Culture of human main pancreatic duct epithelial cells

In VitroCell.Dev.Biol, Animal34:211-216, March1998 © 1998Societyfor In VitroBiology 1071-2690/98 $05.00+0.00 CULTURE OF HUMAN MAIN PANCREATIC DUCT EPITHELIAL CELLS DOLPIdINE ODA, CHRISTOPHER E SAVARD,TOAN D NGUYEN, ERIC R SWENSON, ~,~DSUM P LEE~ Departments of Oral Biology (D 0.) and Medicine (C E S T D N E R S S P L.), University of Washington and I~ Medical Center Seattle, Washington 98108 (Received 22 April 1997: accepted 19 May 1997) SUMMARY Attempts to grow human pancreatic duct epithelial cells in long-term culture have proven difficult We have developed a system of growing these cells for several passages by adapting methods used to culture dog pancreatic duct cells Epithelial cells were enzymatically dissociated from the main pancreatic duct and plated onto collagen-coated cuhure inserts suspended above a human fibroblast feeder layer After primary, culture, the cells were either passaged onto new inserts or plastic tissue culture plates in the absence of collagen Cells grown on the latter plates were maintained in a serum-free medium Primary pancreatic duct epithelial cells grow steadily to confluence as a monolayer in the feeder layer system After primary culture, cells passaged onto new inserts with fresh feeder layer or plastic plates and fed with serum-fiee medium continued to develop into confluent monolayers for up to four passages The cells were columnar with prominent apical microvilli, sub-apical secretory vesicles, and lateral intercellular junctions resembling the morphology of normal in vivo epithelial cells These ceils were also positive for cytokeratin 19 7, and and carbonic anhydrase II, as measured by immunohistochemistry Metabolically, these cells synthesized and secreted mucin, as measured by incorporation of tritiated N-acetyl-D-glucosamine In conclusion, we demonstrated that human pancreatic epithelial cells from the main duct can be successfully grown in culture and repeatedly passagcd using a feeder layer system, with serum-free medium, and in organotypic cultures Key words: tissue culture; pancreatic duct: human an in vitro culture system for normal human pancreatic duct epithelial ceils beyond limited primary culturing has proved more difficult (14,37,39) In this paper, we describe different methods of culturing human pancreatic duct cells in primary culture and over several passages ~hile maintaining a high proliferative rate These methods were modified from techniques used to euhnre and characterize dog pancreatic duct epithelial cells (30,31) The human cells retain many of the morphological, imnmnohistological, and secretory characteristics of normal pancreatic duct epithelial cells INTRODUCTION Pancreatic cancer is the fifth leading cause of cancer death in the United States (1) and fourth in the United Kingdom (16) It is aggressive and uniformly lethal: mortality approaches 100% The most common tumors are those with a ductal phenotype (1) The absence of a good in vitro model for human ductal cells has contributed to the inadequate knowledge of the molecular and genetic events related to pancreatic carcinogenesis Normal pancreatic epithelial cells in culture would provide an excellent opportunity to study the process of carcinogenesis and neoplastic progression where genetic events and markers of cell differentiation could be sequentially studied Furthermore, cultured pancreatic cells with high proliferative capacity would be useful for studying the contribution of these epithelial cells to the composition of the mucin and bicarbonate contained in pancreatic secretions, as well as in the study of various pathophysiologies such as cystic fibrosis and chronic pancreatitis Although a number of cell lines of pancreatic epithelial origin are available, most are of either metaplastic or neoplastic origins (812,24-26,29,32), or have been genetically altered (13) Due to their source, these cells are less than ideal for the study of initial events in carcinogenesis and have many significant differences in morphology and activity compared to nor,naI cells (25) The development of MATERIALSAND METHODS Materials Analytical grade chemicals and tissue culture supplies were obtained from Sigma Chemical Co (St Louis, MO), except where noted Vitrogen, a bovine dermal collagen, was purchased from Collagen Corporation (Fremont CA) Falcon culture and Primaria plates were used (Becton Dickinson Co Franklin Lakes, NJ), and the Transwell inserts (24 mm diameter, um pore size) were purchased from Costar Co (Cambridge, MA) Cell isolation and culture The culture