Organotypical tissue cultures from adult murine colon as an in vitro model of intestinal mucosa

Together with animal experiments, organotypical cell cultures are important models for analyzing cellular interactions of the mucosal epithelium and pathogenic mechanisms in the gastrointestinal tract. Here, we introduce a three-dimensional culture model from the adult mouse colon for cell biological investigations in an in vivo-like environment. These explant cultures were cultured for up to 2 weeks and maintained typical characteristics of the intestinal mucosa, including a high-prismatic epithelium with specific epithelial cell-to-cell connections, a basal lamina and various connective tissue cell types, as analyzed with immunohistological and electron microscopic methods. The function of the epithelium was tested by treating the cultures with dexamethasone, which resulted in a strong upregulation of the serum- and glucocorticoid-inducible kinase 1 similar to that found in vivo. The culture system was investigated in infection experiments with the fungal pathogen Candida albicans. Wildtype but not Δcph1/Δefg1-knockout Candida adhered to, penetrated and infiltrated the epithelial barrier. The results demonstrate the potential usefulness of this intestinal in vitro model for studying epithelial cell-cell interactions, cellular signaling and microbiological infections in a three-dimensional cell arrangement.


Introduction
The intestinal mucosal epithelium acts as a cellular barrier between the gut and the intestinal lumen, controlling the passage of ions, molecules and microorganisms. Both this barrier function and epithelial turnover are strongly dependent on paracrine and exogenous factors, on extracellular matrix compounds and on the epithelial-mesenchymal cell-cell interaction in the intestinal mucosa. A number of primary gastrointestinal diseases such as autoimmune enteropathies and microbiological infections result in damage to this highly organized epithelial barrier (for reviews see Heyman 2005;Rowlands et al. 1999).
In addition to animal experiments, intestinal cell cultures are important models for investigating the function of epithelial cells of the intestinal mucosa under normal and pathological conditions. Because of the complex threedimensional cellular interactions and heterogeneity of mucosal cells in vivo, there are methodical limitations to the use of monolayer in vitro models and cultures with tumor cell lines. For this reason, three-dimensional primary tissue cultures, which are more representative of the in vivo environment, have certain advantages for studies in physiology, pharmacology, developmental biology, tumor biology, or microbiology (Howdle 1983;Trier 1976).
Long-term, three-dimensional intestinal cell and tissue cultures from diVerent species have been widely used in basic research, particularly since the seventh decade of the last century (mouse (Defries and Franks 1977), rat (Altmann and Quaroni 1990;Shamsuddin et al. 1978), man (Autrup 1980;Menard and Arsenault 1987), chicken (Mothes et al. 1985) and hamster (SchiV 1975)). Interestingly, these complex in vitro models are now undergoing a renaissance in combination with new analytical methods and again play an increasing role in intestinal research, including investigations of epithelial homeostasis, cellular diVerentiation and organization (Abud et al. 2005;Quinlan et al. 2006). However, only a few groups have worked with adult intestinal tissue (Autrup et al. 1978a, b;Defries and Franks 1977;Finney et al. 1986;Moorghen et al. 1996;Shamsuddin et al. 1978).
In the present study, we established an organotypical explant culture of adult mouse colon that allowed the investigation of cellular interactions in a three-dimensional cell arrangement. We analyzed this in vitro model using immunohistochemistry, in situ hybridization and electron microscopy.
Two types of experiments were performed to validate the cultivation system. First, the inXuence of dexamethasone on the gene expression of the serum-and glucocorticoid-inducible kinase 1 (SGK1) in the cultures was compared with respective in vivo experiments . The SGK1 plays an important role in the activation of ion channels located in apical membranes of epithelial cells, especially in the lung, kidney and gut. Expression of the kinase is known to be upregulated by glucocorticoids (Firestone et al. 2003) and in inXammatory bowel disease (Waldegger et al. 1999).
Second, we veriWed the quality of the epithelial barrier in the tissue culture by means of in vitro infection with Candida albicans. This fungus is an opportunistic human pathogen usually found on the skin and on the mucosa of the gut, oral cavity and vaginal canal and can lead to deeply seated systemic infections (Darouiche 1998;Defries and Franks 1977).

Animals
The animal experiments were conducted in accordance with local guidelines for the ethical use of animals in research at the University of Tuebingen, which conform to international guidelines. Adult male C57/BL6 mice were sacriWced by carbon dioxide inhalation for histological analysis and tissue culture experiments.

BrdU labeling
BrdU (10 M) was added to the culture medium of proliferating cultures for 24 h. Thereafter, tissue cultures were immediately Wxed. Detection of BrdU-labeled cells was carried out by immunohistochemistry.

