Dog pancreatic duct epithelial cells long term culture and characterization

American Journal ofPathology, Vol 148, No 3, March 1996 Copyrigbt © American Society for Investigative Pathology Dog Pancreatic Duct Epithelial Cells: Long-Term Culture and Characterization Dolphine Oda,* Christopher E Savard,t Toan D Nguyen,t Lydia Eng,t Erik R Swenson,t and Sum P Leet From the Departments of Oral Biology* and Medicine,t University of Washington and Veterans Affairs Medical Center, Seattle, Washington Epithelial ceUls, isolatedfrom a normal dog pancreatic duct, were grown on coUagen-coated culture inserts suspended above a feeder layer of myofibroblasts The cels were examined by transmission electron microscopy, immunohistochemistry, cytogenetics, and flow cytometry In addition, the constitutive and agonist-stimulated mucin secretion of these ceUls was studied using a [3HJN-acetyl-D-glucosamine labeling assay, and the stimulation of intracelular cAMP was measured CeUls grown on inserts with afeeder layer developed into confluent monolayers consisting of strictly polarized columnar epithelial ceUs with prominent microviUi, intercelular junctions, and normal chromosomal characteristics They could be passaged repeatedly witbout a detectable alteration in their morphology The ceUs could also be grown on organotypic cultures, resulting in further differentiated cells simulating in vivo morphology Immunohistochemistry demonstrated the presence of carbonic anhydrase II in these ceUls CeUs treated with vasoactive intestinal peptide, epinephrine, and dibutyryl-cAMP denmonstrated a marked increase in mucin secretion compared with controls In paraUlel experiments, VIP and epinephrine significantly increased intracelular cAMP In conclusion we have developed a pancreatic epithelial ceU preparation with morpbology, cytokinetics, chromosomal, and DNA analyses characteristic of normal ceUls Similar to normal columnar epithelial ceUs, these pancreatic duct cels secreted mucin constitutively and responded to agonist by increasing secretion via a cAMP-mediated patbway They also contained carbonic anhydrase, wbich indicates that the ceUs are capable of secreting bicarbonate (Am J Pathol 1996, 14&977-985) Various methods for culturing the different types of epithelial cells lining the gastrointestinal tract, including pancreatic duct epithelial cells, have been developed.5 However, a practical and representative model of cultured nonmalignant and nontransformed pancreatic duct epithelial cells, to be used for functional studies, still remains to be established.6 In vitro research on pancreatic duct cells has relied primarily on organ culture7 or isolated duct fragments of the rat8-13 and guinea pigi'0 The primary culture of pancreatic duct epithelial cells from some species has been successful, but these cells tend to have very limited proliferative capabilities.14 18 Longer-term culture of rat19 and hamster20 pancreatic duct epithelial cells has been reported Harris and Coleman21 have described the culture of cells isolated from fetal human pancreas that survived for up to five passages Many of the longer-lived cell lines of pancreatic duct developed to date have been derived from neoplastic origins.22 26 Although these cells have been used as models for normal pancreatic duct cells in the study of cellular activity, there are usually many significant differences in cellular morphology and behavior of these cells compared with normal cells.24 A long-term culture method for normal pancreatic duct epithelial cells is highly desirable for studying cellular physiology and the kinetics of mucin and bicarbonate secretion In addition, such a cell model system could be used to investigate the pathophysiology of pancreatic diseases such as cystic fibrosis, pancreatitis, and cancer Our laboratory has developed a method for growing normal dog gall bladder epithelial cells in longterm culture.27 We have modified these methods to Supported by the Medical Research Service of the Department of Veterans Affairs Accepted for publication November 1, 1995 Address reprint requests to Dr Sum P Lee, Gastroenterology, Mailstop GI-l1l, VA Medical Center, 1660 South Columbian