2 4 A1 Cells At 37 0c example essay topic

Permeability of HydrophilicSupervisors: Vladan Milovic Professor Per ArturssonSUMMARYInvestigations of the integrity and transport characteristics of 2/4/A1 cellshave been done in this report. The cell line was isolated from rat fetalintestinal epithelial cells and transfected with thermolabile SV40 large Tantigen. These cells proliferated at 33 ^0C, but eliminated the antigen and ceasedproliferating at a non-permissive temperature (39^0C). At 39^0C 2/4/A1 cellsstarted to differentiate but simultaneously the cells also underwent massivecell death. When cultured at 37^0C these cells formed confluent and tight monolayers thatseemed to have paracellular transport characteristics similar to that of thehuman intestine.

Transmission electron microscopy confirmed the development ofmultilayers at 33^0C, monolayers at 37^0C and defects in the cell layer due toapoptosis at 39^0C. Different immunostainings of ZO-1, E-cadherin and vinculin confirmed formationof tight and adherence junctions. Transepithelial resistance reached a plateauof 25-35 Ohm. cm2, which was similar to the small intestine. In transport studies2/4/A1 cell line monolayers selectively restricted the permeation of hydrophilicpermeability markers proportional to molecular weight and discriminated moreaccurately between the molecules of intermediate molecular weight compared toCaco-2 cells.

These results indicated that 2/4/A1 cells could be used as a model forhydrophilic drug absorption. INTRODUCTIONThe small intestine plays a crucial role in the absorption of drugs andnutrients. Exogenous substances cross a series of barriers during the processof intestinal absorption: (1) the aqueous boundary / mucus layer, (2) a singlelayer of epithelial cells, and (3) the lamina propria, which contains the bloodand lymph vessels that then transport the absorbed drugs to other parts of thebody (Artursson 1991). The cell monolayer is comprised of two parallel barriers: the cell membrane andthe tight junctions. Most drugs are absorbed by a passive diffusion across thecell membrane by the transcellular route, or across the tight junctions betweenthe cells - the paracellular route. Drug transport can also be carrier mediated,when the drug utilizes transporters located in the cellular membrane.

Transcytosis is another kind of active transport, in which macromolecules can betransported across the intestinal epithelial cell in endocytosed vesicles. The hydrophilic and charged drugs are absorbed after passing through theparacellular route, the water-filled channels between the cells (Artursson1991). Rates and extent of the paracellular transport are, therefore, highlyinfluenced by the structure and size of the tight junctions as well as by thesize of the molecules. Only small and hydrophilic drugs can pass between thecells rapidly and completely; permeation of larger molecules can be limitedproportionally to their size and lipophilicity (Hillgren et al.

1995). Simple assay methods are needed for drug absorption studies. Excised intestinaltissue, isolated cells, membrane vesicles and in vivo models have distinctlimitations, which have been previously discussed in detail (Audus et al. 1990;Artursson 1991; Hillgren et al. 1995). The most suitable method for the study ofdrug intestinal transport appeared to be the use of cultured intestinalepithelial cells.

This model has several advantages over conventional drugabsorption models: (a) it is less time-consuming; (b) it enables rapidevaluation of methods for improving drug absorption; (c) it allows anopportunity to use human rather than animal tissues; (d) it can minimizeexpensive and sometimes controversial animal studies. Human colorectal carcinoma cell line Caco-2 is nowadays the most widely used andthe best explored model for drug intestinal transport (Hidalgo et al. 1989;Artursson 1990; Artursson & Karlsson 1991). This cell line displays spontaneousenterocytic differentiation in culture and forms a polarized monolayer withapical brush borders and well differentiated tight junctions (Hidalgo, 1989). Drug transport studies across the Caco-2 cell monolayers showed a satisfactorycorrelation with other in vitro absorption models, e.g. rat intestinal segments (Artursson et al. 1993) and in vivo drug absorption (Lennern"as et al., 1995),although a considerable variability has been reported, being related toheterogenity, a number of sub populations, and number of passages (Walter &Kissel, 1995).

