In VitroCell.Dev.Biol. Anilna133:104-109.February1997 © 1997Societyfor In VitroBiology 1071-2690/97 $05.00+ 0.00 ISOLATION AND LONG-TERM CULTURE OF GALLBLADDER EPITHELIAL CELLS FROM WILD-TYPE AND CF MICE RAHUL KUVER.~ CHRISTOPHER SAVARD, TOAN D NGUYEN, WILLIAM R A OSBORNE ANDSUM P LEE Departments of Medicine (R K., C S T D N., S P L ), and Pediatrics (W R A ), University of Washington and VA Medical Center, Seattle, Washington 98195 (Received November 1995; accepted March 1996) SUMMARY Mice with targeted disruption of the cfir gene show pathophysiologic changes in the gallbladder, which correlate with hepatobiliary disease seen in cystic fibrosis patients As gallbladder epithelium secretes mucin, and as this epithelium consists of a relatively homogenous cell type, study of CFTR function in these cells would be beneficial to delineate the complex cellular functions of this protein The size and anatomic location of the murine gallbladder makes such studies difficult in vivo Therefore, the need exists for in vitro models of gallbladder epithelium We describe a method to isolate and culture murine gallbladder epithelium from wild-type and CF mice Ceils were grown in a monolayer on porous inserts over a feeder layer of fibroblasts These nontransformed cells can be successively passaged and maintain a well-differentiated epithelial cell phenotype as shown by morphologic criteria, characterized by polarized columnar epithelial cells with prominent microvilli and intercellular junctions Organotypic cultures showed columnar cells simulating in vivo morphology This culture system should be valuable in delineating cellular processes relating to CFTR in gallbladder epithelium Key words: CFTR; cell culture; gallbladder; cystic fibrosis; epithelium; murine pleiotropic cellular roles of CFTR The gallbladder is abnormal in CF patients, showing hypoplasia, thick mucus, and calculi (1) In UNC mice dying from intestinal blockage in the early postnatal period, the gallbladder was often ruptured when examined postmortem (39) Even in apparently healthy cfir ( - / - ) mice, polymorphonuclear cell infiltration throughout the gallbladder wall suggested ongoing inflammation (39) In vivo electrophysiologic studies are difficult to perform on the nmrine gallbladder, however, due to its size and location Therefore, the need exists to culture murine gallbladder epithelium in order to advance these studies INTRODUCT1ON Mouse models of cystic fibrosis have been created by targeted disruption of the cfir gene (7,11,31,39) These models have been valuable for delineating CFTR-related organ pathology In one such model, created at the University of North Carolina (UNC), homozygous cfir ( - / - ) mice showed significant pathology related to the intestinal tract, with death occurring in the postnatal period from complications relating to intestinal blockage in a syndrome resembling meconium ileus (40) The trachea (15), nasal epithelium (14), jejunum (13), oviduct (24), colon (8), small intestine (42), pancreatic acinar cells (10), and pancreatic duct epithelium (12) have been studied either in vivo or in cell culture in this and other mouse models These studies have provided a detailed understanding of the electrophysiologic role of CFTR in these cell types CFTR possesses diverse cellular functions (32) Negative regulation of an epithelial sodium channel (41), regulation of the outwardly rectifying chloride channel via transport of ATP (38), and its originally demonstrated role as a cAMP-regulated apical membrane chloride channel (36) point to the complex nature of this molecule Intracellular CFTR functions involving the biosynthetic pathway (5,22), endosomal acidification (3), and membrane recycling (4) are under active investigation A central question is the relationship between defective or absent CFTR and the hyperviscous, abundant mucus found in various organ ductal systems The gallbladder is an ideal organ for studying the MATERIALSAND METHODS Materials 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 (Franklin Lakes, NJ) and the Transwell inserts (24-mm diameter, 3-p.m pore size) were obtained from Costar (Cambridge, MA) Cell isolation and culture Mice with targeted disruption of the cftr gene created at the University of North Carolina (39) were maintained at the University of Washington Animal Care Facility For the initial isolation, cfir ( - / - ), cfir heterozygous ( + / - ) and wild-type cfir ( + / + ) littermates were used The isolation procedure was repeated months