of human panerea'dc duct epithelial cells was modified from methods previously developed to grow nontransforreed dog pancreatic duct epithelial cells (31) Portions of normal human pancreas were obtained from organ donors or removed during surgery, with approval from the Human Subjects Committee at the University of Washington and the Virginia Mason Medical Center The main duct was dissected from the surrounding pancreatic tissue under aseptic conditions, cut open, and submerged in 025% trypsin/0.1% ethylenedinitrilo-tetraacetie acid (EDTA) at 37" C The epithelial cells dissociated in sheets from the duct within 20 to 35 rain while very few fibroblasts were released in this short time The released cells were centrifuged, resuspended in medium, and plated o n t o collagen-(Vitrogen) coated Transwell inserts (0.5 ml of part collagen:l part medium) The inserts were then placed above a confluent feeder layer of 1To whom correspondence should be addressed at Gastroenterology Mailstop GI-III, VA Medical Center, 1660 South Columbian Way, Seattle Washington 98108-1597 211 212 ODA ET AL human gallbladder myo-fibroblasts (31) In most cases, the cells isolated from a 1-2 cm length of duct were plated onto two inserts The cells were fed Eagle's minimum essential medium (EMEM) with 10% fetal bovine semrn (FBS), mM L-glutamine, 20 mM N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (EDTA), 100 IU/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) Cells were passaged when confluent using 0.05% trypsin/0.02% EDTA treatment for 10 rain After primary culture, some of the cells were plated onto Primaria tissue culture plates (positively charged plastic plates) and fed with mammary- epithelial growth medium (MEGM), a serum-free medium containing epidermal growth factor (10 ng/ml), insulin (5 ug/ml), hydroeortisone (0.5 ug/ml), and bovine pituitary extract (4 ui/ml) (Clonetics Corp., San Diego, CA) Cells were frozen in EMEM medium containing 10% dimethylsulfoxide Organot)pic culture The organotypic culture technique yields cells with well-differentiated morphology (3.27) This technique was performed as previously described (31) In brief, trypsinized epithelial cells were plated onto the top of a solid gel consisting of type rat-tail collagen (Collaborative Biomedical Products, Bedford, MA) and human dermal fibroblasts (3) After epithelial cell attachment, the gels were submerged in supplemented medimn containing 10% FBS and fed daily After to d, the gels were lifted to the liquid/air interphase by transferring them to metal grids placed on organ culture plates, and the gels were fed exclusively through the basal surface After d, the gels were fixed in Hollande's fixative for thin section light microscopy or prepared for electron microscopy Electron microscop): For transmission electron microscopy, confluent cells were washed with phosphate-buffered saline (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 Irnmunohistochemistry staining The Hollande's fixed cells were paraffinembedded and thin sectioned The sections were utilized for immunohistochemistry using antibodies against various cell markers, as described in Gown and Vogel (15) Markers for epithelial cells were assayed using antibody clone K4.62 (ICN Pharmaceuticals Costa Mesa CA) for cytokeratin 19, OVTL12/30 (Dako, Carpinteria, CA) for cytokeratin 7, 3513Hll for cytokeratin 8, and 341~E12 for cytokeratins 1, 5, 10, and 14 (15) Vimentin and desmin markers were also tested Sheep polyclonal antibody against human carbonic anhydrase lI was purchased from Biodesign International (Kennebunk, ME) Detection of primary antibodies was done by using either biotinylated antimouse or anti-sheep IgG antibody (Vector, Burlingame, CA), followed by Elite ABC (Vector) and stained by diaminobezidine (DAB) and NiCI2 (15) The sections were lightly counterstained with methyl green Sections of pancreatic tissue were used as controls Mucin synthesis and secretion The mucin assay was performed as detailed in Kuver et al (21) In brief, human pancreatic epithelial cells were grown on Transwell inserts until confluent, and p.Ci per well of ZH-N-acetyI-Dglucosamine (ICN Pharmaceuticals) was added to the lower cmnpartment (basolateral side) After 24 h, the apical medium was removed and the cells harvested by trypsinization The incorporation of radioactivity into intraeellular glycoproteins and the release of labeled compounds into the apical medium was measured by scintillation counting after protein precipitation by 10% trichloroacetic acid/l% phosphotungstic acid In parallel experiments confluent human