JOURNAL OF PATHOLOGY, VOL.
162: 255-260 (1990)
BILIARY EPITHELIAL CELLS FROM THE LIVER OF PATIENTS WITH PRIMARY BILIARY CIRRHOSIS: ISOLATION, CHARACTERIZATION, AND SHORT-TERM CULTURE RUTH JOPLIN*, A. J. STRAIN? AND J. M. NEUBERGER*
The Liver Unit, Queen Elizabeth Hospital and Departments of Medicine* and Riochemistryt , University of Birmingham, Birmingham, U.K. Received 10 May 1990 Accepted 8 June I990
SUMMARY Since biliary epithelial cells of the middle-sized interlobular bile ducts are targets for lymphocyte-mediated damage in patients with primary biliary cirrhosis (PBC), we have developed a method for isolating and maintaining these cells in short-term tissue culture. Intrahepatic biliary epithelial cells were isolated from small segments of liver removed at the time of transplantation. Cells were separated from a collagenase digest by immunomagnetic separation using Dynabeads coupled to a monoclonal antibody (HEA 125) specific for a biliary epithelial cell surface antigen. The yield was approximately 3 x 1O5 cells/g of liver. The isolated cells were characterized morphologically and ultrastructurally using light and electron microscopy, and immunocytochemically using HEA 125 and anti-cytokeratin, anti-vimentin and anti-asialoglycoprotein receptor antibodies. By these criteria cells were judged to be identical to biliary epithelial cells from normal liver. The cells could be maintained in short-term tissue culture for up to 4 weeks without loss of biliary epithelial cell markers. Availability of interlobular biljary epithelial cells will be of value in future investigations of the pathogenetic mechanisms of PBC. KEY
WORDS-Biliary epithelial cells, immuno-isolation, cell culture, primary biliary cirrhosis.
INTRODUCTION The pathogenesis of primary biliary cirrhosis (PBC) remains unknown. Current evidence suggests that the disease is due to an immune-mediated attack on the middle-sized interlobular bile ducts. This leads to progressive damage of biliary epithelium and consequent cholestasis with resultant irreversible liver failure. Even in end-stage livers not all portal tract bile ducts are affected: some portal tracts show no bile ducts while others show intact or damaged ducts. Numerous abnormalities of lymphocyte function have been identified in patients with PBC,’ but the extent to which the disease relates to these abnormalities or to abnormal antigenic expression by bile ducts is not clear. Epithelium lining middle-sized Addressee for correspondence: Ruth Joplin, Liver Research Laboratories, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, U.K.
0022-341 7/90/11025546 $05.00 0 1990 by John Wiley & Sons, Ltd.
interlobular bile ducts, the target of attack, is immunologically distinct from that lining septa1 ducts and extrahepatic biliary epithelium.’ We have recently developed a method for the isolation of intrahepatic bile duct cells from normal human liver to investigate lymphocyte recognition and cytotoxicity responses to biliary epitheli~m.~ This involves an immunomagnetic separation method using a monoclonal antibody to a biliary epithelial cell surface antigen. In this study, the technique has been applied to the isolation of cells from the liver of patients with PBC.
MATERIALS AND METHODS Isolation and culture of cells
Biliary epithelial Cells Were isolated from the liver of patients with PBC (n= 8), taken at the time of
