Simple and efficient method for isolation and cultivation of endoscopically obtained human colonocytes

Jakob B. Seidelin,1 Thomas Horn,2 and Ole H. Nielsen1

Departments of 1Medical Gastroenterology C and 2Pathology, Herlev Hospital, University of Copenhagen, DK-2730 Herlev, Denmark

Submitted 20 December 2002 ; accepted in final form 26 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Few comparative and validated reports exist on the isolation and growth of colonoscopically obtained colonic epithelium. The aim of this study was to develop and validate a simple method for the cultivation of colonoscopically obtained colonocytes. Forty patients, who underwent routine colonoscopy and where the diagnosis of irritable bowel syndrome was later reached, were included. Seven colon biopsies were taken and incubated at varying time periods of 10-120 min and temperatures of 4-37°C in a chelating buffer. The epithelium was then harvested and cultivated under three different conditions: 1) on a collagen coating, 2) embedded in a collagen gel, or 3) embedded in a gel put on a porous well insert. The effect of conditioned medium (CM), insulin, transferrin, selenium, and the oxygen content was assessed. Viability was tested by the metabolic dimethylthiazol-diphenyl-tetrazolium bromide assay, by flowcytometry, by phase contrast microscopy, and by transmission electron microscopy. Incubation at 21°C for 75 min gave an optimal yield of 3 x 106 (2.0-3.8 x 106) viable epithelial cells in intact crypts per seven biopsies. Embedding of crypts in a collagen gel put on a porous membrane was superior to the other methods applied [P < 0.003; median viability 71% (62-100%) compared with preculture values] after 24 h, which was a 160% increase in viability compared with coat-cultivated cells. CM had similar viability supporting effects to FCS. Other supplements had no effects. A simple method is presented, which makes cultivation of colonocytes obtained at endoscopy possible for up to 72 h.

apoptosis; colonocytes; primary culture; viability


EPITHELIAL CELLS OF THE COLON play a key role in the patophysiology of a wide variety of large-bowel disorders, ranging from inflammatory disorders to colon cancer. Thus it seems pivotal for large-bowel research to investigate pathophysiological mechanisms at the epithelial level. This has, however, been hampered by the low success of cultivating human colonocytes. Previous methods have therefore usually used surgical specimens to obtain sufficient quantities of cells (1, 24). The need for surgical specimens evidently narrows the applicability, because diseases and/or disease stages that are unlikely to result in colectomy will not be investigable.

Cultivation of whole biopsies has been used, but it carries a high risk of infections and various cell types are seen (22). Cultivation of isolated epithelial cells from biopsies has also been reported (7, 8, 16). Either enzymatic liberation of the epithelium or a chelation-induced epithelial detachment was applied in these studies. All studies employed colonocyte growth on a collagen I-plated surface and had a significant decrease in viability within the first 24 h of cultivating as determined by either metabolic or apoptosis assays (8).

It is well established that detachment from the basal membrane is the major cause of the cell death in cultured colonocytes (11, 20). Because colonocytes in vivo grow in three-dimensional structures consisting of sheets of surface epithelium and crypts, one would expect that a three-dimensional collagenous structure around them would also be preferable in vitro. Colonocytes obtained from surgical specimens have previously been grown in collagen gels (24). The preservation of the epithelial lining structure would additionally permit examinations of specific crypt cell populations in vitro, e.g., by using stained probes, because crypt position depicts the differentiation of cells (17).

The aim of this study was therefore to develop an improved method for the isolation, maintenance, and cultivation of colon epithelial crypts by the use of colonoscopically obtained biopsies. Colonic crypts are highly organized along the crypt axis in a hierarchical manner that might have important implications on epithelial cell function and differentiation (17). It was therefore an important goal to develop an isolation and culture method that gave a pure fraction of epithelial cells, which remained organized in the same three-dimentional structure as seen in vivo, i.e., in crypts.


    METHODS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Patients. Forty patients with the diagnosis of irritable bowel syndrome (14) were included. All patients had a routine colonoscopy or sigmoidoscopy performed as a part of their planned examination program. Patients under the age of 18 or pregnant patients were excluded. No patients received anti-inflammatory treatments at the time of endoscopy. Patients undergoing colonoscopy received 5-10 mg diazepam and 50-100 mg fentanyl intravenously, whereas no medication was administered in those who underwent sigmoidoscopy.