Way, Seattle, WA 98108 977 978 Oda et al AJP March 1996, Vol 148, No culture well differentiated pancreatic duct epithelial cells from normal dog that retain the characteristics of normal cells These epithelial cells provide an excellent opportunity to study the morphological and biochemical characteristics of pancreatic duct cells We have used these cells to study the process of mucin and bicarbonate secretion, the two major functions of the pancreatic duct cells, along with the cellular modifications induced by chemical carcino- gens.28 Materials and Methods Chemicals of analytical grade were obtained from Sigma Chemical Co (St Louis, MO) except where noted Vitrogen, a bovine dermal collagen, was purchased from Celtrix Labs (Palo Alto, CA) Falcon culture plates were from Becton Dickinson Co (Franklin Lakes, NJ), and the Transwell inserts (24 mm diameter, p.m pore size) were obtained from Costar Co (Cambridge, MA) Cell Isolation and Culture The pancreatic epithelial cell culture technique was initially described in Oda et al.28 Briefly, the accessory pancreatic duct of a normal dog, which is the largest excretory duct in the dog pancreas,29 was dissected from a freshly removed pancreas, and epithelial cells were dissociated by trypsin The cells were seeded onto Vitrogen-coated plates (1 part Vitrogen to part medium) and fed bronchial epithelial growth medium (BEGM from Clonetics Corp., San Diego, CA) for a week to inhibit fibroblast growth The cells then were passaged to Vitrogen-coated Transwell inserts (0.5 ml of diluted Vitrogen per insert), and the inserts were suspended above a confluent feeder layer of cultured human gall bladder myofibroblasts The cells were maintained at 370C in 5% carbon dioxide/95% air They were fed twice weekly (4 ml per well) with Eagle's Minimum Essential Medium (EMEM) containing 10% fetal bovine serum, mmol/L L-glutamine, 20 mmol/L HEPES, 100 lU/ml penicillin, 100 ,ug/ml streptomycin plus ,ug/ml insulin from bovine pancreas, ,ug/ml human transferrin, and ng/ml sodium selenite (ITS supplement, Sigma Chemical Co., St Louis, MO) Confluent cells were passaged using 0.25% trypsin/EDTA For cryopreservation, the epithelial cells were frozen in medium containing 10% dimethylsulfoxide Organotypic Cultures The organotypic culture technique was performed as previously described.30 In brief, trypsinized epithelial cells were plated onto a freshly prepared collagen and fibroblast matrix After cell attachment to the collagen matrix, the matrix was gently released from the sides and bottom of the dish for the collagen to contract evenly The cells were submerged in supplemented Dulbecco's Modified Eagle's medium31 and fed every day for to days They were then raised to liquid-air interface and were fed from below After days, the cells were fixed in Hollande's or half-strength Karnovsky's fixative for thin section light microscopy or electron microscopy, respectively Mucin Secretion Assay Mucin secretion was measured using a method adapted from Kuver et al.32 Pancreatic duct epithelial cells were grown to confluency on Transwell inserts The cells were labeled for 24 hours with ,uCi per well of [3H]N-acetyl-D-glucosamine (ICN Biomedicals, Irvine, CA), a mucin precursor,33 added to the lower compartment (basolateral side) The cells were washed, and activators or inhibitors were added, in triplicate, to the lower compartment of the wells After a 4-hour incubation, ml of the medium from the upper compartment (apical side) was removed and centrifuged at 500 x g for 10 minutes to pellet detached cells, and 0.5 ml of the supernatant was mixed with ml of 10% trichloroacetic acid/1% phosphotungstic acid and protein precipitated overnight at 40C After centrifuging the samples at 1500 x g for 10 minutes, the pellets were washed twice, resuspended in 0.5 ml of water, and counted in 10 ml of scintillation fluid The average dpm of each treatment was divided by the average dpm of three untreated wells The results of a treatment in each assay were expressed as the percentage of the control, with the control as 100% Each treatment was repeated in at least three different assays The values are presented as the mean of multiple assays ± standard error (SE) Statistical significance of differences between treatments and control was assessed using Student's paired t-test cAMP Assay Confluent cells on inserts were washed with phosphate-buffered saline (PBS), serum-free EMEM was added, and the cells were returned to the incubator After 30 minutes, activators were added, in duplicate, to the lower compartment After 20 minutes at Pancreatic Duct Cell Culture 979 AJP March 1996, Vol 148, No 37°C, the cells were quickly scraped from the insert with a flat-edged spatula, resuspended in ml of deionized water, and pipetted into a test tube containing ml of cold 5% perchloric acid After 15 minutes at 40C, the samples were centrifuged at 1500 x g for 10 minutes, and potassium bicarbonate (400 ,ul of 30% w/v) was added to the supernatant of each sample to precipitate potassium perchlorate After 45 minutes at 4°C, the samples were spun at 1500 x g for 10 minutes, and the supernatant was lyophilized overnight The dried sample was resuspended in 1.2 ml of Tris-EDTA buffer, and the cAMP concentration was determined by radioimmunoassay using the cAMP RIA assay kit from Diagnostic Products Corp (Los Angeles, CA) Results are expressed as picomoles of cAMP per well Immunohistochemistry Staining Thin sections of Hollande's fixed, paraffin-embedded cells grown on organotypic culture were used for immunohistochemistry following the methods of Gown and Vogel.34 For controls, sections of normal dog pancreas containing ducts were fixed in Hollande's solution and embedded in paraffin Thin sections of fixed tissue were processed along with the cultured cell samples Detection of primary antibodies was done by using an antibody cocktail (antirabbit and anti-mouse antibodies linked to biotin), avidin-peroxidase, and diaminobenzidine staining method (BioTek reagents) using an automated TechMate 1000 (BioTek, Santa Barbara, CA) Polyclonal antibodies against carbonic anhydrase (CA) 11 and IV were raised in rabbits after isolation of CA 11 from dog red blood cells and CA IV from dog kidney.35 Cytogenetic Analysis Cells were grown in culture media for 48 hours before chromosome preparation, and then colcemid (10 ,tg/ml) was added for hour Chromosomes were prepared in a standard fashion using KCI (0.075 mol/L) as a hypotonic and methanol/acetic acid (3:1, v/v) as a fixative After staining by Gbanding, 10 cells were analyzed and two cut karyotypes were established Transmission Electron Microscopy Confluent cells were washed with PBS and fixed in half-strength Karnovsky's reagent The cells were post-fixed in 2% osmium tetroxide with 0.1 mol/L cacodylate buffer, dehydrated in graded ethanols, and embedded in Epon (Pella, Redding, CA) Thin sections were stained with uranyl acetate and lead tartrate and photographed in a Philips EM410 transmission electron microscope Flow Cytometry Cells were plated at x 105 cells per well The culture medium was changed every days On days 2, 4, 9, and 10 after plating, duplicate wells were washed and trypsinized Staining and flow analysis were done as described by Rabinovitch et al.36 Cell pellets were resuspended in ml of 4',6-diamidino2-phenylindole staining solution with 10% dimethylsulfoxide (v/v) for 10 minutes The samples were evenly mixed and stored frozen at -70°C After thawing, the stained cell suspension was analyzed with an epi-illumination flow system designed by GOHDE (ICP22A; Phywe AG, Gottingen, Germany, now Ortho Diagnostic Systems, Westwood, MA) Results Epithelial cells were dissociated in small sheets from the dog pancreatic duct sections by treatment with trypsin/EDTA The cells attached to Vitrogen in primary culture when plated with EMEM containing 10% fetal bovine serum After epithelial cell attachment, a few isolated fibroblasts were apparent When fed BEGM, a serum-free, low calcium medium, the epithelial cells continued to divide slowly (Figure 1A) Meanwhile, the fibroblasts stopped dividing, became detached from the Vitrogen, and were then removed during medium changes After a week in BEGM, the epithelial cells were passaged to Vitrogen-coated Transwell inserts suspended above a myofibroblast feeder layer The cells, now fed EMEM, required the