256
R. JOPLIN ET AL.
Table I-Immunostaining characteristics of livers from patients with primary biliary cirrhosis* and biliary epithelial cells isolated and grown in culture
HEA125
Primary antibody CK-I ASGP-R
Immunohistochemistry Biliary epithelium Proliferating ductules Hepatocytes
+ +-
+ + +
Immunocytochemistry Isolated biliary epithelial cells
+
+
Vimentin
-
-
+
-
-
-
-
*The staining pattern of normal liver samples was similar (with the exception of proliferating ductules. which are absent in normal liver)
orthotopic liver transplantation. All patients had histologically confirmed disease (stage I11 or IV). Normal adult human liver ( n= 6) for comparison was available from donor livers which underwent graft reduction prior to paediatric liver replacement;4 donor organs had been perfused and maintained at 4°C for up to 8 h in Wisconsin5 or Marshall' surgical preservation fluid. The method used to isolate cells is described in detail elsewhere.' In brief. 30 g of liver was finely diced and incubated with I mg/ml collagenase (Sigma type IA). Four ml of digest containing lo7 cells/ml were layered onto 3 ml each of 1.04 g/ml and 1.09 gjml iso-osmotic Percoll. After centrifugation at 800 g for 30 min, a non-parenchymal fraction. which included biliary epithelial cells, Kupffer cells, lymphocytes. and other non-parenchymal cells. was harvested at the differential density interphase. Biliary epithelial cells were isolated from this population by incubation initially with 5 pgiml HEA 125 (Progen Biotechnik GMBH, Heidelberg) and then with lo7 sheep anti-mouse IgG 1 coated Dynabeads (Dynal U.K. Ltd.). The antibodylabelled cells were then extracted by magnetic separation. Immuno-isolated cells were cultured in a growth factor-enriched medium containing Dulbecco's modification of Eagles' medium, 10 per cent fetal bovine serum, 10 ng/ml epidermal growth factor, 5 pg/ml insulin, 0.4 pg/ml hydrocortisone, 10 ng/ml cholera toxin, 24.3 pg/ml adenine, 5 pg/ml transferrin, and 2 x M tri-iodo-thyronine. They were cultured in 25 cm' tissue culture flasks on a feeder cell layer of irradiated 3T3 fibroblasts as described previously.'.'
Characterization of cells
Cells were stained for expression of specific proteins at intervals between 24 h and 3 weeks. The cultures were fixed with 70 per cent ethanol, and after washing with phosphate-buffered saline, they were incubated with primary antibodies for 1 h at 25°C. HEA 125 was used at 1:10 dilution; anti-CK1, anti-vimentin. and anti-factor VIII, (Dakopatts Ltd.) at 1:50 dilution; and hepatocyte-specific antiasialoglycoprotein receptors (anti-ASGP-R, provided by Miss Barbara Wojcicka and Dr Ian McFarlane, Liver Unit, Kings College Hospital, London) at ]:I00 dilution. Sites of primary binding were visualized using an immunoperoxidase technique (Vector Stain Elite. Vector Laboratories). For transmission electron microscopy freshly immuno-isolated cells were pelleted by centrifugation at 200 g for 1 min, then fixed with 2.5 per cent glutaraldehyde in 0.1 M cacodylate buffer for I h. After washing twice in 0.1 M cacodylate buffer with 0.05 per cent sucrose for 4 h, the cells were osmicated in 1 per cent OsO, for 4 h , dehydrated through graded alcohols, and embedded in Epon resin. Sections showing gold interference colour were cut using a Porter-Blum microtome and were stained with uranyl acetate and lead tartrate. They were examined using a Jeol 100 x electron microscope. Specijicity of monoclonal antibodies for biliarjs epithelial cells HEA 125 recognizes an antigen present in the cytoplasm and on the surface of many different epithelial cell types, but which in liver reacts only with
257
BILIARY EPITHELIAL CELLS
Fig. I-The immunohistochemical staining pattern of HEA125 in (a) normal liver and (b) PBC liver, and (c) CK-I expression in PBC liver
biliary epithelial cells.8 To assess the specificity of HEA125 in PBC livers, 5 mm3 samples were snapfrozen in liquid nitrogen and stored at - 70°C until use. Five-micrometre sections were cut, acetonefixed, and endogenous peroxidase activity quenched by incubating at 25°C for 10 min in 1 per cent hydrogen peroxide. Serial sections were stained with monoclonal antibodies to CK-1 and vimentin (150 dilution), HEA125 (working dilution 1:lo), and ASGP-R (1:lOO). Sites of primary binding were visualized using an immunoperoxidase technique (Vector Stain Elite, Vector Laboratories).