Ethics. The Scientific Ethics Committee of Copenhagen County approved the study. All patients gave their informed consent before participation, and the project fulfilled the Helsinki II declaration with later amendments.

Colonoscopies. Colonoscopies were made using videoendoscopy technology (Olympus Optical, CF/GIF-system, Tokyo, Japan). Biopsies were taken with disposable Precisor XL forceps (Bard, Billerica, MA).

Isolation of human colonic epithelial cells. All cell cultivation materials were obtained from Invitrogen (Carlsbad, CA) unless otherwise stated. Five to seven biopsies were obtained from colon transversum or descendens in each patient. The biopsies were discharged from the forceps into the transport media that consisted of sterile filtered ice-chilled PBS without Mg2+ or Ca2+ containing 50 IU/ml penicillin, 50 µg/ml streptomycin, and 0.5 mg/ml gentamycin and held under these conditions until further processing (5-20 min). All subsequent treatment was made in media containing the abovementioned antibiotics. The biopsies were washed three times in ice-chilled transport media. They were then chelated in 1 mM EDTA and 1 mM EGTA in PBS without Mg2+ or Ca2+ to liberate the epithelial cells from the lamina propria. Chelation was done under different conditions by varying the incubation duration from 10 to 120 min and the incubation temperatures at 4, 21, and 37°C. The chelating buffer was replaced by ice-chilled PBS, and crypts and surface epithelium were liberated from the biopsies by vigorous shaking of the tube by hand. The PBS-containing crypts and surface epithelium was transferred to a new tube and centrifuged at 40 g for 2 min. This gentle spinning gave a loose pellet consisting of sheets of surface epithelium and crypts devoid of single cells.

Assessment of the efficiency of the isolation procedure. The liberated crypts were evaluated by phase contrast microscopy (Leica Labovert FS, Leica AG, Solms, Germany; Kappa CF 15 Camera, Kappa Opto-electronics, Gleichen, Germany). Immediately after isolation and before spinning, photomicrographs were taken. The efficiency of the isolating procedure was evaluated semiquantitatively by the quantity of crypts liberated and by scoring the crypt architecture and amount of apoptotic morphology (cell membrane blebbing and cell shrinkage, see Fig. 1 and Table 1 for details). The optimal conditions were applied in the subsequent experiments.



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Fig. 1. From left to right: a well-preserved crypt without evidence of apoptosis; next: a crypt with several apoptotic cells throughout the crypt axis, although primarily in the crypt neck zone; next: typical high yield of cells incubated at optimal conditions. Original magnification, x40.

 

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Table 1. Chelation time and temperature

 

Cultivation of human colonic epithelial cells. The crypts from a single patient were subsequently stratified to the following culture protocols: 1) either cultivation in an atmosphere of 5% CO2 or in an atmosphere of 5% CO2 and 70% O2 both at a relative humidity of 90%; 2) either cultivation on a collagen coating [bovine dermal collagen (Cellon S.A., Strassen, Luxembourg)], in a collagen gel (Collagen R, Serva Electrophoresis, Heidelberg, Germany) on the bottom of the culture well, or in a collagen gel in a culture well insert with a 0.02-µm pore size Anopore membrane (NUNC, Naperville, IL); 3) either with supplementation of 15% FCS (Sigma-Aldrich, St. Louis, MO), but without further additives, with supplementation of 15% FCS, insulin (1 µg/ml), transferrin (0.55 µg/ml), sodium selenite (0.67 ng/ml), and EGF (5 ng/ml; all delivered from Invitrogen), or with supplementation of 15% conditioned medium (CM). CM was made according to the method developed by Panja (15): 10 biopsies were grown at a concentration of 250 µg/ml tissue in DMEM growth medium overnight to generate a supernatant enriched with secreted growth factors and cytokines. In contrast to previous studies (15), the conditioned medium was subsequently used in heterologous and not autologous colonocytes because of the limited amount of tissue available from each individual. All cells were grown on standard 24-well plates (Techno Plastic Products, Trasadingen, Switzerland) in a final volume of 400 µl of growth medium (DMEM). The gel was made with 3/4 DMEM and 1/4 collagen R (final collagen concentration, 1.0 mg/ml). The osmolarity was adjusted by adding Hanks' balanced salt solution, and pH was adjusted to 7.4 with 1 M NaOH, and the final FCS/CM concentration was set to 15%. The mixture was kept at 4°C to avoid premature polymerization. After the addition of the colonocytes, the gel (100 µl) was allowed to polymerize by incubation for 5 min at 37°C, and the growth medium was finally added to adjust the total final volume to 400 µl.