presence of the myofibroblast feeder layer for continued rapid cell proliferation in this system After primary culture, the epithelial cells attached but did not divide on plastic or Vitrogencoated plates when fed with the EMEM only Cells grown above a myofibroblast feeder layer formed confluent monolayers of uniform short columnar cells consistent with epithelial cell morphology in culture (Figure B) The cells had distinct apical and basolateral surfaces with numerous well developed microvilli on the apical surface The cells had highly lobed nuclei, numerous mitochondria, rough endoplasmic reticulum, and well developed Golgi bodies Cilia, found in some cells of the dog main pancreatic duct in situ,37 were not seen in the cultured cells, but a microtubule assembly associated with the basal body of a cilium (nine sets of three microtu- 980 Oda et al AJP March 1996, Vol 148, No M." I I ok ;p c q -1 , "W' Figure A: Phase contrast microscope picture of a spreading colony ofprimary cultured normal dog pancreatic duct epithelial cells growing in BEGM Magnification, X 200 B: Electron micrograph of the cells grown on collagen after one passage above the myofibroblastfeeder layer showing lateral intercellular interdigitations (5) and intercellular junctions (arrowhead) Magnification, X 8000 bules) was located near the apical surface in one electron micrograph suggesting their presence The myofibroblasts in the feeder layer were separated from the epithelial cells by the barrier of the collagencoated polycarbonate membrane of the Transwell However, large molecules such as bovine serum albumin were able to pass freely through the collagen-coated membrane by diffusion (results not shown) The confluency of a monolayer was verified by the ability of the epithelial layer to act as a barrier to the diffusion of the medium from the upper to lower compartment These pancreatic epithelial cells could be maintained in culture for over a year and more than 40 passages without discernible changes in microscopic morphology The cells could be stored in liquid nitrogen and thawed without changes in their morphology When grown in organotypic culture, the dog pancreatic duct epithelial cells maintained strict polarity with a higher density of well developed microvilli on the apical surface (Figure 2) The cells grew in monolayers consisting of tall columnar cells with basally located nuclei and prominent apical secretory granules (Figure 2C) Electron microscopy showed cells with numerous lateral intercellular junctions (both tight junctions and desmosomes) and prominent interdigitations between the cells Later passages (up to passage 40) grown in organotypic culture maintained the same morphology as early passages Using standard immunohistochemical techniques, the cells were positive to antibody 35,BH1 1, which is a marker for cytokeratin of simple epithelial cells,34 and AE1/AE3, a marker for acidic and basic keratins.38 The cells were negative to antibodies for vimentin and desmin, markers for mesenchymal and muscle cells, respectively The CA 11 antibody labeled the cytoplasm of the cultured cells with the strongest labeling in the subapical cytoplasm (Figure 3) CA IV antibody weakly labeled the apical surface only (data not shown) Control pancreatic tissue demonstrated the presence of CA 11 within the cytoplasm of the epithelial cells lining the ducts The strongest CA 11 binding was in the subapical cytoplasm (Figure 3) Also, it was noted that some epithelial cells were very positive for CA 11 compared with adjacent cells Antibodies against CA IV, on the other hand, weakly labeled the apical surface of the epithelial cells in the control tissue Cytogenetic analysis showed that cells in culture for 10 passages retained a karyotype of 39 diploid pairs of chromosomes in each cell, the normal chromosome complement of dog somatic cells The flow cytometric profiles of normal cells were examined using trout erythrocytes as an internal standard The cells had a single Gl peak Results of flow cytometry of the cells at different days after plating (Figure 4) showed that 43% (±2) of the cells were in S phase on day followed by a marked reduction in S phase Pancreatic