RESULTS Distribution of antibod):binding sites in normal and PBC livers The immunohistochemical staining characteristics are summarized in Table I. HEA 125 staining was limited to biliary epithelium in normal liver (Fig. la) and in livers from patients with PBC, it was expressed by bile duct epithelium and proliferating ductules (Fig. 1b). In both normal and PBC liver, CK-1 was expressed by hepatocytes and biliary epithelial cells lining all bile ducts (Fig. Ic). Biliary
258
R. JOPLIN ET AL.
(d) Fig. 2-Biliary epithelial cell (BEC)preparations from PBC liver. (a)48 h culture showing large and small colonies (BEC) surrounded by 3T3 feeder cells. (b)Seven-day culture-3T3 feeder cells have been displaced by colony expansion due to proliferation of BEC. Fiveday cultures stained for (c) HEA125 expression and (d) CK-1 expression. Note the different staining patterns of HEA125 (a predominantly membrane protein) and CK-1 cytoplasmic expression (Cf
epithelial cells were consistently negative for vimentin and ASGP-R in both normal and PBC liver.
viability as assessed by trypan blue dye exclusion was 96 per cent ( + 5 per cent). Hepatocyte contamination of the fraction, as assessed by light microscopy, was less than 1 per cent. The freshly immuno-isolated cells formed a mixed population Isolation and culture of cells of single cells, small clusters, and large ovoid The average yield of non-parenchymal cells har- accumulations of cells which morphologically vested from the Percoll preparations was 3 x lo6 resembled the immuno-isolated biliary epithelial ( + lo6) per g of tissue processed and the mean cells obtained from normal livers. Owing to the
BILIARY EPITHELIAL CELLS
Fig. 3-Electron microscopy of immuno-isolates. Accumulations of cells with typical epithelial cell morphology to which Dynabeads can be seen adhering (arrows). x 5000
aggregate nature of the isolate, accurate quantitation of the yield was difficult. We determined that approximately 10 per cent of the non-parenchymal fraction could be extracted by immuno-isolation, giving an estimated net yield of 3 x lo5 biliary epithelial cells/g of liver processed. Following culture, cells formed colonies (Fig. 2a) which expanded and gradually displaced the feeder layer (Fig. 2b). Phase-contrast microscopy of the colonies demonstrated a typical epithelial cell cobble-stone morphology. Phenotypic analysis of the cultures demonstrated maintenance of HEAl25 and CK- 1 expression for at least 3 weeks (Figs 2c and 2d). Cultures were uniformly negative for vimentin, factor VIII related antigen. and hepatocyte-specific ASGP-R for a minimum of 7 days. After 4 weeks, the cells became large, vacuolated, and differentiated, features associated with loss of proliferative capacity , Electron microscopy
Transmission electron microscopy of the isolates revealed clusters of cells with Dynabeads adhering to the plasma membrane (Fig. 3). The cells demonstrated polarity, nuclei being situated in the basal region, while the apical surface exhibited numerous
microvilli. The cytoplasm contained a paucity of mitochondria and endoplasmic reticulum, and junctional complexes could be seen between adjacent cells in individual clusters. DISCUSSION These results show that biliary epithelial cells can be isolated with high purity from PBC livers. Owing to the aggregate nature of the immuno-isolate, accurate quantitation of the yield was difficult, although it appeared generally to be higher from PBC livers than from normal livers. This may relate to the different preservation procedures used for normal livers. although the viabilities of the initial isolates did not differ (96+2 per cent normal vs. 96+ 5 per cent PBC). Alternatively, it may relate to the contribution of proliferating ductules to the yield. PBC is characterized by destruction of the epithelial lining of interlobular bile ducts which is accompanied by proliferation of bile ductules. In view of the immunohistochemical pattern of staining apparent from this study, it may be that the latter contributed significantly to the yield. Monoclonal antibodies raised against biliary epithelial cells immuno-isolated from PBC livers showed a range of specificities; some antibodies recognized