later with another cohort of mice, with identical results Mice were more than 20 d old Mice were killed by cervical dislocation and washed with 90% ETOH, and a laparotomy was performed under sterile conditions The gallbladder was exposed, removed from the common bile duct, and punctured, and bile was aspirated Each gallbladder was cut longitudinally, then washed with phosphate-buffered saline (PBS) multiple times Five milliliters of 0.25% trypsin/ 0.1% EDTA was added, followed by incubation for 20 at 37° C with frequent vortexing Ten milliliters of Eagle's minimum essential medium containing 10% fetal bovine serum, mM L-glutamine, 100 IU penicillin/ml, ~To whom correspondence should be addressed at Division of Gastroenterology, Box 356424, University of Washington, 1959 NE Pacific Street, Seattle, Washington 98195 104 MOUSE GALLBLADDER EPITHELIAL CELL CULTURE 105 inserts was between 60 000 and 250 000 cells per well When confluent, each well contained approximately million cells The insert allowed separate access to both the apical and basolateral sides of a confluent monolayer For cryopreservation, the epithelial cells were frozen in medium containing 10% dimethyl sulfoxide In parallel experiments, cells were plated onto Vitrogen-coated 35-mm plastic plates and fed with conditioned medium obtained from confluent monolayers of human gallbladder myofibroblasts and mixed 1:1 with MEM Organotypic cultures This culture method permits greater morphologic differentiation of ceils, allowing comparison to in vivo nmrphologic characteristics (26) The culture method features an air-liquid interface, with the apical FIG Phase-contrast light micrograph of mouse gallbladder epithelial cells in monolayer culture Shown is a subconfluent monolayer of cfir (+/+ ) ceils grown on Vitrogen with human gallbladder myofibrublast-conditioned media Original magnification )< 20 100 p,g streptomycin/ml, 20 mM HEPES, 1.0 g glucose/1, supplemented with ~tg insulin/ml from bovine pancreas, p,g human transferrirdml, ng sodium selenite/ml {ITS supplement), X vitamins solution, and X nonessential amino acid solution (Sigma) was then added This medium will subsequently be referred to as MEM Trypsin was inactivated by the fetal bovine serum The samples were transferred to a tube and centrifuged, and the superuatant was discarded The pellet was resuspended in ml of medium and pipetted onto a 35-mm plate Many sheets of cells were visible which were separate from the gallbladder wall fragments These gallbladder wall fragments were manually removed with forceps, and the medium with suspended cells was pipetted onto Vitrogen-coated inserts above a confluent layer of human gallbladder myofibroblasts The seeding density of the gallbladder epithelial cells was approximately X 105 per insert One insert was plated out from each gallbladder The remaining cells in the 35-mm plate were more than 95% viable by Trypan blue exclusion MEM was added into the apical and basolateral compartments (2 ml each side) and the inserts were placed in a 37 ° C humidified incubator (5% CO2/95% air) Twenty-four hours after plating, the unattached epithelial cells were removed during replacement of the medium, and the attached ceils became confluent d after plating The myofibroblast cell line was isolated from a normal human gallbladder after a 1-h treatment with 0.5% trypsin/0.2% EDTA, which enabled isolation of both epithelial cells and myofibroblasts Myofibroblasts preferentially attached to and grew on plastic culture plates, whereas gallbladder epithelial cells did not Therefore, gallbladder myofibroblasts were cultured on 60-ram plastic culture plates in MEM The seeding density was approximately '1 X 105 cells per 60-mm culture dish The MEM was replaced every d until cells were confluent which required approximately week Once confluent, these cells were trypsinized with 0.25% trypsin/0.1% EDTA; then MEM was added, and the ceils were plated onto 35-mm six-well plates at a seeding density of x 105 cells per well Confluency was achieved in approximately week, at which point the cells stopped dividing and could be maintained in culture for up to weeks Vitrogen-coated inserts containing mouse gallbladder epithelial cells were placed above these wells, as described earlier The mouse gallbladder epithelial cells and the human gallbladder myofibroblasts were fed twice weekly (2 ml apical/2 ml basolateral compartments) with Dulbecco's modified Eagle's medium (Gibco-BRL, Gaithersburg, MD) with 10% fetal bovine