Assessment of cell numbers. Cell numbers were determined by measurement of the DNA content after Hoechst 33258 staining. The cells were lysed and sonicated to liberate the DNA and incubated with the DNA stain, and the fluorescence thereafter was measured (DyNA Quant 200 Fluorometer, Amersham Biosciences Europe, Freiburg, Germany). A standard of known DNA concentration was included to calculate the DNA concentration (calf thymus DNA, Amersham Biosciences Europe).

Viability assessment by the dimethylthiazol-diphenyl-tetrazolium bromide test. Viability was assessed by the reduction of dimethylthiazol-diphenyl-tetrazolium bromide (MTT) by mitochondrial succinate dehydrogenase to the colored formazan during4hof incubation, as previously described in detail (9). Viability was assessed after 24 h of culture and in one experiment at 0, 24, 48, 72, and 96 h of culture.

Viability assessment by flowcytometry. Apoptotic nuclei (sub-G0) have less DNA-content than 2n diploid (G0 or G1) or 4n diploid nuclei (G2) of normal cells, and thus stain less intensively with DNA dyes. Viability was detected by measuring the fraction of viable cells in the G0/1 and G2 peak by flowcytometry, as earlier described in detail (13, 21). In brief, the gel was digested by collagenase (200 units/ml; Sigma-Aldrich) to solubilize colon epithelial cells. The cells were then spun down (500 g; 5 min) and incubated at 4°C for 3 h in a nuclear extraction buffer containing the fluorescent DNA stain propidium iodide (0.1% sodium citrate, 0.1% Triton X-100, 50 µg/ml propidium iodide). The propidium iodide fluorescence of the individual nuclei was determined by flowcytometry (FACScan, BD Biosciences, San Jose, CA). Viability was assessed at 0, 24, and 48 h of culture.

Viability assessment by electromicroscopy and assessment of cell types. The colonocyte viability and relative numbers of different cells were assessed by transmission electron microscopy (EM). Cells were fixed in gel (2.5% glutaraldehyde) overnight, postfixed in 2% osmium tetroxide for 1 h, and embedded in Epon resin. For orientation semithin 1-µm sections were stained with toluidine blue. Subsequently ultrathin sections were mounted on 150 mesh copper grids and stained with uranyl acetate-lead citrate before examination under a Philips transmission EM EM210 (Amsterdam, the Netherlands). Photomicrographs were taken at x2,000-3,200. More than 50 cells were counted per single experiment and classified by type (e.g., absorptive, goblet, and stem cells).

Determination of purity of isolated cells by immunohistochemistry. The isolated cells were embedded in a gel as described above, and then they were fixed in 10% buffered formalin, embedded in paraffin in a standard manner, and stored until analysis. This was done after 0, 24, and 48 h of culture.

Immunostaining was performed with a panel of cell type-specific markers. These were epithelial markers: anti-cytokeratin 18 (CK18; clone DC10, DakoCytomation; Glostrup, Denmark), anti-CK20 (clone KS20.8; DakoCytomation), and the pan-specific anti-CK antibody (clone AE1/AE3; Immunotech, Paris, France); mesenchymal cell marker: anti-vimentin antibodies (clone Vim3B4; DakoCytomation); and endothelial cell marker: anti-CD34 antibodies (clone My10; BD Biosciences). The antibodies were used in the following concentrations: CK18: 1:400; CK20: 1:400; CK-PAN: 1:300; Vimentin: 1:400; CD34: 1:100. A two-step method was employed using the EnVision+ visualization system (DakoCytomation). In brief, 5-µm-thick sections were cut, deparaffinized, and hydrated in graded ethanol-water washes and pretreated with microwaves and 3% H2O2. The primary antibodies were applied overnight at 4°C following washes. The EnVision+ system was then applied for 30 min. After washes, the substrate diaminobenzidine was applied to yield a colored product. The slides were counterstained with Mayer's hematoxylin.