Caco-2 cells however, form monolayers that resemble colonic rather than smallintestinal epithelial cells. Due to its well-formed tight junctions, Caco-2 cellmonolayers have a transepithelial electrical resistance of 260 Ohm. cm2 which issimilar to the transepithelial electrical resistance of the colon rather than ofthe small intestine (Hillgren et al. 1995). Therefore, there is a need toinvestigate drug intestinal transport in a model which has apparent transportcharacteristics corresponding to the human intestine, and several studies havebeen attempted to characterize a cell line that can be used for this purpose. A novel intestinal epithelial cell line (2/4/A1) is derived from the rat fetalintestinal epithelial cells conditionally immortalized with thermolabile SV40large T antigen, pzipSVtsa58 (Paul et al. 1993).

According to the originalreport, these cells form more leaky monolayers, with paracellular transportcharacteristics similar to that of the human intestine. When cultured at 32^0Cthese cells continually proliferate and display few markers of intestinaldifferentiation. However, after being switched to a non-permissive temperature (39^0C), these cells cease proliferating and exhibit a more markedlydifferentiated phenotype. They form a polarized monolayer covered with a fewmicrovilli; tight junctions are also present (Paul et al.

1993; Hochman,personal communication). The 2/4/A1 cell line has been preliminary investigated in this laboratory. Itappeared that cells grown at 39^0C underwent massive apoptotic cell deathsimultaneously with differentiation, and that those grown at permissivetemperature continued proliferating and form multilayers. However, when grown atan intermediate temperature (37^0C), the cells underwent apoptosis to a lesserextent, but maintained their proliferative capacity sufficiently to form tightand continuous monolayers. The aim of this study was to investigate permeability of paracellular markermolecules across the 2/4/A1 cell line monolayers and to look at thecharacteristics of the cell line.

MATERIALS AND METHODSCell culture2/4/A1 cells were expanded in flasks at 33^0C, in RPMI 1640 medium supplementedwith 2% fetal calf serum, 10 mM Hepes, 2 mM L-glutamine, 200 mg / ml geneticin, 1mg/ml BSA, 2 mg / ml dexamethasone, 20 ng / ml EGF, 50 ng / ml IGF-I, 10 mg / ml insulin,10 mg / ml transferrin and 10 ng / ml selenic acid (ITS premixTM, CollaborativeResearch), with 5-6% CO2 and 95% humidity. The cells were seeded on Transwell polycarbonate filter inserts (O 6.5 mm) coated with ECL extracellular matrix (entactin-collagen IV-laminin; Promega,Madison, Wisconsin, USA), at a density of 100,000 cm2 in a serum-free RPMI 1640medium supplemented with 10 mM Hepes, 2 mM L-glutamine, 200 mg / ml geneticin, 1mg/ml BSA, 2 mg / ml dexamethasone, 20 ng / ml EGF, 10 mg / ml insulin, 10 mg / mltransferrin and 10 ng / ml selenic acid. Transport studiesParacellular markers of different size and molecular weight labelled with 14C orfluorescein were used: mannitol (MW 182), fluorescein (MW 376), lucifer yellow (MW 450), polyethylene-glycol 4000 (MW 4000), and dextran (MW 50,000). Theexperiments were performed at 37^0C in Hank's Balanced Salt Solution pH 7.2 under'sink conditions'. When PEG 4000 was used unlabelled PEG 4000 was also added tothe donor solution to limit possible drug metabolism. The labelled markermolecules, 250 ml, were added to the apical side of the monolayer and after 20,40, 60 and 80 minutes the inserts were moved to new wells and 500 ml samplestaken from the basolateral solution.

