Proc. Nail. Acad. Sci. USAVol. 88, pp. 5282-5286, June 1991Physiology/Pharmacology

Polarized intestinal hybrid cell lines derived from primary culture:Establishment and characterizationGREGORY W. APONTE*, ANDREW KEDDIE, GUNNEL HALLDtN, ROBERTA HESS, AND PATRICK LINK

Department of Nutritional Sciences, University of California, Berkeley, CA 94720

Communicated by Howard A. Bern, March 14, 1991 (received for review December 30, 1990)

ABSTRACT A cell culture system has been developed thatproduces stable gastrointestinal (GI) polarized cell lines capa-ble of maintaining hormone secretion. A spontaneously trans-formed rat mucosal epithelial cell was selected for hypoxan-thine/guanine phosphoribosyltransferase deficiency and trans-fected with a plasmid conferring hygromycin resistance (BRIE291 cells). Fusion of these cells with dispersed small intestinalepithelia cells resulted in hybrid cell lines that retained char-acteristic properties of the native GI cell more effectively thanthe transformed tumorigenic parental cell line. Hybrid hBRIE380 cells are uniformly cuboidal with microvilli, contain villin,are contact inhibited, are anchorage dependent, require serumsupplementation for growth, and are more sensitive to virusinfection than the parental BRIE 291 cells. Fusion ofBRIE 291with dispersed pancreatic islet cells has resulted in a variety ofpancreatic-hormone-producing cell lines. One of these, hybridhBRIE 291-i2, forms confluent monolayers capable of synthe-sizing insulin-like immunoreactivity. These studies demon-strate that functionally polarized GI cells can be generatedfrom primary cultures of nondividing committed epithelial cellsby somatic cell hybridization and make feasible the selectionand maintenance of specific GI epithelial cell types in confluentmonolayer cultures.

Epithelial cells of the gastrointestinal (GI) tract exhibit struc-tural and functional asymmetry between apical and basolat-eral surfaces and thus provide a model system to study themechanisms involved in the sorting of proteins to specificdomains. The GI epithelium, unlike Madin-Darby caninekidney (MDCK) cells that have been used extensively tostudy protein trafficking (1), exhibits a diversity of pheno-types all of which maintain distinct apical and basolateralplasma membranes (2). This potentially allows comparativestudies of protein sorting in specific cell types (i.e., endo-crine, absorptive, and exocrine). For example, many GIendocrine cells are "receptosecretory cells" that are incontact with the external and internal environment of theorganism. These gut endocrine cells possess a long thincytoplasmic process covered by a tuft of microvilli thatreaches out into the gut lumen. It has been proposed thatthese "open-end" cells recognize chemicals in the gut lumenthrough these luminal processes and release hormones intothe tissue spaces from their basolateral surfaces by exocy-tosis (3-5). Peptide-containing GI cells often display bipolardistribution of hormone-containing secretory granules. Be-cause these cells contact the lumen (apical plasmalemma) ofthe intestine and the endothelia of the capillary walls (baso-lateral plasmalemma), they most likely have plasma mem-branes specialized to receive specific chemical signals orig-inating from the external (or apical surface) and internal (orbasolateral surface) environments of the organism. Thesecells may respond directly to such signals by exportingregulatory peptides or other proteins to either or both mem-

brane surfaces. It has not been possible to examine themechanism by which these GI cells direct the flow of theirpeptide-containing mature secretory granules to the basolat-eral versus apical membranes to date.

Investigations of properties characteristic of any one GImucosal cell phenotype require the establishment of specificepithelial cell lines derived from a mixed population ofshort-lived nonproliferating cells derived from the GI epithe-lium. In addition cells must maintain polarity. None of thesefeatures are attainable in primary cell culture. Althoughattempts have been made to transform primary cell culturesto obtain GI epithelial cell lines (6-8), no continuous cell lineshave yet been selected from any of these normal or trans-formed intestinal cells. We have demonstrated that termi-nally differentiated (nonreplicating) GI cells can be immor-talized when fused with transformed cells of similar tissueorigin. These cells, which form confluent monolayers andappear to be stable over long-term culture, retain polarizedcharacteristics of in situ GI epithelium and can be selected forhormone secretion.

