute hepatic failure has not been established. We have been trying to construct hybrid artificial liver. The system should stably possess at least the abilities to detoxify substances causing hepatic coma and to supply plasma proteins. Excised whole liver ( l ) , liver slices(2), and isolated hepatocytes (3) lose liver function very rapidly. It was shown that isolated hepatocytes recovered their function during monolayer culture and survived f o r u p t o s e v e r a l weeks. However, hepatocytes change physiologically as well as morphologically during monolayer culture. Therefore, we tried to immobilize and cultivate hepatocytes three-dimensionally, because the hepatocytes are expected to maintain almost the same physiological state as in vivo. MATERIALS AND METHODS
Cell isolation Hepatocytes were isolated from male SD rats weighing 250-300 g by perfusion of the liver with collagenase, essentially as The isolated cells were suspended at described by Seglen ( 4 ) . 2.0 x l o 6 cells/ml in basal medium, which was Williams medium E(WE) containing 1 0 % fetal bovine serum, 1 pM insulin, 1 0 pM dexamethasone. Immobilization of hepatocytes A mixture of 0 . 5 ml cell suspension and 0.5 m l 2% sodium alginate was dropped into 0 . 1 M CaC12(pH 7.2) through a 23-gauge syringe needle. The resulting globular gels ( 1 .5-2.0 mm in diameter) were washed with the basal medium. Culture conditions Inocula of 1.0 x l o 6 entrapped cells were seeded in 1 ml of basal medium in a Falcon plastic culture dish(3.5 cm in diameter). The same number of isolated cells was cultured as a
IMMOBILIZED HEPATOCYTES AS L l V E R SUPPORT
551
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
monolayer in 1 ml of the basal medium in the dish. These two cultivations were carried out at 37'C under a gas mixture of 5% C02, 45% N2, and 50% 0 2 .
RESULTS AND DISCUSSION Isolated rat hepatocytes were immobilized within various gel matrix and cultivated three-dimensionally. Hepatocytes immobilized within collagen, fibrin, and Ca-alginate showed high Among these activities of ammonia removal and urea synthesis. three materials, Ca-alginate was chosen a s the best entrapping material because it gels under very gentle conditions and the resulting gel has good mechanical intensity, and it is easy to sterilize alginate with an autocrave. Therefore , the hepatocytes entrapped within Ca-alginate were used in the following experiments. Besides ammonia, hepatocytes immobilized within Ca-alginate could remove other toxic substances causing hepatic coma, such as indole, phenol, bilirubin, and short chain fatty acids. Although free hepatocytes lost those activities within 2 days, immobilized hepatocytes maintained those activities for more than 7 days. These results suggest that entrapped cells maintained function of microsomal drug metabolizing system, cytochrome P450. Synthesis and secretion of proteins are indispensable function in hybrid artificial liver. First, we tried to examine whether entrapped hepatocytes can respond to hormon and retain Tyrosine t h e a b i l i t y of d e n o v o p r o t e i n synthesis. aminotransferase(TAT) is one of the liver-specific enzymes and is reported to be induced in the presence of dexamethasonefDEX) and dibutyryl-cAMP(DBcAMP) in monolayer culture(5). Entrapped hepatocytes cultured for 1 , 3 , 5, 7 days were incubated with or without DEX and DBcAMP for 6 hours and homogenized with teflon glass homogenizer. Intracellular TAT activity was measured according to the method described by Granner and Tomkins(6). The addition of hormons increased intracellular TAT activity to 2.5-5 times the control value. Stimulation of TAT synthesis was These observed even in immobilized cells cultured for 7 days. results indicate that immobilized hepatocytes can respond to
MIURA ET AL.
5 52 Table I
Effect of cycloheximide on secretion of prothrombin, cholinesterase, and LCAT in cultured hepatocytes
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
Activities Entrapment culture Monolayer culture CycH(-) ,CycH(+) CycH(-) CycH(+) 2.710.6 Prothrombin (unit/1o6 cells) Cholinesterase 13724 (uunit/1o6 cells) LCAT 0.3 3T0.04 (munit/lO6 cells)
Means
2
SD (n=3), N.D.;
0.7+0.2
5 722 5
N.D.