Prior to the experiments samples of 50 mlwere taken from the apical solutions for measurements of the initialconcentration (C0). All solutions were preheated to 37^0C, and a heating platewas used when the wells were moved. Transport was measured over time (days 1-10) and compared with the values obtained from Caco-2 monolayers used as standard. The radioactivity of the samples was determined using a standard liquidscintillation technique. The apparent permeability coefficient was calculated asdescribed before (Artursson 1990), using a Microsoft Excel 4.0 software package (Macintosh Power PC computer and Microsoft Office software) and templatesmodified by K. Palm. Electrophysiological measurementsTransepithelial electrical resistance, short circuit current andpotential difference were measured by an in-house computer-based automaticsystem using a single unit Transwell diffusion chamber (Grasj"o & Karlsson,unpublished results).

Development of electrical parameters in 2/4/A1 cells wasstudied over time (days 1-10). The data was processed using a Lab View softwarepackage modified by Grasj"o et al. Cell morphology2/4/A1 cells were routinely monitored under phase-contrast microscope each day. At appropriate time points nuclei were stained with DAPI (4,6-diamidino-2-phenylindolole, Molecular Probes, Leiden, Holland). The percentage of apoptoticnuclei was quantified according to the method of Aharoni et al. (1995).

Cellsgrown on filters at different temperatures were examined by transmissionelectron microscopy (TEM) after fixation in glutaraldehyde and dehydration with1% osmium-tetroxide and 1% uranyl acetate. The presence of actin was assessed bydirect immunofluorescence with rhodamine-conjugated phalloidin. Development oftight junctions were studied by indirect immunofluorescence to ZO-1 protein, andadherence junctions by immunostaining to E-cadherin and vinculin. Immunohistology slides were processed under laser scanning confocal microscope (Leica, Heidelberg, Germany) and images were obtained by Silicon graphicssoftware package. MaterialsIf not otherwise indicated, cell culture media and supplements were purchasedfrom Life Technologies AB, T"aby, Sweden.

Mouse monoclonal antibodies to SV40large T antigen were from Oncogene Science, Uniondale, New York, USA, andrhodamine-conjugated phalloidin from Molecular Probes, Leiden, Holland. Rabbitpolyclonal antibodies to ZO-1 were obtained from Zymed Laboratories Inc., SanFrancisco, USA, and mouse monoclonal antibodies to human E-cadherin fromTransduction Laboratories, Lexington, Kentucky, USA. Mouse monoclonal antibodiesto human and rat vinculin were from Serotec, Oxford, UK. StatisticsNumerical data is expressed as the mean + SD of four to six experiments. One-wayANOVA (corresponding to unpaired one-tailed Students t-test) was used to comparemeans. A 95% probability was considered significant.

RESULTSGrowth of 2/4/A1 cells2/4/A1 cells seeded on ECL coated filter supports showed different growth ratedependent on the temperature. At 33^0C 2/4/A1 cells proliferated rapidly, growingexponentially until day 4 after seeding and forming multilayers consisting ofimmature enterocytes. Growth was significantly reduced at 37^0C and the cellsformed monolayers. There was a decrease in cell number at 39^0C and 10 days afterseeding only 15% of the initial number of cells remained attached to the matrix.

Apoptosis, as calculated per 1000 cells, was present at 33^0C to a negligibleextent, although the proportion of apoptotic cells raised steadily at 39^0C. After 10 days no nuclei without apoptotic morphology were noted at thistemp erature. Number of apoptotic cells did not differ at the remaining twotemperatures (Figure 1). As estimated qualitatively by the immunohistochemical detection of SV40 large Tantigen, the presence of the antigen was a prerequisite for growth in 2/4/A1cells. SV40 large T antigen was present in the entire nuclei at 33^0C, lessprominent at 37^0C, and poorly stained in the nuclei at 39^0C (Figure 2).

Figure 2. Expression of SV40 large T antigen in 2/4/A1 cells seeded at 33^0C, 37^0C and 39^0C. Bar indicates 10 mm. Figure 3. Bar indicates 5 mm.

Figure 4. Vertical sections of 2/4/A1 cell layers seeded to 33^0C (A,C,E) and 37^0C (B,D,F) stained to ZO-1 (A,B), E-cadherin (C,D) and vinculin (E,F). Barindicates 5 mm. Development of tight and adherence junctionsAs estimated by the appropriate antibodies, ZO-1 protein was present in 2/4/A1cells grown at all temperatures.