Statistics. Nonparametric statistics were applied. In comparing groups from the individual experiments, the Wilcoxon's signed rank test was used. The Spearman correlation coefficient was calculated for the DNA measurement experiments. Values were shown with 95% confidence intervals or given as medians and ranges, and a significance level of 0.05 (2{alpha}) was chosen.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
Isolation procedure. The isolated crypts were evaluated on the basis of relative numbers of crypts liberated and the quality of these crypts in terms of crypt structure preservation and absence of apoptotic cells. In general, the shorter and the colder the duration of the chelation, the more intact were the crypts. The yield, however, had an inverse relationship, i.e., the longer and warmer incubation conditions, the more crypts were liberated (Table 1). No change of crypt architecture was, however, revealed when incubation was performed at 21°C for <75 min, whereas the yield dramatically increased at incubation times >60 min. On the other hand, even a 10-min incubation at 37°C gave crypts with prominent apoptotic features (Fig. 1). Incubation at 4°C for 2 h gave very low yields. As a result, an incubation time of 75 min at 21°C was found to be optimal.

Phase-contrast imaging of the cells released after 75 min at 21°C revealed almost only epithelial cells arranged in crypts and only few single cells. Immunohistochemistry of the released cells showed almost all cells (>99%) to be positive for CK18 and CK20 (Fig. 2). No vimentin or CD34-positive cells were found. EM revealed crypts to contain all cell types normally found in the colon crypts: goblet cells were predominant (45% of all cells) in the middle part of the crypt, whereas secretory cells dominated the mouth and plateau zones of the epithelium. Less-differentiated cells were encountered at the crypt base. No mesenchymal-like cells were found in the EM sections.



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Fig. 2. Immunohistochemistry of isolated epithelial cells at 0 h. Anti-cytokeratin-18 (CK-18) staining. Epithelial cells are CK-18 positive. No cells are unstained. Disruption of crypt architecture is due to preparation techniques.

 

Determination of cell content. There was a correlation between crypt numbers and the DNA content [see Fig. 3; correlation coefficient 0.85 (0.68-0.93); P < 0.0001]. No interpatient variability was found, and a simple crypt counting was, in this setting, an estimate of the relative cell number. From the DNA measurements, it could be calculated that the yield was 3 x 106 (2.0-3.8 x 106) epithelial cells per five biopsies at the chosen chelation conditions.



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Fig. 3. DNA content vs. crypt counts. A correlation is demonstrated [correlation coefficient 0.85 (0.68-0.93); P < 0.0001]. The figure includes data from 6 patients and 24 wells. 95% Confidence intervals are shown as well as the linear regression line. 10XF, magnification.

 

Culture methods. Embedding of the epithelial cells in a three-dimensional collagen structure gave a considerably higher viability of the cells than growing them on a collagen coating (40% better viability; P < 0.02). The viability was even higher in cells embedded in a collagen gel and grown on a porous membrane (60% higher viability; P < 0.01; Fig. 4). The median viability of the colonocytes at 24 h of culture was 71% [62-100%; median (range)] of the preculture value. Cells grown in CM had viability comparable with those grown in FCS-supplemented medium, and no statistical significant difference was found between those two. The median viability was 78% (77-100%) after 24 h (Fig. 5). Neither growth factor supplementation nor increased oxygenation gave a viability benefit (Fig. 6). This was equally found on cells cultured on coated wells, in gels, and in gel plus membrane insert (data not shown). Viability was stable within the first 72 h and decreased only substantially from 72 to 96 h. Thus viability was 58% (46-100%) at 48 h, 55% (40-60%) at 72 h, and 25% (22-36%) at 96 h (Fig. 5). Flowcytometry of propidium iodide-stained nuclei confirmed the results found by MTT (Fig. 7).



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Fig. 4. Viability assessed by the dimethylthiazol-diphenyl-tetrazolium bromide (MTT) assay. Culture on a porous membrane and in a 3-dimensional collagen type I gel confers increased viability after 24 h (n = 10). OD, optical density.