3.7k1.5 74211
0.4920.11
0
N.D. 0.15+0.06
not detectable
hormones and retain the ability of protein synthesis through 7 Next, we examined the ability of immobilized days of culture. hepatocytes to synthesize and secrete three kinds of plasma proteins, prothrombin, 1ecitin:cholesterol acyltransferase(LCAT), and cholinesterase, because the levels of plasma proteins drop drastically at hepatic failure. Hepatocytes were cultured in basal medium for 2 4 hours and were incubated further for 2 4 hours in the absence or presence of 10 pM cycloheximide.Table 1 summarize the results. Immobilized hepatocytes could synthesize and secrete the three kinds of plasma protein. The addition of cycloheximide inhibited secretions of three kinds of plasma proteins. Therefore, it was confirmed that immobilized hepatocytes did not simply release three preexisting kinds of plasma proteins but synthesize them de novo and excreted them into the medium. In terminal differntiated hepatocytes, two types o f functions, growth-related and liver-specific functions, are reciprocally controlled by cell density(7). We tried to examine whether immobilized hepatocytes maintain a differentiated state as shown in vivo. Induction of serine dehydratase(SDH) by DEX and glucagon was used a s a marker of liver-specific functions. Induction o f glucose-6-phosphate dehydrogenase(G6PDH) by epidermal growth factorfEGF) and insulin was used as a marker of growth-related functions. The induction of SDH was stimulated by increasing cell density. In contrast, the induction of G6PDH
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
IMMOBILIZED HEPATOCYTES AS LIVER SUPPORT
3
553
5 Cell deniity [ X 10 cells/ml gel)
Figure 1. Cell density-dependent control of serine dehydratase (SDHf and glucose-6-phosphate dehydrogenase(G6PDH) induction in hepatocytes immobilized within Ca-alginate. Circles indicate SDH activities induced by DEX and glucagon. Triangles indicate G6PDH activities induced by EGF and insulin.
Figure 2. Effect of adrenerqic agonists on qluconeoqenesis from lactate in cultured hepatocytes. Hepatocytes were seeded at 1 x lo6 cells/ml gel and 1 x lo5 cells/cm2 as entrapment ( A ) and monolayer (B), respectively. CONT, control(n0 hormon); EPI, epinephrine; PRO, prOpranOlOl.
was strongly stimulated by decreasing cell density. Thus, liver-specific functions and growth-related functions were reciprocally controlled by cell density in entrapment culture. These results suggest that immobilized hepatocytes maintain the terminal-differentiated state as in vivo. Gluconeoqenesis from lactate, alanine, fructose, and galactose were observed in immobilized hepatocytes. A s shown in Fig. 2, in monolayer culture, qluconeogenesis from lactate was stimulated by the adrenergic agonist epinephrine. This stimulative effect was antagonized by t h e p -blocker propranolol. It was reported that the effect of epinephrine on gluconeogenesis was d, -adrenergic action in freshly isolated hepatocytes(8). Thus, conversion from an A - to a P -adrenergic action occurred
Artif Cells Blood Substit Immobil Biotechnol Downloaded from informahealthcare.com by TIB/UB Hannover on 01/03/15 For personal use only.
5 54
MIURA ET AL.
during monolayer culture. Also, gluconeogenesis in immobilized hepatocytes was stimulated by epinephrine. But this stimulated effect was not antagonized by A-blocker. These results indicate that hepatocytes immobilized and three-dimensionally cultured were able to maintain&-adrenergic response as shown in vivo. Therapeutic effect of immobilized hepatocytes on the hepatic failure was investigated in vitro by using monolayer cultured hepatocytes damaged with D-galactosamine. When immobilized hepatocytes were co-cultured with damaged monolayer-cultured hepatocytes, immobilized hepatocytes showed stimulative effect on TAT induction in damaged hepatocytes. These characteristics of hepatocytes immobilized within Caalginate and three-dimensionally cultured are regarded as significant when immobilized hepatocytes are applied to a hybrid artificial liver support.
REFERENCES 1. 2. 3.
J. J. Otto, J. C. Pender, J. H. Cleary, D. M. Sensenig, and C. S. Welch. Surgery, 4 3 , 3 0 1 - 3 0 9 ( 1 9 5 8 ) H. Sakamoto, I. Koshino, N. Yoshikawa, Y. Tsuji, Y. Nakajima, Y. Shinada, M. Matsusita, T. Mizutani, Y. Kasai. Jpn. J. Artif. Organs, 7 , 1 0 7 8 - 1 0 8 1 ( 1 9 7 8 ) B. Eiseman, L. Norton, and Kralios. Surg. Gynecol. Obstet., 142,
4. 5.
6.
21-28
(1976)
P. 0. Seglen. Methods Cell Biol., 1 3 , 2 9 - 8 3 ( 1 9 7 6 ) T. Nakamura, C. Noda, and A . Ichihara. Biochem. Biophys. Res. Commun. 99, 7 7 5 - 7 8 0 ( 1 9 8 1 ) U. K. Granner and G. M. Tomkins. Methods Enzymol. 1 7 , 633-637
(1970)
7.
T. Nakamura, K. Yoshimoto, Y. Nakayama, Y. Tomita, and A . Ichihara. Proc. Natl. Acad. Sci. USA, 8 0 , 7 2 2 9 - 7 2 3 3
8.
J. A. Gracia-Sainz and S . M. T. Hernandez-Stmayor. Proc. Natl. Acad. Sci. USA, 82, 6 7 2 7 - 6 7 3 0 ( 1 9 8 5 )
(1983)