260
R. JOPLIN ET AL.
interlobular ducts and ductules of all sizes, while advantage of producing a population of intraothers had specificity for septal and interlobular hepatic biliary epithelial cells which is not only ducts alone, suggesting that cells from both inter- greatly depleted of non-biliary epithelial contamilobular and septal bile ducts were present in the nants, such as fibroblasts, but is also devoid of initial isolate. excessive contamination by cells from major bile Despite the probable presence of both biliary ducts. Furthermore, it might be anticipated that the epithelial cells and proliferative cells, the immuno- isolate contains cells representative of those which isolates had morphological characteristics typical of in vivo may be involved in the disease process. biliary epithelial cells isolated from normal l i ~ e r . ~ . ~ They also maintained expression of epithelial specific proteins in tissue culture, continuing to ACKNOWLEDGEMENTS express HEA125 and CK-1 for at least 3 weeks; the cells were negative for factor VIII-related antigen, The support of the Wellcome Trust for funding vimentin, and ASGP-R for at least 7 days. this study is gratefully acknowledged. We wish to The chief aim of the work was to identify and thank Mr P. McMaster, Mr J. A. C. Buckels, Mr I. generate isolates of biliary epithelium for sub- Badger, Mr R. Lee, and Mr N. Zundel for help in sequent use as targets to investigate autologous obtaining hepatectomy specimens; and Dr L. L. lymphocyte reactions and attempt to elucidate Franchi and Mr A. Murdoch, Department of further the pathogenesis of primary biliary cirrhosis. Anatomy, University of Birmingham for advice and Although it could be argued that the biliary epithelial technical assistance with electron microscopy. We cells isolated in this study must represent those which are also grateful to Miss Barbara Wojcicka, and Dr had escaped lymphocyte-mediated damage during Ian McFarlane for providing antibody to asialoprogression of the disease, it must be remembered glycoprotein receptors. The secretarial assistance of that even in the late stages of the disease, portal Miss M. Calcutt is gratefully acknowledged. tracts are involved with continuing inflammatory activity suggesting that stimulation by antigen is still occurring. REFERENCES The immuno-isolation method described in this paper generates a heterogeneous sample of cells, 1 Shaffner F. Popper H. In: Popper H, SchaKner F. eds. Progress in Liver Disease. Vol7. Clinical-pathologic Relations in Primary Biliary and it may be anticipated that some of these cells in Cirrhosis. New York: Grune and Stratton, 1982; 529-554. vivo are involved in the disease process. Alternative 2 Okada Y, Jinnon K, Shousuke M, er a / . Blood group antigens in the intrahepatic biliary tree. I. Distribution in normal liver. J Heparol 1988; isolation approaches such as biliary tree perfusion’ 6 63-70. are likely to yield cells from major ducts distal to the 3 Joplin R. Strain AJ, Neuberger JM. Immuno-isolation and culture of biliary epithelial cells from normal human liver. In Virro Cell Dev Biol foci of damage. Thus, those cells which are pri1989;ZS 1189-1192. marily involved in the pathophysiology of the dis- 4 de Hemptinne 9. Saliuoni M. Yandza TC. er a / .Indication, technique ease might be poorly represented. In many epithelial and results of liver graft volume reduction before orthotopic transplantation in children. Trans Proc 1987; 1 9 3549-3551. cell isolation systems, explants are used as the Kalayoglu M, Sollinger HW. Stratta RJ, er a / . Extended preservation source of cells. With only a very small segment of 5 ofliver for clinical transplantation. Lancer 1988; I: 617-619. initial tissue, a highly selective population may 6 Ross H. Marshall VC. Escott ML. 72-hr canine kidney preservation continued perfusion. Transplanrarion 1976; 21: 498-501. expand which is not representative of all the cell 7 without Allen-Hoffman BL, Rheinwald JG. Polycyclic aromatic hydrocarbon types within a larger tissue block. In addition, mutagenesis of human epidermal keratinocytes