Its distribution, however was uneven in themultilayers at 33^0C, reaching an intensively stained network at 37^0C. At thenon-permissive temperature the ZO-1 pattern was discontinuous, indicatingloosening of cell-to-cell contact preceding cell death (Figure 3, A-C). Adherence junctions were also present at all temperatures. E-cadherin formed adotted network distributed diffusely in the cytoplasm at both 33 and 39^0C; thepattern was located more closely near the cellular membrane at 37^0C (Figure 3,D-F). Actin filaments were well developed at all three temperatures, showingstress fibers at 33^0C and being distributed evenly at 37^0C in the cell membrane. At 39^0C the actin network indicated broadening of extracellular spaces anddefects in the monolayer (Figure 3, G-I).

ZO-1 protein was located diffusely across the membrane at 33^0C. On the contrary,at 37^0C ZO-1 was located exclusively in the upper pole of the cell-to-celljunctions, indicating that normal tight junctions are formed at 37^0C. At 39^0Cthe ZO-1 formed a discontinuous pattern located at the upper pole of themonolayer, but with clear defects in the staining pattern indicating defects inthe cellular layer. E-cadherin and vinculin were located below the ZO-1 band,forming a dotted network of filaments accumulated around the cell membrane (Figure 4).

Transmission electron microscopy confirmed the development ofmultilayers at 33^0C, monolayers at 37^0C, and defects in the layer due toapoptosis at 39^0C (Figure 5). Tight junctions occurred at all temperatures,although those at 37^0C were longer and appeared tighter than those at 33^0C. Atall temperatures, at least within the time interval studied, the brush bordermembrane surface remained undifferentiated, with few microvilli and withoutvisible brush borders. These data imply that 2/4/A1 cells may be presumably usedas a model of paracellular transport, in which the influence of brush borderenzymes and transcellular transport systems does not interfere with theparacellular pathway. This data indicates that well developed tight and adherence junctions occur when2/4/A1 cells are grown at 37^0C. We therefore decided to evaluate 2/4/A1 cellsgrown at 37^0C as a model for paracellular transport of hydrophilic drugs acrossthe small intestine.

Transepithelial resistanceTEER reached a plateau of 25-35 Ohm. cm2 after four days in culture. Restingpotential and short circuit current were low throughout the time studied, andwere consistent with the cellular morphology (Figure 6). Figure 6. Transepithelial resistance, resting potential and short circuitcurrent of 2/4/A1 cell line monolayers seeded at 37^0C. Experiments wereperformed in Hanks balanced salt solution at 37^0C. N=6.

Figure 5. Transmission electron microscopy of 2/4/A1 cells seeded at (a) 33^0C, (b) 37^0C and (c) 39^0C. Bar indicates 5 mm. Transport studiesTransport experiments were studied 1, 2, 4, 6 and 10 days after seeding. 2/4/A1cell line discriminated well between the paracellular markers of increasingmolecular weight, maintaining such a selective permeability throughout theinvestigated period. Papp values for molecules with molecular weight around 400were about 4.5x10-6 cm /'s and correlated well to the human intestine (Figure 7).

When compared to Caco-2 cell line, 2/4/A1 cells had 40 to 250 times higher Pappvalues and discriminated more accurately between the molecules of intermediatemolecular weight (Figure 8). Transport of mannitol and PEG-4000 in a calcium-free medium showed a two-fold increase in comparison to normal values (Figure 9). Since the adherence junctions can not function properly without calcium, thisdata indicates that the permeation of the markers is restricted mainly to theparacellular pathwayFigure 7. Permeability of hydrophilic marker molecules across 2/4/A1 cell linemonolayers. N=6.

Figure 8. Permeability of hydrophilic marker molecules across 2/4/A1 cell linemonolayers (A) and Caco-2 cell line monolayers (B). Note that Papp values differaprox. 100-fold. N=6.