gallbladder myo-fibroblasts grown on plastic were treated similarly Protein was measured using a modified Lowry assay (Sigma) Each cell type was assayed in duplicate or more and the assay repeated twice• The values are presented as the mean of multiple inserts _+ standard error (SEM) Statistical significance of differences between the different cell types was assessed using Student's unpaired t-test RESULTS Enzymatically dissociated cells from human main pancreatic duct attached and grew in primary cuhure on collagen-coated Transwell inserts in the presence of a myo-fibroblast feeder layer The quantity of harvested cells varied fronl pancreas to pancreas, and most cells were released in large sheets, making accurate cell counts difficult The primary cultured cells reached confluence in to d The cells " / < - " ~ ~ - k " ,k': FIG (A) Phase contrast microscope picture of a spreading colony of primary cultured human pancreatic duct epithelial cells on collagen Extending from this colon) are collagen strands, which tend to form as the ceils grow Original magnification = × 200./B) Electron micrograph (EM) of the cells grown on collagen after three passages above myo-fibroblast feeder layer showing apical microvilli Space bar = ~tM Magnification = :x:5600 were subsequently passaged, in the form of single cells and small cell clusters, at a seeding density of 200 000 to 400 000 cells per insert and became confluent in - wk Both single cells and cell groups appeared to attach and grow equally They were successfully passaged up to four times, after which the cells stopped dividing In a few of the isolations, cells with apparent fibroblast characteristics quickly appeared and grew faster than the epithelial cells In these cases, the cells were not used for further experiments Frozen epithelial cells from early passages remained viable after thawing The cells were small, cuboidal, and uniform They grew into small colonies that coalesced at confluence Because the Transwell insert makes the photographing of attached cells difficult, some primary ceils were plated onto collagen-coated culture dishes Fig A shows primary, cells grown on collagen, a few days after plating, forming a HUMAN PANCREATICCELL CULTURE 213 A 13 fd :-? ~ I FiG Human pancreatic duct epithelial cells grown in organotypicculture after two passages on inserts Layer below epithelial cells is collagen matrix containing fibroblasts (A) Thin section showing palisaded cells growingin a monolayerand the immunohistochemical labeling of cytokeratin 19 (light microscope X 1200) Space bar = 10 g.'~/.(B) The tall colunmar cells have well-developedmicrovillion the apical surface, lateral intercellular junctions (arrow), and secretor'; vesicles [electron micrograph (EM) × 5600] Space bar = ~tM (C) The apical surface exhibiting secretory, vesicles and extensive glyeocalyxsurrounding microvilli Some vesicles appear to be in contact with the apical plasma membrane fEN X 21 000) Space bar = 0.5 la.l~l colony that is dividing and spreading out Over this short time period, these cells have the same visual attributes as the cells grown on inserts Alternatively, after primary culture, the cells could be grown on Primaria plates when fed with MEGM Cell attachment was enhanced by using MEGM plus 1% FBS for the first 24 h, after which they were fed with serum-free MEGM Under these conditions, the cells could sustain a fourth passage By passage four the cells became larger and vacuolated, and later started to degenerate By transmission electron microscopy (Fig B), cells grown above feeder layers after three passages retained apical micro-villi, normal appearing mitochondria and endoplasmie reticulum, varying degrees of lateral interdigitation, and intercellular junctions Basal lamina was not observed, and some cells displayed large vacuoles at later passages When grown on a collagen-fibroblast matrix of the organotypic culture, the cells grew as a sirigle layer of columnar cells, mostly palisaded and polarized, but at times folding into small projections (Fig A) By transmission electron microscopy (Fig B), the olganotypieally grown cells more closely resembled the in vivo morphology of pancreatic duct ceils than the ceils cultured on inserts (i.e., the organotypic culture cells were more columnar and had extensive apical microvilli, prominent lateral interdigitations, and intercellular junctions) The microvilli were surrounded by a distinct glycoealyx Most ceils displayed numerous apically located secretory granules, some of which appear to be in physical contact with the plasma membrane (Fig 6) Using