in culture. Proc Narl Acad Sci USA 1984;81: 7802-7806. explant culture is associated with problems of conMomburg F, Moldenhauer G , Hammerling GH, Moller P. Immunotamination by other cell types such as fibroblasts 8. histochemical study of the surface expression of a M , 34.000 human which tend to overgrow the epithelial cell growth. epithelium-specific glycoprotein in normal and malignant tissues. Cancer Res 1987;47: 2883-2891. While other isolation procedures may produce cells 9. Demetris AJ, Marks BH, Saidman S. er a / . Isolation and primary which are more amenable to maintenance in longcultures of human intrahepatic bile ductular epithelrum. I n Virro Cell Der Biol1988; 24: 464-470. term tissue culture, immuno-isolation has the
162: 255-260 (1990)
BILIARY EPITHELIAL CELLS FROM THE LIVER OF PATIENTS WITH PRIMARY BILIARY CIRRHOSIS: ISOLATION, CHARACTERIZATION, AND SHORT-TERM CULTURE RUTH JOPLIN*, A. J. STRAIN? AND J. M. NEUBERGER*
The Liver Unit, Queen Elizabeth Hospital and Departments of Medicine* and Riochemistryt , University of Birmingham, Birmingham, U.K. Received 10 May 1990 Accepted 8 June I990
SUMMARY Since biliary epithelial cells of the middle-sized interlobular bile ducts are targets for lymphocyte-mediated damage in patients with primary biliary cirrhosis (PBC), we have developed a method for isolating and maintaining these cells in short-term tissue culture. Intrahepatic biliary epithelial cells were isolated from small segments of liver removed at the time of transplantation. Cells were separated from a collagenase digest by immunomagnetic separation using Dynabeads coupled to a monoclonal antibody (HEA 125) specific for a biliary epithelial cell surface antigen. The yield was approximately 3 x 1O5 cells/g of liver. The isolated cells were characterized morphologically and ultrastructurally using light and electron microscopy, and immunocytochemically using HEA 125 and anti-cytokeratin, anti-vimentin and anti-asialoglycoprotein receptor antibodies. By these criteria cells were judged to be identical to biliary epithelial cells from normal liver. The cells could be maintained in short-term tissue culture for up to 4 weeks without loss of biliary epithelial cell markers. Availability of interlobular biljary epithelial cells will be of value in future investigations of the pathogenetic mechanisms of PBC. KEY
WORDS-Biliary epithelial cells, immuno-isolation, cell culture, primary biliary cirrhosis.
INTRODUCTION The pathogenesis of primary biliary cirrhosis (PBC) remains unknown. Current evidence suggests that the disease is due to an immune-mediated attack on the middle-sized interlobular bile ducts. This leads to progressive damage of biliary epithelium and consequent cholestasis with resultant irreversible liver failure. Even in end-stage livers not all portal tract bile ducts are affected: some portal tracts show no bile ducts while others show intact or damaged ducts. Numerous abnormalities of lymphocyte function have been identified in patients with PBC,’ but the extent to which the disease relates to these abnormalities or to abnormal antigenic expression by bile ducts is not clear. Epithelium lining middle-sized Addressee for correspondence: Ruth Joplin, Liver Research Laboratories, Queen Elizabeth Hospital, Edgbaston, Birmingham B15 2TH, U.K.
0022-341 7/90/11025546 $05.00 0 1990 by John Wiley & Sons, Ltd.
interlobular bile ducts, the target of attack, is immunologically distinct from that lining septa1 ducts and extrahepatic biliary epithelium.’ We have recently developed a method for the isolation of intrahepatic bile duct cells from normal human liver to investigate lymphocyte recognition and cytotoxicity responses to biliary epitheli~m.~ This involves an immunomagnetic separation method using a monoclonal antibody to a biliary epithelial cell surface antigen. In this study, the technique has been applied to the isolation of cells from the liver of patients with PBC.
MATERIALS AND METHODS Isolation and culture of cells
Biliary epithelial Cells Were isolated from the liver of patients with PBC (n= 8), taken at the time of