 


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Fig. 5. Cells grown on membranes had a significantly better viability within the first 48 h compared with cells grown on a collagen plating (P < 0.02; n = 6). Conditioned medium (CM) conferred viability at least as good as fetal calf serum (P > 0.05). Medians and ranges are shown.

 


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Fig. 6. Viability assessed by the MTT assay. No beneficial effect of insulin, transferrin, selenium (ITS), and EGF was observed. No beneficial effect of high O2 partial pressure was observed (n = 12).

 


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Fig. 7. Flowcytometry of propidium iodide-stained nuclei. No apparent decrease in viability was seen within the first 24 h (B) of culture, whereas viable cells decreased to 43% of that at 0 h (A) at 48 h (C). These values are comparable with the results from the MTT assays. Representative experiment of 3 experiments.

 

Immunohistochemistry at 24 and 48 h showed that >99% of cells were CK18 or CK20 positive. No cells of mesenchymal origin were found. EM confirmed no major change in the relative number of different cell types.

Viability confirmed by transmission EM. No significant morphological changes were found in colonocytes cultured for 24 h, compared with those freshly isolated. Cultured cells were all epithelial and revealed normal morphology (Fig. 8). Cells were detached from the basal lamina, and cultures did not contain mesenchymal cells. However, as expected, random sections revealed more apoptotic cells in the cultivated specimens than in the noncultivated.



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Fig. 8. Photomicrographs of transmission electron microscopy pictures of cells grown for 24 h. Magnification x3,400. Cells are without signs of apoptosis and show colonocytes with intact zonula occludentes, tight junctions, and microvilli.

 


    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study provides a new efficient method for the short-term monoculture of colonoscopically obtained colonic epithelial cells organized in preserved crypt structures. The method is optimized in terms of epithelial cell-isolation technique, culture technique, and culture medium additives. The method is compared with previously published methods.

The aim of the isolation procedure is to obtain a pure fraction of viable colonocytes. Any potential apoptosis-inducing condition should therefore be minimized. Isolation has been performed by enzymatic digestion of the connective tissue (4) and by chelation, which disrupts Ca2+-dependent cell-matrix interactions (24). Chelation has previously been shown to be superior to enzymatic digestion with regards to the purity of epithelial cells and has additionally been shown to cause less apoptosis (10). It is shown in the present study that chelation time and temperature must be optimized to yield crypts without apoptosis features. The optimization is restrained by the inverse relationship between these two variables as shown in Table 1. However, the combination of a long incubation time (i.e., 75 min) and a low incubating temperature (21°C) gives both a high yield and a high viability. As detachment from the basement membrane induces apoptosis (20), the time and temperature should be kept low when cells are in suspension. Even when the cells are embedded in the unpolymerized collagen gel, mechanical stresses such as vigorous mixing should be avoided, because crypt tears will result in decreased viability due to apoptosis (data not shown). The gentle handling of the crypts can, on the other hand, result in some variation in the cell number of each well. It is here shown that simply counting crypts gives a good estimate of cell numbers compared with DNA content measurements. The cells obtained by this method are a highly pure fraction of colonic epithelial cells with very low contamination of mesenchymal cells.

From the present study, it is clear that the gel embedding is substantially superior to collagen coating alone. Culture with crypts embedded in a collagen gel and on porous membrane well insert is further advantageous to culture on the bottom of the well. A viability of 46-100% within the first 48 h was achieved with this new method, which is substantially higher than other culture methods (16). This may be due to a three-dimensional epithelium-matrix interaction and an improved exchange of metabolites on all sides of the crypts, because crypts will fall to the bottom of the well before the gel has polymerized. The collagens used in this study contain mainly collagen type I of nonhuman origin. Other more basement membranelike substances, e.g., Matrigel, which contains collagen type IV, have previously been tried without beneficial effects on the cultivation process (19). Accordingly, the collagen I/{beta}1-integrin interactions seems to provide the required survival signaling in the colonocyte and inhibit detachment-induced apoptosis; such an effect has not been shown for collagen type IV (20). Medium was not changed in this experiment during the cultivation period. Medium shifting may, however, improve viability during long-term cultivation, i.e., >96 h.