serum, mM L-glutamine, 100 IU penicillin/ml, 100 ~tg streptomycin/ml, mM sodium pyruvate, 4.9 g glucose/1 supplemented with ~tg insulin/ml from bovine pancreas, ~g human transferrin/ml, ng sodium selenite (ITS supplement)/ml, X nonessential amino acids solution and X vitamins solution (Sigma), hereafter referred to as DMEM Whereas DMEM was used from Passage onwards, the cells could be maintained beyond Passage in MEM Care was taken during feeding to not contaminate the epithelial cells in the upper compartment of the insert with the myofibroblasts from the bottom compartment The cells were passaged when they became confluent, usually 6-10 d after plating, with 0.25% trypsin/0.1% EDTA to release the cells When being passaged, the seeding density for new A B FiG Electron micrographs of (A) cfir (+ / +) and (B) cfir ( - / - ) mouse gallbladder epithelial cells grown on Vitrogen-coated inserts over a feeder layer of human gallbladder myofibroblasts Both cell types are at Passage Apical microvilli, lobed nuclei, mitochondria, and apically localized secretory granules are evident Original magnifications: (A) X 8000; (B) X 21 000 106 KUVER ET AL o f mr FIG Light micrograph of cfir ( + / + ) mouse gallbladder cells in organotypic culture The cells are at Passage Tall, columnar cells with basally localized nuclei are evident Original magnification × 40 surface of cells facing the air and the basolateral surface bathed in medium It is not a method for maintaining cells in long-term cuhure Full details of the technique is provided elsewhere and was performed as described (26) In brief, mouse gallbladder epithelial cells, maintained on inserts as described earlier, were trypsinized with 0.25% trypsin/0.1% EDTA, resuspended in medium, and plated at a seeding density of × 10~ cells onto a freshly prepared matrix consisting of seven parts rat-tail collagen Type (Collaborative Biomedical Products, Bedford MA) at mg/ml, and one part fetal bovine serum containing human neonatal foreskin fibroblasts (26) The collagen-fibroblast matrix was preincubated at 37 ° C for 15 in order to solidify After h to allow epithelial cel] attachment to the collagen matrix, the matrix was gently scraped from the sides and bottom of the dish to allow A the collagen to contract evenly This resulted in a matrix approximately i cm in diameter and 1.5 mm thick The ceils were submerged in medium (26) and fed daily After to d, they were transferred to metal grids placed on 60-mm organ tissue culture plates with culture medium in the center well In this system, the epithelial cells were exposed to 5% CO2/95% air on the apical surface and received all their nutritional support vectorally from the basolateral side as the medium diffused through the collagen-fibroblast layer After d, the cells were fixed in Hollande's or half-strength Karnovsky's fixative for thin-section light microscopy or electron microscopy, respectively Transmission electron microscopy Confluent cells were washed with PBS and fixed in half-strength Karnovsky's reagent The cells were postfixed in 2% osmium tetroxide with 0.1 M cacodylate buffer, dehydrated in graded ethanols, and embedded in Epon (Pella Inc., Redding, CA) Thin sections were stained with uranyl acetate and lead tartrate and photographed in a Philips EM410 transmission electron microscope Flow cytor,vetry Cells were plated at X 105 cells per well The culture medium was changed every d At Days 2, 4, 9, and 14 after plating, duplicate wells were washed and trypsinized Staining and flow analysis were done as described (35) Cell pellets were resuspended in ml of 4',6-diamidino-2-phenylindole-staining solution with 10% dimethyl sulfoxide (vol/vol) 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) Western blotting Cells were harvested with 0.25% trypsin/0.1% EDTA, washed, pelleted, and solubilized in 0.125 M Tris-HC1, 4% sodium dodecyl sulfate (SDS), 20% glycerol, 10% 2-mercaptoethanol, and protease inhibitors Proteins (200 ~g/lane) were then separated by SDS-polyacrylamide gel electrophoresis and blotted onto polyvinylidene membrane (Immobilon-P, Millipore Corp., Bedford, MA) CFTR was detected with sequential 1-h incubations with a 1:2000 dilution of a polyclonal CFTR antibody, a 1:40 000 dilution of biotin-goat-anti-rabbit antibody (Vector Labs, Burlingame, CA), and streptavidin-horseradish peroxidase Peroxidase activity was demonstrated with enhanced chemiluminescence (Amersham, Arlington Heights, IL) The CFTR polyclonal antibody was raised in rabbits against