256
R. JOPLIN ET AL.
Table I-Immunostaining characteristics of livers from patients with primary biliary cirrhosis* and biliary epithelial cells isolated and grown in culture
HEA125
Primary antibody CK-I ASGP-R
Immunohistochemistry Biliary epithelium Proliferating ductules Hepatocytes
+ +-
+ + +
Immunocytochemistry Isolated biliary epithelial cells
+
+
Vimentin
-
-
+
-
-
-
-
*The staining pattern of normal liver samples was similar (with the exception of proliferating ductules. which are absent in normal liver)
orthotopic liver transplantation. All patients had histologically confirmed disease (stage I11 or IV). Normal adult human liver ( n= 6) for comparison was available from donor livers which underwent graft reduction prior to paediatric liver replacement;4 donor organs had been perfused and maintained at 4°C for up to 8 h in Wisconsin5 or Marshall' surgical preservation fluid. The method used to isolate cells is described in detail elsewhere.' In brief. 30 g of liver was finely diced and incubated with I mg/ml collagenase (Sigma type IA). Four ml of digest containing lo7 cells/ml were layered onto 3 ml each of 1.04 g/ml and 1.09 gjml iso-osmotic Percoll. After centrifugation at 800 g for 30 min, a non-parenchymal fraction. which included biliary epithelial cells, Kupffer cells, lymphocytes. and other non-parenchymal cells. was harvested at the differential density interphase. Biliary epithelial cells were isolated from this population by incubation initially with 5 pgiml HEA 125 (Progen Biotechnik GMBH, Heidelberg) and then with lo7 sheep anti-mouse IgG 1 coated Dynabeads (Dynal U.K. Ltd.). The antibodylabelled cells were then extracted by magnetic separation. Immuno-isolated cells were cultured in a growth factor-enriched medium containing Dulbecco's modification of Eagles' medium, 10 per cent fetal bovine serum, 10 ng/ml epidermal growth factor, 5 pg/ml insulin, 0.4 pg/ml hydrocortisone, 10 ng/ml cholera toxin, 24.3 pg/ml adenine, 5 pg/ml transferrin, and 2 x M tri-iodo-thyronine. They were cultured in 25 cm' tissue culture flasks on a feeder cell layer of irradiated 3T3 fibroblasts as described previously.'.'
Characterization of cells
Cells were stained for expression of specific proteins at intervals between 24 h and 3 weeks. The cultures were fixed with 70 per cent ethanol, and after washing with phosphate-buffered saline, they were incubated with primary antibodies for 1 h at 25°C. HEA 125 was used at 1:10 dilution; anti-CK1, anti-vimentin. and anti-factor VIII, (Dakopatts Ltd.) at 1:50 dilution; and hepatocyte-specific antiasialoglycoprotein receptors (anti-ASGP-R, provided by Miss Barbara Wojcicka and Dr Ian McFarlane, Liver Unit, Kings College Hospital, London) at ]:I00 dilution. Sites of primary binding were visualized using an immunoperoxidase technique (Vector Stain Elite. Vector Laboratories). For transmission electron microscopy freshly immuno-isolated cells were pelleted by centrifugation at 200 g for 1 min, then fixed with 2.5 per cent glutaraldehyde in 0.1 M cacodylate buffer for I h. After washing twice in 0.1 M cacodylate buffer with 0.05 per cent sucrose for 4 h, the cells were osmicated in 1 per cent OsO, for 4 h , dehydrated through graded alcohols, and embedded in Epon resin. Sections showing gold interference colour were cut using a Porter-Blum microtome and were stained with uranyl acetate and lead tartrate. They were examined using a Jeol 100 x electron microscope. Specijicity of monoclonal antibodies for biliarjs epithelial cells HEA 125 recognizes an antigen present in the cytoplasm and on the surface of many different epithelial cell types, but which in liver reacts only with
257
BILIARY EPITHELIAL CELLS
Fig. I-The immunohistochemical staining pattern of HEA125 in (a) normal liver and (b) PBC liver, and (c) CK-I expression in PBC liver
biliary epithelial cells.8 To assess the specificity of HEA125 in PBC livers, 5 mm3 samples were snapfrozen in liquid nitrogen and stored at - 70°C until use. Five-micrometre sections were cut, acetonefixed, and endogenous peroxidase activity quenched by incubating at 25°C for 10 min in 1 per cent hydrogen peroxide. Serial sections were stained with monoclonal antibodies to CK-1 and vimentin (150 dilution), HEA125 (working dilution 1:lo), and ASGP-R (1:lOO). Sites of primary binding were visualized using an immunoperoxidase technique (Vector Stain Elite, Vector Laboratories).