Duct Cell Culture 981 AJP March 1996, Vol 148, No B A '' , diSSll|i - 41 t,~ Figure Dog pancreatic duct epithelial cells grown in organotypic culture after passages in regular culture Tbe tall columnar cells have well developed microvilli on the apical surface and lateral intercellular junctions The layer below the epithelial cells is collagen matrix containing fibroblasts Magnification, X 400 (A), X 4200 (B) C: Apical surface uith secretory vesicles and microvilli after 10 passages in regular culture Magnification, x>36, 000 activity in confluent cells to approximately 10% of the cells The cells were labeled by [3H]N-acetyl-D-glucosamine (443,000 dpm/mg total protein ± 49,700 SE, n = 7) and demonstrated a constitutive release of radiolabeled trichloroacetic-acid-precipitable mucin from the apical surface (16,790 dpm in the upper compartment in hours ± 2788 SE, n = 9).28 When confluent cells were treated with ,umol/L vasoactive intestinal peptide (VIP) or 0.1 ,umol/L epinephrine, there was a significant increase in mucin secretion from the apical surface as compared with untreated control cells (Figure 5) Removal of the myofibroblast feeder layer before labeling the epithelial cells did not alter this stimulation of mucin secretion The amount of radiolabeled glycoprotein in the bottom compartment of the transwell inserts (basolateral release) of untreated cells was almost equal to that found in the apical medium, but the amount of basolateral release did not change after the addition of VIP or epinephrine Apically directed mucin secretion was stimulated also by dibutyryl-cAMP (dbcAMP), a membrane-permeating analogue of cAMP When mmol/L deoxyadenosine, an adenylate cy- 982 Oda et al AJP March 1996, Vol 148, No T 4010 30o0 - 0 20 V 10 i Figure Immunohistochemistry using polyclonal antibodies against CA II of A) cultured dog pancreatic duct cells grown on organotypic ctulture (A) and nornal dog pancreas tissue (B) Magnification, _ X 1000 clase inhibitor, was added along with VIP or epinephrine, mucin secretion was reduced to control levels (Figure 6), suggesting that generation of intracellular cAMP was essential in mediating accelerated mucin secretion caused by these agents In contrast, deoxyadenosine had no inhibitory effect on the db-cAMP-induced stimulation of mucin secretion In corroboration of the above findings, VIP and epinephrine both induced a large generation and accumulation of intracellular cAMP in pancreatic duct epithelial cells within 20 minutes of exposure (Table 1) In all experiments, the cell monolayers retained confluency as indicated by the complete absence of medium diffusion from the upper to the lower chamber Figure 7The effect of different agonists on mucin glycoprotein secretion from the apical surface of dog pancreatic duct epithelial cells Tbe agonists had no effect on basolateral release of label Each treatment was done in triplicate in each assay and repeated in at least three different assays Values are averages of each treatment, and the error bars represent standard error All treatments were statistically different from control (P < 0.05) function and to create a model for studying cellular pathophysiology, we have established an in vitro culture system for growing well differentiated pancreatic duct epithelial cells Morphologically, this cultured cell line shares the distinguishing features of normal main pancreatic duct cells in situ,37,39,40 most rou, oS 200- Discussion V 150- c" To understand better how the epithelial cells lining the ducts of the pancreas contribute to pancreatic 100- 50S0 X 3000 , T mm -w - 0- -2500 40- - -2000 co 1- -1-1 - - - - - o 0 + + + : _/ 30- A -1500 _ -1000 _ 0- ; i 20- _ II II o C II 10- 1I - 1.4 U @ 500 10 12 days Figure Percenttage of cells in Sphase (solid line) and the number of cells per well (dashed line) versus dais after plating Tbe cells became corifluent by the 8th day after plating Figure The effect of mmol/L deoxyadenosine (DOA) on mucin secretion from the apical surface of agonist-treated cells is compared with untreated control cells Each treatment was done in triplicate in each assay and repeated in at least two different assays Values are means of each