the 13 amino-acid C-terminal sequence of human CFTR (22) B FIG Electron micrographs of (A) cfir (+/+ ) and (B) cfir ( - / - ) mouse gallbladder ceils in organotypic culture Both cell types are at Passage A higher density of apical microvilli when compared to cells grown on inserts, and numerous apical secretory granules are evident Original magnifications: (A) X 9000; (B) X 16 000 MOUSE GALLBLADDEREPITHELIAL CELL CULTURE Measurement of monolayer resistance [29] Wild-type mouse gallbladder epithelial cells were cultured on Vitrogen-coated inserts as described earlier and monolayer eonfluenee was confirmed with an inverted microscope The membrane supporting the cell monolayer was then excised and mounted in a modified Ussing chamber with a circular aperture of 0.95 cm2 Both sides of the monolayer were bathed in Ringer's solution (115 mM NaC1, 3L.2mM CaC12, 1.2 mM MgCI2,0.4 mM KHzPO~, 2.5 mM K2HPO4, 25 mM HCO3, and 10 mM glucose) warmed to 37° C with a circulating water jacket and gently mixed and aerated with a constant inflow of 95% air/5% CO2 The two compartments of the Ussing chamber, corresponding to the apical and basolateral surfaces of the monolayer, were next connected to an automatic voltage clamp (model DVC-1000, WPI, Sarasota, FL) through agar bridges and Ag-AgC12 electrodes After equilibration, a constant current of 100 pA was circulated across the mooolayer and the resulting potential difference between the two compartments recorded The resistance of the monolayer was calculated with Ohm's law To adjust for resistance not generated by the monolayer, instrument calibration was performed with a Vitrogen-coated membrane 25 20- ¢13 Mouse gallbladder epithelial cells were dissociated in small sheets from the gallbladder by treatment with trypsin/EDTA The cells attached to Vitrogen-coated inserts in primary culture when plated over a confluent layer of human gallbladder myofibroblasts Cells became confluent in approximately d, and could be successfully passage& In parallel experiments, mouse gallbladder epithelial cells grown on Vitrogen-coated plastic plates and fed with conditioned medium showed attachment and growth, albeit at a slower rate A subconfluent monolayer of cells from cfir ( + / + ) mouse gallbladder is shown in Fig When the feeder layer was replaced with mouse-derived flbroblasts [PA317 cells (27)], the cell density was decreased dramatically and cells did not become confluent Cells grown on inserts above a myofibroblast feeder layer formed confluent monolayers of uniform, short columnar cells consistent with epithelial cell morphology in cuhure (Fig 2) The cells had distinct apical and basolateral surfaces with numerous well-developed microvilli on the apical surface The cells had lobed nuclei, numerous mitochondria, rough endoplasmic reticula, and well-developed Golgi bodies In addition, multiple apically localized secretory vesicles were noted These vesicles were morphologically identical to mucin granules which stained with periodic acid-Schiff/Alcian Blue, pH 2.5, in a previously characterized canine gallbladder epithelial cell line (30) No difference in morphology was noted between cfir ( + / + ) (Fig A) and cfir ( - / - ) (Fig B) mouse gallbladder epithelial cells The confluency of the monolayer was verified by the ability of the epithelial layer to act as a barrier to diffusion of the medium from the upper to lower compartment Co~ffluent monolayers also generated an electrical barrier between their apical and basolateral surfaces Indeed, when mounted in Ussing chambers, monolayers of wild-type gallbladder epithelial cells exhibited a transepithelial resistance of 163 + 11 DJcm2 (mean + SE, N = 12) These cells have been maintained in culture for more than months and 20 passages without discernible changes in microscopic morphology The cells could be stored in liquid nitrogen and thawed without changes in morphology When grown in organotypic culture, the cells maintained strict polarity with a higher density of well-developed microvilli on the apical surface The cells grew in monolayers of tall columnar cells with basally located nuclei as seen under light microscopy (Fig 3) Electron microscopy (Fig 4) showed cells with numerous lateral in- 1000 O \'k 15- ,, TX\ ,,,' ; " 750 -° ,, 500 = 250 ~ 10- 5- 0 RESULTS ]07 10 15 Day FIG Contactinhibition of monolayers with eonfluency The percentage of cells in S phase decreased as the cell numbers plateaued Closed symbols represent cfir ( + / + ) cells; open symbols represent