MATERIALS AND METHODSIntestinal Cells. Dispersed cells for each fusion experiment

were obtained from the duodenum from one Sprague-Dawley(300-400 g) rat with medium containing Eagle's basal me-dium (BME), collagenase IV (100 mg/500 ml), Pronase (100mg/500 ml), and 0.1% bovine serum albumin, 5:1 (vol/wetweight) (Sigma) by incubating at 370C for 15 min. Tissue wasthen washed with 2 mM EDTA in 20 mM Hepes-bufferedsaline (pH 7.2) and incubated again with the BME/enzymesolution. After repeated washing of the cells, loose pelletswere collected, washed with sterile ice-cold BME/1% bovineserum albumin and then with Hank's balanced salt solution(HBSS), and enriched for epithelial mucosa by counterflowelutriation as described (9). Dispersed rat mucosal cells wereplated on culture dishes containing Iscove's modified Dul-becco's medium (IMDM) (GIBCO), enriched with 10%o (vol/vol) fetal calf serum (FCS; HyClone), and examined forspontaneous transformation by colony formation after 1week. A cell line that maintained epithelial characteristicswas selected and karyotyped (10) to use as a future parentalfusion partner for hybridization with the primary culturedintestinal and pancreatic islet epithelia. These cells, Berkeleyrat intestinal epithelial clone 139 (BRIE 139), have undergone>50 passages in a period of 20 months.

Establishment of BRIE 291 Cell Line. Experiments wereperformed with clone BRIE 139. Stock populations weremaintained as monolayers in tissue culture flasks (ComingT-25) in BME containing 10o FCS and 90% (vol/vol) IMDM(containing glucose at 4.5 mg/ml with pyruvate omitted).This BME was supplemented before use with sodium peni-cillin G (60 ,g/ml), streptomycin (50 ug/ml, GIBCO), and

Abbreviations: GI, gastrointestinal; FCS, fetal calf serum; VSV,vesicular stomatitis virus; ILI, insulin-like immunoreactivity.*To whom reprint requests should be addressed.

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L-glutamine (100 Ag/ml, Sigma). HAT medium was preparedby adding 100 tiM hypoxanthine, 0.4 jiM aminopterin, and 16gM thymidine to BME (Sigma). Parent BRIE 139 cells andderivatives were routinely screened by culture methods formycoplasma. After 16 passages, a subclone of BRIE 139 wasisolated for hypoxanthine-guanine phosphoribosyltrans-ferase deficiency (HGPRT-) by stepwise increases inthioguanine (10-100 jig/ml, Sigma) over the course of 3months. To eliminate fibroblast contamination in selectivemedium, cells were transfected with the plasmid pML 272 (akind gift from M. Botchan, University of California at Berke-ley) containing the hygromycin B-resistance gene (Hmr)(11-14), using a calcium phosphate DNA coprecipitatemethod (15). Clone BRIE 291 (BRIE 139HGPRT-/Hmr) wasselected as the immortal parent fusion partner (and universalhybridizer).

Establishment of Intestinal-BRIE 291 Hybrids. Approxi-mately 1 x 106 dispersed cells were plated on collagen-coatedtissue culture dishes as described. After attachment (2 h),BRIE 291 cells were layered on top and allowed to adhere foranother 2 h and then fused using 40% (wt/vol) polyethyleneglycol (PEG, Mr = 1300-1600; American Type CultureCollection) at 370C. Immediately after cell fusion, PEG wasremoved and replaced with 50% (vol/vol) conditioned me-dium from stock BRIE 139 cells in 10% FCS/IMDM, for 48h, followed by replacement with HAT medium with hygro-mycin B (300 ,ug/ml, Boehringer Mannheim). After 21 days,HAT medium had killed all cells except HGPRT-/Hmr cellsthat had fused with wild type. Single cells were cloned fromthe resulting colonies and karyotyped (10). Hybrid clonehBRIE 380 was selected for characterization.

Establishment of Pancreatic Islet-BRIE 291 Hybrids. Pan-creatic islets of Langerhans were isolated as described (16).Cells collected from dispersed islets were washed in 10%FCS/IMDM, centrifuged, resuspended in 10% FCS/IMDM,and counted. Islet cells were then mixed with BRIE 291 cellsin a 15-ml centrifuge tube at 1:2 (4 x 106 islet cells to 8 x 106BRIE 291 cells). Cells were then centrifuged and washedtwice with Ca2 -free Eagle's minimal essential medium(MEM) with glutamine. After the second centrifugation, thecell pellet was gently resuspended in 1 ml of45% PEG (Mr =1300-1600) in MEM at 37°C for 1 min. This solution was thengently diluted with 10 ml ofMEM (Ca2+-free), and cells werecentrifuged, then resuspended in MEM, and allowed to sit atroom temperature for 15 min. Cells were then dispensed intotissue culture dishes containing 10% FCS/IMDM kept in a5% C02/95% air atmosphere overnight at 37°C. SelectiyeHAT medium with hygromycin (300 ,g/ml) was then added,with changes every 4 days until colonies appeared. Thesecolonies were then isolated in 10% FCS/IMDM, grown toconfluency, and karyotyped. Single cells were then clonedand characterized for insulin secretion by radioimmunoassay(RIA) using anti-insulin Gp537 (ICN) as described (17).