treatment, and the error bars represent standard error Only the db-cAMP plus DOA treatment was statistically different from control(P < 0.05) Pancreatic Duct Cell Culture 983 AJP March 1996, Vol 148, No Table Intracellular cAMP of Control and AgonistTreated Cells as Measured by Radioimmunoassay Agonist Control cells only ,umol/L VIP ,umol/L epinephrine cAMP (pmol) 6.7 43.8 37.8 ± ± ± 34* 18* Values are in picomoles of cAMP per confluent monolayer Each treatment was done in duplicate in each assay and repeated in at least three different assays Values are means of multiple assays + SE *Significantly different from control by Student's t-test (P < 0.05) notably the presence of well developed microvilli, intensive lateral interdigitations between the cells, and secretory vesicles concentrated at the apical surface The cultured dog epithelial cells were passaged repeatedly without visible morphological changes This cell line formed confluent, electrically leak-proof, polarized monolayers All the cultured cells appeared to have the structure of simple epithelial cells Whole tissue or tissue explants of the pancreatic duct have been used extensively by others to study epithelial cell function However, when using these samples, it is often difficult to isolate the response of the epithelial cells to an external stimulus when the stimulus can have multiple effects simultaneously on the various cell types located in the subepithelial layer As the cultured dog cell line consists exclusively of the simple epithelial cell type found in the main pancreatic duct, these cells not have complicating intercellular interactions and can be useful for isolating specific responses to various stimuli As the cells required the presence of a feeder layer of myofibroblasts for continued proliferation, fibroblast-derived factor or factors were diffusing through the support membrane to act as trophic factor(s) for the epithelial cells Feeder layers or conditioned media from feeder layers have been used by other researchers to grow other types of epithelial cells.41-43 It is a common observation that many malignant and normal cells, grown under regular cell culture conditions, not maintain the morphology that is characteristic of these cells in vivo Organotypic culture allows these cells to develop morphology more closely resembling that found in vivo30'31; eg, cultured squamous epithelial cells grow in stratified layers in organotypic culture but not in monolayer culture When grown in organotypic culture, the dog pancreatic cells retained the single-layered columnar and polarized architecture of pancreatic duct epithelial cells The cells had the enhanced morphology characteristic of mature dog main pancreatic duct epithelium,37 such as a higher density of microvilli, taller columnar shape, and an increase in the number of secretory vesicles Important functions of pancreatic epithelial duct cells include the ability to secrete bicarbonate44 and mucin Carbonic anhydrases are enzymes that support high rates of bicarbonate production.45 We have demonstrated by immunohistochemistry that CA 11 and CA IV are present in the cultured columnar epithelial cell in a pattern similar to that seen in whole tissue Others have shown CA activity in pancreatic tissue using a histochemical reaction.46 47 The presence of CA 11 and CA IV, localized respectively to the cytoplasm and cell surface, strongly suggest that these cells have the ability to secrete bicarbonate In addition, these epithelial cells could also synthesize and secrete mucin When the cells were exposed to specific agonists, there was an increase in mucin secretion The addition of VIP or epinephrine to the basolateral side of the monolayer produced an increase in labeled glycoprotein release from the apical surface but not from the basolateral surface The stimulation of mucin secretion by VIP and epinephrine was controlled, at least in part, by the cAMP second messenger pathway, as both of these agonists caused a rapid increase in intracellular cAMP The inhibition of this intracellular cAMP production by an adenylate cyclase inhibitor completely blocked the agonist-induced stimulation of mucin secretion from