standard immunohistoehemical techniques, the cells expressed the cytokeratins 19 (Fig A), 7, and which are markers of simple epithelial cells (14,15); hm~eve~: there was some labeling by the antibody 3413E12, which is a marker for squamous epithelial ceils The cells showed no binding of antibodies against vimentin and desmin, markers for mesenchymal and muscle ceils, respectively The carbonic anhydrase lI (CA II) antibody specifically labeled the cytoplasm of the cultured cells with the strongest signal in the sub-apical cytoplasm (Fig 3) Control pancreatic tissue demon- 214 ODA ET AL ~4t, i • v'II :n ' : ,4, , FIG Immunohistochemistry using polyelonal antibodies against carbonic anhydrase II on cultured human pancreatic duct cells grown on organotypic culture after two passages on inserts Space bar = 10 p.M Magnification = X 1200 TABLE INCORPORATION OF 3H-N-ACETYL-D-GLUCOSAMINE BY CELLS IN CULTURE~ Cell Type D P M / m g protein ± SEM (n) Human Pancreatic Duct Epithelial Cells Human Gallbladder Myo-fibroblast Cells 2674 _+ 186 632 _+ 46 (n = 8) (n = 4) °Incorporation of tritium-labeled N-acetyl-Dglucosamineinto glycoproteins of cultured cells After protein precipitation, the glyeoprotein labeling of the pancreatic cells was found to be more than four times higher than labeling of human myo-fibroblast cells based on equal cellular protein amounts Pancreatic duct cell labeling was significantly different from the fibroblasts as determined by Student's unpaired t test (P < 0.01) SEM = standard error strated the presence of CA II within the cytoplasm of the epithelial cells lining the ducts, as others have previously reported (14) The strongest CA II labeling was in the sub-apical cytoplasm Some of the epithelial cells of both the cultured cells and control pancreatic tissue samples were very positive for CA II compared with adjacent cells The tritium labeled N-acetyl-D-glucosamine was incorporated into the intracellular glycoproteins of the pancreatic cells at significantly higher levels (P < 0.01) than myo-fibroblast cells (Table 1) The pancreatic cells showed an average release of labeled glycoprotein from the apical surface of 433 DPM per ml medium + 252 (SEM) after 24 h (n = 6) The myo-fibroblasts had no measurable release (scintillation counts equal to background) of labeled glycoprotein in 24 h (n = 2) DISCUSSION Alterations in the biological function of pancreatic duct cells have been implicated in the development of pancreatic cancer, pancreatitis, and cystic fibrosis (17) Of the many questions raised concerning the etiology of these diseases at the cellular level, few have been answered One of the reasons is the lack of an appropriate isolated duct cell preparation (23) Dependable human in vitro models are essential for understanding the etiology and pathogenesis of these diseases, for instance the cellular and subcellular changes that induce normal cells to become cancerous The development of in vitro models has been limited by the difficulty of growing isolated duct cell preparations from human pancreas (14), thus the majority of studies to date have used cell cultures derived from hamster (18,20,36), guinea pig (38), rat (6,33), or dog (31) The greatest success achieved so far with human pancreatic duct epithelial ceils have been with cancer cell lines (8-12,2426,29,32), with cell aggregates, or muhicellular spheroids derived from normal or malignant cells (4,10.34.41) Vila et al (39) described the primary culture of adult human pancreatic duct cells; however, these primary" epithelial cells were unable to proliferate after passaging Recently, Furukawa et al (13) used human papilloma virus genes to genetically alter primary human pancreatic duct epithelial cells, in order to maintain these cells in long-term culture This paper describes alternative methods for culturing normal human pancreatic duct epithelial cells beyond primary culture by using: (a) a collagen-coated insert system together with myo-fibroblast feeder layers, and (b) a serum-free system with cells grown on plastic These techniques allowed the cells to greatly proliferate from the limited number of cells originally isolated from small pancreatic duct sections and to be passaged up to four times, without using chemical or genetic alteration The ceils have morphological characteristics of well-differentiated, polarized epithelial cells with prominent microvilli Organotypic culture allows the epithelial cells to be nourished from their basolateral side only and to have a close association with fibroblasts, thereby replicating the in vivo interactions between