RESULTS Distribution of antibod):binding sites in normal and PBC livers The immunohistochemical staining characteristics are summarized in Table I. HEA 125 staining was limited to biliary epithelium in normal liver (Fig. la) and in livers from patients with PBC, it was expressed by bile duct epithelium and proliferating ductules (Fig. 1b). In both normal and PBC liver, CK-1 was expressed by hepatocytes and biliary epithelial cells lining all bile ducts (Fig. Ic). Biliary
258
R. JOPLIN ET AL.
(d) Fig. 2-Biliary epithelial cell (BEC)preparations from PBC liver. (a)48 h culture showing large and small colonies (BEC) surrounded by 3T3 feeder cells. (b)Seven-day culture-3T3 feeder cells have been displaced by colony expansion due to proliferation of BEC. Fiveday cultures stained for (c) HEA125 expression and (d) CK-1 expression. Note the different staining patterns of HEA125 (a predominantly membrane protein) and CK-1 cytoplasmic expression (Cf
epithelial cells were consistently negative for vimentin and ASGP-R in both normal and PBC liver.
viability as assessed by trypan blue dye exclusion was 96 per cent ( + 5 per cent). Hepatocyte contamination of the fraction, as assessed by light microscopy, was less than 1 per cent. The freshly immuno-isolated cells formed a mixed population Isolation and culture of cells of single cells, small clusters, and large ovoid The average yield of non-parenchymal cells har- accumulations of cells which morphologically vested from the Percoll preparations was 3 x lo6 resembled the immuno-isolated biliary epithelial ( + lo6) per g of tissue processed and the mean cells obtained from normal livers. Owing to the
BILIARY EPITHELIAL CELLS
Fig. 3-Electron microscopy of immuno-isolates. Accumulations of cells with typical epithelial cell morphology to which Dynabeads can be seen adhering (arrows). x 5000
aggregate nature of the isolate, accurate quantitation of the yield was difficult. We determined that approximately 10 per cent of the non-parenchymal fraction could be extracted by immuno-isolation, giving an estimated net yield of 3 x lo5 biliary epithelial cells/g of liver processed. Following culture, cells formed colonies (Fig. 2a) which expanded and gradually displaced the feeder layer (Fig. 2b). Phase-contrast microscopy of the colonies demonstrated a typical epithelial cell cobble-stone morphology. Phenotypic analysis of the cultures demonstrated maintenance of HEAl25 and CK- 1 expression for at least 3 weeks (Figs 2c and 2d). Cultures were uniformly negative for vimentin, factor VIII related antigen. and hepatocyte-specific ASGP-R for a minimum of 7 days. After 4 weeks, the cells became large, vacuolated, and differentiated, features associated with loss of proliferative capacity , Electron microscopy
Transmission electron microscopy of the isolates revealed clusters of cells with Dynabeads adhering to the plasma membrane (Fig. 3). The cells demonstrated polarity, nuclei being situated in the basal region, while the apical surface exhibited numerous
microvilli. The cytoplasm contained a paucity of mitochondria and endoplasmic reticulum, and junctional complexes could be seen between adjacent cells in individual clusters. DISCUSSION These results show that biliary epithelial cells can be isolated with high purity from PBC livers. Owing to the aggregate nature of the immuno-isolate, accurate quantitation of the yield was difficult, although it appeared generally to be higher from PBC livers than from normal livers. This may relate to the different preservation procedures used for normal livers. although the viabilities of the initial isolates did not differ (96+2 per cent normal vs. 96+ 5 per cent PBC). Alternatively, it may relate to the contribution of proliferating ductules to the yield. PBC is characterized by destruction of the epithelial lining of interlobular bile ducts which is accompanied by proliferation of bile ductules. In view of the immunohistochemical pattern of staining apparent from this study, it may be that the latter contributed significantly to the yield. Monoclonal antibodies raised against biliary epithelial cells immuno-isolated from PBC livers showed a range of specificities; some antibodies recognized