cgqr( - / - ) cells Boxes and solid lines correspond to percent cells in S phase; circles and dashed lines correspond to the number of cells/well (× 1000) Each point is a mean + SE of duplicate confluent inserts tercellular junctions (both tight unctions and desmosomes) and prominent interdigitations between the cells No consistent morphologic difference was noted between cfir ( + / + ) (Fig A) and cfir ( - / - ) (Fig B) mouse gallbladder epithelial cells Preliminary cytogenetic analysis showed that cells retained a karyotype of 40 diploid pairs of chromosomes in each cell, the normal chromosome complement of mouse somatic cells (not shown) The flow cytometric profiles of cfir ( + / + ) and cfir ( - / - ) cells were examined with trout erythrocytes as an internal standard Results of flow cytometry of the cells at different days after plating (Fig 5) showed that the percentage of ceils in S phase gradually decreased to zero as time in culture increased, indicating contact inhibition with confluency Western blotting with a polyclonal antibody made against the Cterminal sequence of human CFTR confirmed the presence of a broad band at n r 140-180 kDa in cfir (+ / + ) mouse gallbladder epithelial cells (Fig 6) This band was absent in cells from cfir ( - / - ) mice, thus confirming the phenotype of these cells DISCUSSION Gallbladder epithelium consists of a relatively homogenous population of columnar epithelial cells which secrete mucin both constitutively and in response to certain secretogogues (21) Culture of canine (30), human (2,17,18,19), and bovine gallbladder epithelial cells (33) has been described Except for the canine cultures, these primary cell cultures not allow successive passaging leading to long-term culture Other investigators have cultured neoplastic gallbladder cells (34); the shortcomings of neoplastic or transformed cells are well known One group has reported primary culture of murine gallbladder epithelium but did not report serial passaging (37) We describe a method to isolate and culture murine gallbladder epithelium from wild-type and CF mice 108 KUVER ET AL 200 116 97 Q 66 FIG Western blot of gallbladder epithelial cells from cfir ( + / + ) and cfir ( - / - ) mice with a polyclonal rabbit CFTR antibody Ceils were at Passage 10 Lane 1: cfir ( + / + ) mouse gallbladder epithelial cells Lane 2: cfir ( - - ] - - ) nlouse gallbladder epithelial cells Lane 3: Molecular weight makers A prominent diffuse band at Mr 140-180 kDa was present in wild-type cells and absent in cells from cfir ( - / - ) mice These nontransformed cultures, which have been serially passaged without change in microscopic morphology, provide a model system for studying the cellular pathophysiology of cystic fibrosis These cells maintain morphologic criteria of epithelial cells for multiple passages The presence or absence of CFTR was confirmed with a polyclonal CFTR antibody on Western blot analysis Thus, these cells maintained phenotypic differentiation with respect to CFTR expression Immunocytochemistry for standard epithelial cell markers was not done due to the lack of availability of antibodies directed against murine epitopes In addition, the widely available antibodies are mouse monoclonal antibodies, which in murine tissues lead to high background staining The morphologic criteria as shown on monolayer and organotypic culture with both light and electron microscopy, however, supports a well-differentiated epithelial cell phenotype for these cells Although the phenotypic expression of CFTR on Western blot was noted in cfir ( + / + ) cells at Passage 10, it remains to be tested whether these cells will retain CFTR and other epithelial cell phenotypic characteristics with further passaging The presence of pathologic changes in the gallbladder in CF patients (1), as well as in the UNC CF mouse model (39), indicates a correlation between human and murine CF pathophysiology for this organ Inspissated secretions in the hepatobiliary ductal system of patients with CF is well described, paralleling changes seen in the airways, pancreatic ducts, and intestines The relatively homogenous environment of the epithelium of the gallbladder, with a predominant columnar epithelial cell type which secretes mucin, provides an ideal model system for researchers to investigate the relationship between the complex intracellular functions of CFTR and the pleiotropic clinical manifestations of CF The formation of a confluent monolayer makes electrophysiological studies possible