the apical surface Furthermore, a membrane-permeating analogue of cAMP mimicked the effect of VIP and epinephrine, but this stimulation of apical mucin secretion was not affected by the inhibition of intracellular cAMP production The availability of this cell culture preparation offers a unique opportunity to study the biological function of the pancreatic duct cells, as well as the pathophysiology of a number of disease processes We have been able to demonstrate in these cells cystic fibrosis transmembrane-conductance-regulator- and calcium-activated chloride channels,48 which are involved in normal bicarbonate secretion and cystic fibrosis Furthermore, it has been suggested39 that the duct cells closer to the intestine may be more susceptible to chemical transformation We have used these cultured cells to examine the effects of a known carcinogen and found that long-term alterations in morphology and mucin secretion occurred.28'49 A cell culture system such as this has other advantages It allows single variables to be vectorially (apically or basolaterally) applied to a pure epithelial 984 Oda et al AJP Marcb 1996, Vol 148, No cell culture, it is consistent and highly reproducible, it avoids the unnecessary use of live animals, and it reduces costs Although we have not exhaustively studied these cells to senescence, we not believe, at this stage, that this cell culture preparation is immortalized The morphological, cytokinetic, and metabolic characteristics are so well preserved that we believe it will serve well as a cell model for normal pancreatic duct epithelium 14 15 16 17 Acknowledgments We thank Peter Rabinovitch, MD, PhD, for his assistance with flow cytometry, Audrey Wass for the electron microscopy, Marilyn Skelly and Alan Gown, MD, for immunohistochemistry, and Geoffrey Haigh and Doris Martin for their help References Githens S: Pancreatic duct cell cultures Annu Rev Physiol 1994, 56:419-443 Gstraunthaler G: Epithelial cells in tissue culture Renal Physiol Biochem 1988, 11:1-42 Joplin R: Isolation and culture of biliary epithelial cells Gut 1994, 35:875-878 Kedinger M, Haffen K, Simon-Assmann P: Intestinal tissue and cell cultures Differentiation 1987, 36:71-85 Evans GS, Flint N, Potten CS: Primary cultures for 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cells in culture Int J Cancer 1979, 24:407-414 43 Kobayashi K, Kan M, Yamane I, Ishii M, Toyota T: Primary culture of human gallbladder epithelial cells Gastro Japonica 1991, 26:363-369 44 Case R, Argent B: Bicarbonate secretion by pancreatic duct cells: mechanisms and control The Exocrine Pancreas: Biology, Pathobiology, and Diseases Edited by V Go, F Brooks, E Dimagno, J Gardner, E Leventhal, G Scheele New York, Raven Press, 1986, pp 213-243 45 Swenson ER: Distribution and functions of carbonic anhydrase in the gastrointestinal tract The Carbonic Anhydrases Edited by SJ Dodgson, RE Tashian, G Gros, ND Carter New York, Plenum Press, 1991, pp 265-287 46 Buanes T, Grotmol T, Landsverk T, Ridderstrale Y, Raeder MG: Histochemical localization of carbonic anhydrase with pig's exocrine pancreas Acta Physiol Scand 1986, 128:437-444 47 Githens S, Schexnayder JA, Frazier ML: Carbonic anhydrase 11 gene expression in mouse pancreatic duct cells Pancreas 1992, 7:556-561 48 Nguyen TD, Moody MW, Fox NR, Kuver R, Lee SP: Characterization of chloride transport in primary cultures of dog pancreatic duct epithelial cells Gastroenterology 1995, 108:A378 49 Oda D, Savard CE, Bronner M, Lee SP: Pancreatic epithelial cells in culture: a model of carcinogenesis Lab Invest 1994, 70:65A ... M: Isolation, culture, and characterization of human pancreatic duct cells Pancreas 1993, 8:248-254 Sato T, Sato M, Hudson E, Jones R: Characterization of bovine pancreatic ductal cells isolated... (MNNG) on cultured dog pancreatic duct epithelial cells Pancreas 1996 (in press) Pancreatic Duct Cell Culture 985 AJP March 1996, Vol 148, No 29 Evans H, Christopher G: Miller's Anatomy of the Dog, ... mouse pancreatic duct cells Pancreas 1992, 7:556-561 48 Nguyen TD, Moody MW, Fox NR, Kuver R, Lee SP: Characterization of chloride transport in primary cultures of dog pancreatic duct epithelial cells