Virus Infection. hBRIE 380 cells were plated at 2 x 105cells per 35-mm dish, grown 2 days past confluency (8 days),and then infected as described for MDCK cells (14-16, 18)with some modifications. Cells were infected with vesicularstomatitis virus (VSV) ts0.45 (a kind gift from H. P. Moore,University of California at Berkeley), a temperature-sensitive mutant of VSV that accumulates G protein in theGolgi at 37°C. Infected cells were held at 37°C for 40 min in15% C02/85% air. Cells were then transferred to the per-missive temperature of 32°C, allowing synchronous move-ment of G proteins to the plasma membrane. Cells were thenremoved from the incubator at 20, 60, 90, 120, or 180 min andprocessed for routine electron microscopy.

Electron Microscopy. Approximately 1 x 106 cells, of eachcell line examined, were plated on 35-mm tissue culturedishes that contained a 0.29o collagen gel matrix. Cells weregrown for 12 days after confluency in 10% FCS/IMDM

supplemented with insulin (8.0 Ag/ml) and hydrocortisone(1.0 jg/ml). Cells were then fixed for electron microscopyusing McDonald's fixative (19), embedded in Medcast/Araldite 6005 (Pelco), and ultra-thin-sectioned for examina-tion utilizing a Philips electron microscope 300 at 60 keV asdescribed (9).

Insulin Assay. Confluent cells were incubated in fresh 10%FCS/IMDM 12 h before hormone determination. Cells werethen washed twice with HBSS and preincubated withHBSS/20 mM Hepes/1% bovine serum albumin (fraction V)for 30 min. Culture medium was aspirated for RIA and cellswere then incubated for 90 min in fresh medium containingHBSS/Hepes with 5 mM glucose, 25 mM glucose, or 10 mMtheophylline plus 10 mM L-arginine. Experiments on clonespositive for insulin-like immunoreactivity (ILI), as deter-mined by RIA, were repeated four times. Immunocytochem-istry was performed as described (9).

Cytokeratin. The presence of cytokeratin was determinedon confluent BRIE 291, hBRIE 380, HT-29 (a human coloniccarcinoma cell line; American Type Culture Collection), andWI-38 (human lung fibroblasts, American Type Culture Col-lection) cells. These cells were then scraped off dishes andsonicated in phosphate-buffered saline (PBS; 200 ,ul per dish)for 15 sec at 40C. Proteins from cell suspensions wereseparated by SDS/PAGE on a 10% gel as described (20) andtransferred to nitrocellulose sheets (21, 22). Mouse anti-cytokeratin type II IgG1 (Amersham) was used at 1:50dilution.

Villin. To determine the presence of villin, cells grown toconfluency on 8-cm tissue culture dishes were incubated 5min at 24°C in extraction buffer consisting of 20 mM Tris (pH8.8), 2 mM CaC12, 2% (vol/vol) Triton X-100, and proteaseinhibitors [5 mM phenylmethylsulfonyl fluoride, leupeptin (1jig/ml), and aprotinin (1 ,g/ml) (Sigma)] and treated forimmunoblot analysis as described using mouse anti-villinIgGi (Chemicon) at a 1:80 dilution (21, 23). Visualization wasby peroxidase-coupled goat anti-mouse IgG antibodies (Bio-Rad) at a 1:2000 dilution with diaminobenzidine (Sigma) asthe chromagen.