Heterologous-conditioned medium is here shown to be at least as effective as FCS in supporting viability of isolated colonic cells grown in three-dimensional gels. The median viability was higher in the CM-treated cells, but this difference failed to reach statistical significance. Cells grown without either FCS or CM have a poor viability. These ill-defined additions seem to contain growth factor(s) or other mediators that enhance viability. It has recently been shown that Winglass/INT-1 signaling may be pivotal for stem cell survival in the intestinal epithelium (2, 23). The source might be myoepithelial cells close to the epithelial cells, thus emphasizing that enterocyte monoculture requires both extracellular matrix proteins and compartment-specific mediators. The effect of CM was, however, not as profound as that found by Panja (15). The intestinal epithelial cells used in that study were, however, very different from those isolated in this study, mainly by being passaged several times after isolation. They may most likely represent a stem cell-like cell type with a high dividing potential and could thus be more responsive to CM-derived mitogens. The crypt cells used in this study are mainly differentiated cells without a dividing potential, and stem cells may account for <5%, which could explain the weaker effect of CM (17). Addition of EGF, insulin, transferrin, or sodium selenite did not enhance viability in this study. Only a few comparative studies on culture media for colonocytes have been performed (5, 6, 18, 19, 25). Various growth factors have been used; however, the effects have only been poorly investigated. The supplements include transferrin and EGF (5), insulin (4, 5, 19), and/or hydrocortizone (4, 19, 24). EGF has been shown to be beneficial in three studies (5, 6, 19), to impair proliferation in one study (5), and to have no effects in other studies (18, 25). Conflicting results have also been found with insulin supplementation (5, 18, 19). Other growth factors have been shown to have no effects: amphiregulin, {beta}-regulin, heregulin, pentagastrin, bombesin, hepatocyte growth factor, or insulin-like growth factors 1-3 (19, 25). Lastly, transforming growth factor-{beta} has been shown to be beneficial (6). In the present study, EGF, insulin, transferrin were applied at concentrations similar to the ones mentioned in the above studies.

It is essential to recognize that isolation procedures have profound effects on the viability in subsequent cultures and that variations of these procedures may account for some of the inconsistencies mentioned above, which refer to data from studies that have employed widely differing isolating procedures. Thus many of the growth factors mentioned above are known to inhibit or retard detachment-induced apoptosis (3). This could explain why some of the growth factors enhance viability in some studies, because they could be retarding detachment-induced apoptosis. In addition, some isolation procedures let a substantial fraction of nonepithelial cells pass on with the epithelial cells, most likely fibroblasts and myofibroblasts, which are known to be supported by some of the mentioned growth factors (12).

In conclusion, a new method is provided that permits growth of highly purified and viable human colonocytes. This method is superior to others in terms of cellular viability (16). Furthermore, the method allows for the cultivation of freshly isolated morphologically well-preserved colonocytes in a three-dimensional structure that resembles in vivo conditions. The method has made it possible to develop a method by which apoptosis can be observed in living colonocytes over time based on loading of the cells with fluorescent caspase-3 substrates. The preserved crypt structure makes categorizing of dying cells possible, i.e., whether apoptosis occurs in the stem cell region or in differentiated cells. Furthermore, ultrastructural expression analyses with immunogold labeling have been carried out. The method is stable and simple, because it is solely based on routine colonic biopsy samples obtained by endoscopy.


    DISCLOSURES
 TOP
 ABSTRACT
 METHODS
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 DISCUSSION
 DISCLOSURES
 REFERENCES
 
This study was supported by grants from Else and Mogens Wedell-Wedellsborg's Foundation and E. C. and H. I. Hertz's Foundation.


    ACKNOWLEDGMENTS
 
We thank the laboratory technicians B. Deibjerg Kristensen, A. Petersen, and S. Sørensen for skillful assistance. We are grateful to the endoscopy unit staff for help in obtaining biopsy material.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J. B. Seidelin, Dept. of Gastroenterology C, Herlev Hospital, Univ. of Copenhagen, DK-2730 Herlev, Denmark (E-mail: jakse{at}herlevhosp.kbhamt.dk).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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