Consistent with the presence of tight junctions and desmosomes seen by electron microscopy, confluent monolayers of gallbladder cells produced an electrical barrier The transepithelial resistance of these monolayers, 163 D,/cm z, is similar to the resistance of 136 D-Jcm2 reported for the Necturus gallbladder epithelium (20) and compares favorably with the resistance of 57 D-,/cm2 reported for the prairie dog gallbladder (28) Compared with other cultured cells derived from the digestive tract, this resistance is smaller than the 1,900 D,/cmz generated by colonic T84 cells (29) but is similar to the 173 ~,Jcmz generated by colonic Caco-2 cells (16) Of particular relevance to the secretory dysfunction(s) of cystic fibrosis, this transepithelial resistance will allow confluent monolayers of gallbladder ceils to be used in Ussing chambers for studies of ion transport and permeability We have initiated studies with iodide efflux to assess chloride secretion by these ceils (T D Nguyen and R Kuver, unpublished) Mucin synthesis and secretion is also being investigated (23) Transduction of these ceils with retroviral vectors is being pursued (R Kuver, S P Lee, W R A Osborne, unpublished), as the gallbladder has been advocated as a target organ for gene therapy (25,43) In this report we describe a method for the isolation and long-term culture of primary murine gallbladder epithelial cells from wild-type and cfir ( - / - ) UNC mice The availability of this culture technique will enable studies of the role of CFTR in cellular function and the mechanisms of mucin synthesis and secretion In addition, with the increasing availability of various types of transgenic and knock-out mice, this culture method may have wider applicability than proposed here ACKNOWLEDGMENTS We thank Audrey Wass for the electron microscopy and Peter Rabinovitch, MD, PhD, for assistance with flow cytometry This work was supported by a Research 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Curr Opin Puhn Med 1:435 443; 1995 33 Plevris, J N.; Walker, S W.; Harrison, D J., et al Primary culture of bovine gallbladder epithelial ceils Gut 34:1612-1615; 1993 34 Purdum, P P.; Ulissi, A.; ttylemon, P B., et al Cultured human gallbladder epithelia: methods and partial characterization of a carcinoma-derived model Lab Invest 68(3):345-353; 1993 35 Rabinovitch, P.; O'Brieu K.; Simpson, M., et al Flow cytogenetics Cytogent Cell Genet 29:65: 1981 36 Rich, D P.: Anderson, M P.: Gregory, R J., et al Expression of cystic fibrosis transmembrane conductance regulator corrects defective chloride channel regulation in cystic fibrosis airway epithelial ceils Nature 347:358-363: 1990 37 Saito, K.; Yoshida, K.; Kohno, N., et al Purification and culture of mouse gallbladder epithelial cells in secondary culture using microexplant culture on collagen gel Nippon Shokakibyo Gakkai Zasshi 89(11):2693-2699; 1992 [Not Translated] 38 Schweibert, E M.; Egan, M E.; Hwang, T.-H., et at CFTR regulates outwardly rectifying chloride channels through an autocrine mechanism involving ATP Cell 81:1063-1073: 1995 39 Snouwaert, J N.; Bridgman, K K.; Latour, A M., et al An animal model for cystic fibrosis made by gene targeting Science 257:1083-1088; 1992 40 Snouwaert, J N.; Bridgman K K.; Latour, A M., et at A murine model of cystic fibrosis Am J Respir Crit Care Med 151:$59-$64; 1995 41 Stutts, M J.; Canessa, C M.: Olsen, J C., et al CFTR as a cAMPdependent regulator of sodium channels Science 269:847 850; 1995 42 Valverde, M A.; O'Brien, J A.; Sepulveda, F V., et al Inactivation of the murine cftr gene abolishes cAMP-mediated but not Ca2+-medi ated secretogogue-induced volume decrease in small-intestinal crypts Eur J Physiol 425:434-438; 1993 43 Yang, Y.; Raper, S E.; Cohn, J A., et al An approach for treating the hepatobiliary disease of cystic fibrosis by somatic gene transfer Proc Natl Acad Sci USA 90(10):4601-4605; 1993 ... epithelial cells from wild- type and cfir ( - / - ) UNC mice The availability of this culture technique will enable studies of the role of CFTR in cellular function and the mechanisms of mucin synthesis... sequence of human CFTR confirmed the presence of a broad band at n r 140-180 kDa in cfir (+ / + ) mouse gallbladder epithelial cells (Fig 6) This band was absent in cells from cfir ( - / - ) mice, ... blot of gallbladder epithelial cells from cfir ( + / + ) and cfir ( - / - ) mice with a polyclonal rabbit CFTR antibody Ceils were at Passage 10 Lane 1: cfir ( + / + ) mouse gallbladder epithelial