RESULTSSpontaneous transformations of the dispersed rat mucosalepithelial cells occurred at an approximate frequency of 1:107cells. We selected BRIE 139 cells to develop as the universalhybridizer because they exhibited epithelia-like characteris-tics, such as a cuboidal mosaic morphology and formation ofconfluent monolayers, and expressed cytokeratin. Thesecells have a normal ploidy of 42 chromosomes and havemaintained these characteristics for >18 months of continu-ous culture.BRIE 291 Cells. Clone BRIE 291 (BRIE 139HGPRTJ/Hmr)

was selected as the immortal parent fusion partner. TheBRIE 291 cells assumed confluent cuboidal monolayers whengrown on a type I rat tail collagen gel matrix (24). Newlyconfluent monolayers of cells grown on the gel matrix aver-aged 6 gm thick and formed tight junctions at the apical andapical-lateral plasmalemma. Ten days after confluency, stel-lar- and domed-shaped colonies were observed that had acentral mass five or six cells thick from which ridges of cellsprojected in an irregular star shape. The confluent top cellsgrew at the gel surface and were 6 ,um thick, formed confluentcuboidal monolayers, expressed cytokeratin, and displayedapically oriented tightjunctions between every cell (Fig. 1A).The other cell layers were inside the gel matrix, were thin (3,m), were attenuated, lacked tight junctions, and weresimilar in appearance to the endothelia of the submucosa(Fig. 1A). Like the BRIE 139 cells, the BRIE 291 cells werealso characterized by a smooth even plasma membrane witha thick well-defined layer of cortical filaments (Fig. 2A).

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'44~~~~~~~~.

~~~~~ ~ ~~~~~~~~~~~~~~~~A

4~~~~~~~~~4

FIG. 1. Electron micrographs of 17-day-old monolayers of BRIE 291 cells from a 22-month-old culture (56 passages) (A) and hBRIE 380 from

a 6-month-old (20 passages) culture (B). (Bars = 10 gm.)

Many clathrin-coated-pit-like structures and pinocytotic ves-icles were present. The cells displayed an extensive Golgicomplex and large mats of intermediate filaments (7-11 nm indiameter) scattered throughout the cytoplasm (Table 1).BRIE 291 cells were not contact inhibited, grew on agar

plates (anchorage independent), and appeared relatively in-sensitive to a wide range of serum concentrations in themedium. There was no significant difference in the doublingtimes (18-24 h) of cells grown in 0-10% bovine calf serum for72 h. These characteristics are those expected of a tumori-genic cell line.hBRIE 380 Cells. Although several of the hybrid clones

displayed enterochromaffin-like secretory granules, hBRIE380 cells were selected for characterization because theywere the first of the hybrids that formed confluent monolay-ers and exhibited polarized distribution of microvilli. Thesecells have retained a stable chromosome count between 72and 74 for >13 months of continuous culture (50 passages).Unlike BRIE 291 cells, the hBRIE 380 cells were contactinhibited, anchorage dependent, and would not sustaingrowth in serum-free medium (data not shown). The hBRIE380 cells grown on collagen gel substrate were characterized

'" "

P~~~~~~~~~~~~~~41g-!,INiC

..-A .......................

4.si w S ^K n t ;

FIG. 2. Electron micrograph of apical plasmalemma from BRIE291 (A) and hBRIE 380 (B). Microvilli at the surface of the hBRIE380, when cross sectioned, contained the typical circular array ofmicrofilaments (C). (Bars = 0.25 /m.)

by cuboidal morphology measuring up to 18 ,um high and 12pum wide. They most frequently appeared to grow on thesurface of the gel substrate in monolayers one cell thick (Fig.1B). In contrast to the BRIE 291 cells, there was no layer ofcells within the gel matrix (Table 1). hBRIE 380 cells dis-played numerous apical microvilli when confluent (Fig. 2B).These microvilli were 3 ,um long and contained a core offilaments in cross section (Fig. 2C). The microvilli had attheir base a cortical layer of filaments that contained pinocy-totic vesicles and clathrin-coated-pit-like structures and ves-icles. Below this cortical layer was an organelle-free area1.5-2 gm deep. Extensive bands of tight junctions could beseen running between cells in enfaced sections.

Viral Marker. VSV was found to induce cell rounding inhBRIE 380 cells by 40 min after infection compared to 2 h forBRIE 291 cells. Virus was observed budding from the VSV-infected hBRIE 380 cells 6 h after infection (Fig. 3 A andInset). All observations were made on groups of cells that hadmaintained their cellular junctions (Fig. 3B) and did notexhibit overt cytopathic effects. Virogenic areas were mostfrequently seen in the basal regions of the cells beneath thenuclei. Budding virions were observed in the basal andbasolateral regions of the plasma membrane below the apicalcellular tightjunctions (Fig. 3 Inset). In these cells, the typicalbullet-shaped morphology of the VSV was observed gnd

Table 1. Summary of ultrastructural characteristics betweenBRIE 291 and hBRIE 380 cells grown on a collagen gel matrix

Cell characteristic BRIE 291 hBRIE 380

Cortical layer of filaments ++++ + +Intermediate filaments ++++ + +Golgi development ++ ++ + +Clathrin-coated pit-like and

pinocytotic vesicles ++++ + +Change in cell shape from

elongated to cuboidal + ++++Growth into gel + + + +Lipid-like deposits + + +Apical microvilli + +++Multiple cell layers

(domes) ++++ +

Symbol + or - refers to frequency of observed occurrences(where - means not seen, n > 100).

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110-84-

47-

33-24-

1 2 3 4 56 7