the epithelial ceils and the sub-mucosa The secretory granules seen in the cells closely resemble those seen in our cultured dog pancreatic duct epithelial line (31) The immunohistochemistry studies demonstrated that the cultured cells had cytokeratin 19, 7, and 8, which supports the characterization of these cells as simple epithelial cells of pancreatic origin (14) Important functions of normal pancreatic epithelial duct cells include the ability to secrete bicarbonate (5) and mucin The enzyme carbonic anhydrase is necessary to support the high rates of bicarbonate production associated with normal digestion (35) We have demonstrated by imnmnohistochemistl.'y that CA II is present in these cultured columnar epithelial ceils, in a pattern similar to that seen in whole tissue The presence of CA II strongly suggests that these cells have the ability to secrete bicarbonate, In addition, the mucin assay results suggest that the cultured human cells are metabolically active in nmcin synthesis, since the incorporation of label into the epithelial cells was significantly greater than control myo-fibroblast cells Also, the pancreatic cells had substantial release of mucin from the apical surface A chemical analysis of the mucin released by these cells would be useful since a differenee in chemical composition of secreted muein between normal and cancerous pancreatic ducts has been noted clinically (7,22,28,40) Human epithelial ceils of the larger pancreatic duets in vivo apparently have a higher cell mitotic rate compared to the acinar-associated epithelial cells of the smaller ducts (19) This higher turnover rate, along with the possible reflux of biliary and duodenal contents into the pancreatic ductal system and subsequent biliary pancreatitis (2), may lead to an increased susceptibility to DNA in- HUMAN PANCREATIC CELL CULTURE jury and subsequent alterations in oncogenes or tumor suppressor genes The human cells discussed here were isolated from the main pancreatic duct; therefore, they are an appropriate in vitro model for ductal cancers We have successfully used cultured dog pancreatic duct cells to study constitutive and stimulated mucin secretion by examining the effect of secretagogues and their transduction pathways (31) With this dog cell line, alterations in genetic markers due to chemical carcinogens were monitored by using organotypic culture and flow cytometry (30), Cun'ently, we are using the cultured human cells in a similar manner, since human cells are clearly the preferred model for human diseases However, as commonly observed, human cells tend to be more difficult to culture and have a more limited culture life than animal cells Now, we are working to modify conditions to extend the life and reproductive cycles of the normal human epithelial cells in culture and to further characterize the cells grown in the serum-free medium conditions, as well as to "immortalize" the cells using the viral vector SV40 In conclusion, we have shown that it is possible to grow human main pancreatic duct epithelial ceils and preserve their differentiated morphology and metabolic integrity over several passages ACKNOWLEDGMENTS We thank Audrey Wass for the electron microscopy: Marilyn Skelly and Alan Gown, M.D for immunohistochemistry; the Virginia Mason Medical Center; and Pamela Enayati, Harriet Klinkspoor, Lydia Eng, and Geoffrey Haigh for their valuable assistance This work was supported by a grant from the NIH (DK50246) and in part by the Medical Research Service of the Department of Veterans Affairs REFERENCES Ahlgren, J D.; Hill, M C.; Roberts, I M Pancreatic cancer: patterns, diagnosis, and approaches to treatment In: Ahlgren, J D.; Macdonald, J., eds Gastrointestinal oncology Philadelphia: J B Lippincott Co.; 1992:197 Banerjee, A K.: Steele, R J Current views on the pathophysiolog), of acute biliary pancreatitis Gut 36:803~05: 1995 Blanton, R A.; Perez-Reyes, N.: Merrick, D T., et al Epithelial cells immortalized by human papillomaviruses have premalignant chal~ acteristics in organotypic culture Am J Pathoh 138:673-685; 1991 Carlsson, J.; Nilsson, K.; Westermark, B., et al 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CELLS IN CULTURE~ Cell Type D P M / m g protein ± SEM (n) Human Pancreatic Duct Epithelial Cells Human Gallbladder Myo-fibroblast Cells 2674 _+ 186 632 _+ 46 (n = 8) (n = 4) °Incorporation of tritium-labeled... glycoproteins of cultured cells After protein precipitation, the glyeoprotein labeling of the pancreatic cells was found to be more than four times higher than labeling of human myo-fibroblast cells