Figure 9. Permeability of mannitol (MW 182) and PEG-4000 across 2/4/A1 cell linemonolayers in Hanks balanced salt solution with (left) and without calcium (right). N=4. *, p<0.05; **, p<0.01. DISCUSSIONCell cultures have been broadly used in the studies of drug intestinal transport. It has been generally accepted that the data obtained from the cell culturemodels are easy to interpret, since the influence of adjacent structures (submucosa, luminal enzymes, intestinal transit) has been minimized orcompletely abolished.

However, most of the cell culture models used for thestudies of drug intestinal transport are of cancer origin; there is apossibility that their transport characteristics may differ from the normalintestine. Furthermore, in Caco-2 cells the tight junctions are more similar tothe colon than to the small intestine; also, for instance, T84 cells correspondto crypt cells, with a negligible role in the intestinal transport under in vivoconditions, and the HT-29 cell line can be induced to mimic various cell types,but also showed a number of phenotypic variations and appeared to be a poorlyreproducible model for drug intestinal transport (Artursson 1991; Hillgren etal., 1995). In addition, cells which undergo spontaneous differentiation (e.g. Caco-2) require to be kept in culture for several weeks, which obviouslyincreases the costs of maintenance and experiments. Several attempts tocultivate normal intestinal cells appeared to be unsuccessful.

In order to overcome disadvantages of the use of cancer cell lines in studyingintestinal transport, we evaluated a conditionally immortalized rat fetalintestinal epithelial cell line, 2/4/A1, as a possible alternative model. Suchcells can be isolated from transgenic mice (Whitehead et al,1991), or, as wasthe case in our study, from rat fetal intestinal cells conditionallyimmortalized with a thermolabile SV40 large T antigen (Paul et al, 1993). Thesecells proliferate continuously at a permissive temperature (33^0C), but ceaseproliferating and undergo apoptotic cell death at a non-permissive temperature (39^0C). In the preliminary part of our study, we found out that 2/4/A1 cellspreserved certain proliferative capacity when kept at an intermediatetemperature (37^0C), presumably due to an incomplete elimination of SV40 large Tantigen. Growth rate at the intermediate temperature was significantly reducedin comparison to the growth at a permissive temperature. Furthermore, we haveshown that 2/4/A1 cells at 37^0C developed well differentiated tight andadherence junctions, which differed morphologically from the less developedtight junctions observed at 33^0C.

Our data also showed that intestinal transportsystems located at the brush border membrane of the absorptive cell remainedundifferentiated when 2/4/A1 cells were cultured at an intermediate temperature. Permeability characteristics of the model appeared to be more similar to humanileum than to human colon, as was the case with Caco-2 model: TEER values were25-35 Ohm. cm2, similar to human ileum and slightly lower in comparison to thehuman jejunum (Lennern"as et al 1995). In spite of a great variability of TEERvalues in different clones and passage numbers of Caco-2 cells (Walter andKissel, 1995), TEER values in 2/4/A/1 cells, according to our data, remainedsignificantly lower than in any type or clone of the Caco-2 cells studied.2/4/A1 cell line monolayers also selectively restricted the permeation ofhydrophilic permeability markers proportional to molecular weight and size. Permeability of larger markers (PEG-4000, dextran) was negligible.

This effectcan be attributed exclusively to the selective nature of tight junctions in2/4/A1 cells, since the permeability decreased rapidly after a calcium-freemedium was introduced. Morphologic examination of the tight junctions (byimmunostaining to ZO-1 protein, data not shown) clearly supported the evidencethat tight junctions in 2/4/A1 cells were sensitive to calcium depletion andshowed subsequent impairment in their function.2/4/A1 cell line monolayers discriminated well between the molecules of anintermediate molecular weight, i.e. between 180 and 4000. Since most of thedrugs transported via the paracellular route have molecular weight within thisrange, we conclude that 2/4/A1 cells fulfil the functional criteria to be usedas a model for the hydrophilic drugs that utilize the paracellular pathway whenabsorbed in the human small intestine.

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