~~~ ~ ~ ~ ~ ~ ~ ~ ~ ~~*

!t.

FIG. 3. Electron micrograph of basolateral budding of VSV inhBRIE 380 cells (A and Inset). Polarized budding was observed incells that maintained tight junctions (B), taken from cell in A. (Bars:A, 2 ,um; Inset, 100 nm; B, 0.25 ,um.)

continuity with the plasma membrane was maintained duringbudding (Fig. 3B Inset). Budded virus could also be seenassociated with endocytotic vesicles in adjacent cells, indi-cating that secondary infection had occurred by 6 h afterinfection. No budding was observed under parallel conditionswith BRIE 291 cells.Immunoreactive Markers. BRIE 291 cells fused with dis-

persed pancreatic islet cells resulted in several hybrid cell linesthat secreted ILI ranging from 150 to 250 pg per well inresponse to 5 mM glucose. Hybrid hBRIE 291-i2 was one ofthe earliest of such lines isolated that maintained a cuboidalappearance similar to the parental BRIE 291 cells and releasedILI (375 pg per well) in response to 25 mM glucose. ILIsecretion at this concentration ofglucose was not enhanced bythe addition of 10 mM theophylline and/or 5 mM L-arginine.Immunocytochemical staining revealed that hBRIE 291-i2cells were not glucagon- or somatostatin-immunoreactive.After 3 months of continuous culture, these cells have main-tained 80-85 chromosomes and subcloned colonies have con-tinued to release ILI.To examine whether a marker normally associated with the

mucosal epithelia was preserved in hybrid cell lines grown inlong-term culture, we tested for the presence of villin. Villinimmunoreactivity was detected in the hBRIE 380 cells takenfrom 12-month-old colonies. No immunoreactive villin wasdetected in BRIE 291 cells from a similar passage number(Fig. 4).

DISCUSSION

Early attempts to establish intestinal epithelial cell cultureswith cells dissociated from tissue treated enzymatically or

FIG. 4. Immunodetection of cytokeratin type II and villin incellular extracts analyzed by SDS/PAGE on 10%o gels and trans-ferred to nitrocellulose sheets (13-15, 23). Lanes: 1, molecular massstandards in kDa; 2, cytokeratin in BRIE 291 cells; 3, cytokeratin inhBRIE 380 cells. Lanes 4-8 display immunoreactive villin from thefollowing sources. Lanes: 4, human colonic carcinoma cell lineHT-29; 5, dispersed rat mucosa; 6, BRIE 291 cells; 7, hBRIE 380cells; and 8, human lung cell fibroblasts. Protein (30-60 Ag) wasloaded per lane.

chemically resulted in cells that failed to attach to substrateor to survive. Modification of dissociation procedures andculture conditions has more recently resulted in severallaboratories establishing either primary cultures (9, 25) or celllines from the GI mucosa (8). Although primary culture oftheGI mucosa display some phenotypes specific for the organfrom which they are derived, they do not exhibit differenti-ated characteristics linked to the intestinal cell polarization invivo. These differentiated characteristics have, however,been shown to be expressed in some human colon carcinomacell lines and by animal cell lines derived from chemicallyinduced tumors. Both models have proved to be of greatbenefit in studying some aspects of intestinal cell differenti-ation and function [e.g., the organization of cell polarity,synthesis and transport of microvillar enzymes (26), andstructural proteins (27)], as well as the pharmacology of GIpeptide hormones and the expression of their receptors.Attempts have been made to transform primary cell culturesto obtain cell lines. Long-term suspension cultures of humancolonic cells and rodent cells have been transformed with theoncogenic DNA simian virus and with a chemical carcinogenazoxymethane (7). However, continuous cell lines have notbeen selected from any of these normal or transformedintestinal cells.