260
R. JOPLIN ET AL.
interlobular ducts and ductules of all sizes, while advantage of producing a population of intraothers had specificity for septal and interlobular hepatic biliary epithelial cells which is not only ducts alone, suggesting that cells from both inter- greatly depleted of non-biliary epithelial contamilobular and septal bile ducts were present in the nants, such as fibroblasts, but is also devoid of initial isolate. excessive contamination by cells from major bile Despite the probable presence of both biliary ducts. Furthermore, it might be anticipated that the epithelial cells and proliferative cells, the immuno- isolate contains cells representative of those which isolates had morphological characteristics typical of in vivo may be involved in the disease process. biliary epithelial cells isolated from normal l i ~ e r . ~ . ~ They also maintained expression of epithelial specific proteins in tissue culture, continuing to ACKNOWLEDGEMENTS express HEA125 and CK-1 for at least 3 weeks; the cells were negative for factor VIII-related antigen, The support of the Wellcome Trust for funding vimentin, and ASGP-R for at least 7 days. this study is gratefully acknowledged. We wish to The chief aim of the work was to identify and thank Mr P. McMaster, Mr J. A. C. Buckels, Mr I. generate isolates of biliary epithelium for sub- Badger, Mr R. Lee, and Mr N. Zundel for help in sequent use as targets to investigate autologous obtaining hepatectomy specimens; and Dr L. L. lymphocyte reactions and attempt to elucidate Franchi and Mr A. Murdoch, Department of further the pathogenesis of primary biliary cirrhosis. Anatomy, University of Birmingham for advice and Although it could be argued that the biliary epithelial technical assistance with electron microscopy. We cells isolated in this study must represent those which are also grateful to Miss Barbara Wojcicka, and Dr had escaped lymphocyte-mediated damage during Ian McFarlane for providing antibody to asialoprogression of the disease, it must be remembered glycoprotein receptors. The secretarial assistance of that even in the late stages of the disease, portal Miss M. Calcutt is gratefully acknowledged. tracts are involved with continuing inflammatory activity suggesting that stimulation by antigen is still occurring. REFERENCES The immuno-isolation method described in this paper generates a heterogeneous sample of cells, 1 Shaffner F. Popper H. In: Popper H, SchaKner F. eds. Progress in Liver Disease. Vol7. Clinical-pathologic Relations in Primary Biliary and it may be anticipated that some of these cells in Cirrhosis. New York: Grune and Stratton, 1982; 529-554. vivo are involved in the disease process. Alternative 2 Okada Y, Jinnon K, Shousuke M, er a / . Blood group antigens in the intrahepatic biliary tree. I. Distribution in normal liver. J Heparol 1988; isolation approaches such as biliary tree perfusion’ 6 63-70. are likely to yield cells from major ducts distal to the 3 Joplin R. Strain AJ, Neuberger JM. Immuno-isolation and culture of biliary epithelial cells from normal human liver. In Virro Cell Dev Biol foci of damage. Thus, those cells which are pri1989;ZS 1189-1192. marily involved in the pathophysiology of the dis- 4 de Hemptinne 9. Saliuoni M. Yandza TC. er a / .Indication, technique ease might be poorly represented. In many epithelial and results of liver graft volume reduction before orthotopic transplantation in children. Trans Proc 1987; 1 9 3549-3551. cell isolation systems, explants are used as the Kalayoglu M, Sollinger HW. Stratta RJ, er a / . Extended preservation source of cells. With only a very small segment of 5 ofliver for clinical transplantation. Lancer 1988; I: 617-619. initial tissue, a highly selective population may 6 Ross H. Marshall VC. Escott ML. 72-hr canine kidney preservation continued perfusion. Transplanrarion 1976; 21: 498-501. expand which is not representative of all the cell 7 without Allen-Hoffman BL, Rheinwald JG. Polycyclic aromatic hydrocarbon types within a larger tissue block. In addition, mutagenesis of human epidermal keratinocytes in culture. Proc Narl Acad Sci USA 1984;81: 7802-7806. explant culture is associated with problems of conMomburg F, Moldenhauer G , Hammerling GH, Moller P. Immunotamination by other cell types such as fibroblasts 8. histochemical study of the surface expression of a M , 34.000 human which tend to overgrow the epithelial cell growth. epithelium-specific glycoprotein in normal and malignant tissues. Cancer Res 1987;47: 2883-2891. While other isolation procedures may produce cells 9. Demetris AJ, Marks BH, Saidman S. er a / . Isolation and primary which are more amenable to maintenance in longcultures of human intrahepatic bile ductular epithelrum. I n Virro Cell Der Biol1988; 24: 464-470. term tissue culture, immuno-isolation has the