Cell hybrids between rat intestinal villus cell and mouseteratocarcinoma (EC) cells have led to hybrid cell lines.However, in these experiments the introduction of part of thevillus cell genome in the EC cells does not allow for theexpression of specific morphologic or enzyme traits specificto intestinal cells.

Previous studies utilizing cell lines derived from hybrid-ization of transformed fibroblasts to nondividing cells haveresulted in cell lines with limited divisional potential (28-31).In contrast, the properties that promote replication in theBRIE 291 cells have been conserved when these cells havebeen hybridized with other nonreplicating epithelial cells.None of the isolated colonies, kept in continuous culture,have stopped replicating. It is possible that to improve hybridstability, and the likelihood of immortality, the transformedparent cell line should be of the same species, identical celltype, and tissue origin as its fusion partner.

Although the hBRIE 380 cells appeared polarized, it wasimportant to establish that the plasma membrane proteinsremained segregated. Virus-infected cultured epithelial cellsprovide a convenient system to study sorting processes thataffect the distribution of plasma-membrane-domain-specificproteins (32). Boulan and Sabatini (32) demonstrated thatVSV-infected MDCK cells resulted in budding of the virusparticles from only the basolateral plasmalemma. We foundthis rhabdovirus also budded from the basolateral mem-

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branes of the hBRIE 380 cells. This would suggest that thespecialized sorting apparatus used to ensure that the char-acteristic proteins of the two surface domains have remainedintact or that the VSV G and/or M proteins have retainedspecificity to the basolateral plasma membrane (33). Wechose to examine viral budding rather than the distribution ofthe viral G protein because there is evidence to suggest thatthere is strict polarity by which viral budding takes place anda less stringent distribution of viral glycoproteins to the cellsurface (18). The greater sensitivity to viral infection of thehBRIE 380 cells to infection compared to the BRIE 291 cellsmay be representative of a protective function of the cells insitu. For example, mucosa, in response to infection, couldlose tight junctions and infected cells could slough off thetissue before the virus could bud and reinfect the organism(34).We chose to test whether GI hybrid cell lines that main-

tained polarity could in addition retain the hormone pheno-type from the primary cultured cell. The difficulty in testingthis hypothesis with endocrine cells of the intestine directlyis the very low percentage of endocrine cells from the totalmucosal cell population. It seemed important to first establishthat the property of hormone synthesis and release would beconserved upon hybridization with non-hormone-secretingcells. The pancreatic 8 cell was chosen because of the highabundance of endocrine cells that could be dispersed from theislets for fusion with the BRIE 291 cells and because theseepithelial cells are of similar endodermal origin. There havebeen previous reports of hybrid cell lines from dissociatedhamster pancreatic islet cells with an insulinoma cell lineIn-111-6T6R (35). Although In-111-6T6R consists of a, 83, or 8cells, these hybrids have not been reported to secrete insulin.These findings were unexpected as 80% of islet cells are pcells. The possibility that In-111-6T6R might possess a re-pressor against the insulin gene of normal P cells or thatheterokaryons from fused P cells fail to proliferate in culturemay partially explain why fusions were incapable of express-ing insulin gene function. In contrast, islet cell hybrids withBRIE 291 cells have maintained ILI secretion for a 3-monthperiod.These studies demonstrate that somatic cell hybridization

will generate functionally polarized GI intestinal cell linesfrom nondividing committed primary cultured GI epithelialcells. Previous to this report, studies performed on intestinalcell cultures have shown that increased passages have re-sulted in a progressive dedifferentiation of cells in culture,with progressive loss of brush border antigens (36) andcytokeratin expression (37, 38). We have observed thatmucosal hybrids retain characteristic properties of the nativeGI cell more effectively than the transformed tumorigenicparental cell line. In contrast to BRIE 291 cells, hBRIE 380cells are uniformly cuboidal with microvilli, contain villin, arecontact inhibited, are anchorage dependent, require serumsupplementation for growth, and are more sensitive to virusinfection. In addition the hBRIE 380 cells have retained thesecharacteristics for >15 months of continuous culture. Thismodel can be used in future investigations of protein sortingand expression into specific membrane domains and theinvolvement of cytoskeleton in both the exocrine and endo-crine secretion processes of GI epithelium.

We thank K. Park for her technical assistance, Drs. John Forte,Gary Firestone, and Andrew Stolz for comments on the manuscript,and Drs. Barry Shane and Koong-Nah Chung for generosity withtheir time in developing insightful suggestions for our studies. Thiswork was supported by National Institutes of Health GrantDK38310.

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