In situ hybridization for SGK1
For in situ hybridization, frozen colon samples and tissue cultures were cut into 10 m thick slices with a freezing microtome. The sections were then mounted on Superfrost Plus slides (Langenbrinck, Emmendingen, Germany), dried at 60°C for 30 s and Wxed with 4% phosphate-buVered paraformaldehyde for 20 min. After 3 washes with phosphate-buVered saline (PBS, 0.1 M, pH 7.4), the slides were incubated with TE buVer (100 mM Tris, 50 mM EDTA, pH 8) containing 2 g/ml proteinase K (Sigma) for 10 min at room temperature and rinsed again three times with PBS. In order to reduce nonspeciWc background, the slides were acetylated twice for 5 min with TEA buVer (0.1 M triethanolamine, pH 8.0) containing 0.25% (v/v) acetic anhydride (Sigma). After pre-hybridization with hybridization buVer (50% formamide (Sigma), 10% dextran sulfate, 5 mM EDTA, 20 mM Tris pH 8, 10 mM DTT, 1x Denhardt's solution, 0.05% tRNA, 300 mM NaCl) for 1 h at 63°C, the sections were incubated overnight at 63°C with fresh hybridization buVer containing the denatured DIG-labeled sense or antisense probe (200 ng/ml). DIG-labeled, SGK-1-speciWc, antisense and sense RNA probes were generated by in vitro transcription with a DIG-labeling kit (Roche, Mannheim, Germany). The SGK1 plasmid (pPCRScript, Stratagene, Germany) containing the murine SGK1 sequence (position 20-1373) was kindly provided by Dr. K. Klingel. For in vitro transcription, the plasmid was linearized with NotI (antisense probe) or with SalI (sense probe). To enhance penetration into the tissue sections, the probes were alkali hydrolyzed to an average length of 400 bases. After hybridization, the slides were brieXy rinsed in 2£ SSC at 63°C and then 3 times in 0.1£ SSC for 15 min at 63°C. Detection of DIG-labeled RNA probe was performed according to the protocol of the DIG nucleic acid detection kit (Roche, Basel Switzerland). The tissues were blocked for 30 min with blocking buVer (1% blocking reagent (Roche) in maleic acid buVer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5)) and then incubated with alkaline phosphatase-conjugated antibody solution (anti-DIG antibody (1:2500 Roche) in blocking buVer containing 0.1% Triton ® X-100) for 1 h. Following four washes with maleic acid buVer for 15 min, the slides were equilibrated for 5 min in Tris buVer pH 9.5 (0.1 M Tris, pH 9.5, 0.1 M NaCl, 50 mM MgCl 2 ). Color development was carried out with freshly prepared substrate solution (nitroblue tetrazolium salt and 5-bromo-4-chloro-3-indolyl phosphate (Roche) in Tris buVer pH 9.5). After three washes with PBS, the slides were rinsed in distilled water, dried and coverslipped with Kaiser's gelatine solution.

Electron microscopy
Cultures were Wxed with 2% glutaraldehyde (Sigma) and 5% paraformaldehyde (Sigma) in HEPES buVer (100 mM HEPES, 90 mM saccharose, 10 mM MgCl 2 , 10 mM CaCl 2 , pH 7.0) at 4°C for 2 h. Samples were postWxed with 1% osmium tetroxide in 100 mM phosphate buVer pH 7.2 for 1 h on ice, washed with H 2 O, treated with 1% aqueous uranyl acetate for 1 h at 4°C, dehydrated in a graded series of ethanol, inWltrated with ethanol/resin mixtures and Wnally embedded in Epon (using glycidether 100 from Roth, Karlsruhe, Germany). Ultrathin sections were stained with uranyl acetate and lead citrate (Venable and Coggeshall 1965) and viewed under a Philips CM10 electron microscope at 60 kV using a 30 m objective aperture.
Pharmacological treatment SGK1 expression was analyzed by treating adult mice and tissue cultures with dexamethasone. Animals were injected with dexamethasone phosphate disodium salt (dissolved in 0.9% saline, Sigma) at a dose of 10 g/g body weight for 8 h. Mice injected only with 0.9% saline served as controls.
Cultures were treated with 100 nM dexamethasone for 8 h after 14 days of cultivation in vitro.
Infection experiments were performed in the absence of antibiotics. Thousand cells in 1 l YPD medium with 0.1 mM uridine were pipetted onto each organotypical tissue culture that was cultured for 10 days as described above. After 8 or 24 h, the cultures were Wxed with 4% formaldehyde (Sigma) in PBS and embedded in paraYn. For Candida staining, the Grocott staining protocol was carried out on 5 m thick paraYn sections (Grocott 1955).

Three-dimensional tissue culture
Three-dimensional tissue samples of adult mouse colon were cultured on membrane inserts for up to 4 weeks ( Fig. 1a, d). After 3 days in vitro, the main part of the epithelium had degenerated and was subsequently completely renewed. After this epithelial reorganization, a high-prismatic epithelium covered the surface of the tissue culture and formed crypt-like structures (Fig. 1a-c). Within the Wrst 6 days of cultivation, half the explants exhibited good tissue preservation as shown by an epithelium-speciWc surface structure of the tissue culture. At this time point, a number of proliferating epithelial cells could be identiWed by BrdU proliferation assay (Fig. 2c). Between the third and fourth week in culture, the quality of epithelial cell formation decreased due to the reduced regeneration capacity of the epithelium and a loss of epithelial cells. At this time, no BrdU-positive cells could be detected in the epithelium (data not shown). For this reason, we only used explants cultured between 6 and 14 days for further analysis. Immunohistochemical characterization of tissue cultures were performed in comparison to native mouse gut (Fig. 2). Epithelial cells were immunostained for cytokeratin (Fig. 2b). Alkaline phospha-tase activity at the apical side of epithelial cells was detected by histochemistry (Fig. 1e). DiVerentiated goblet cells, in particular, could be shown by Alcian blue staining and trans-  (Figs. 1f, 3d, e). The Lamininimmunopositive basal membrane indicated the border between epithelium and connective tissue (Fig. 2d). Transmission electron microscopic views identiWed microvilli structures at the apical surface of high-prismatic epithelial cells (Fig. 3a, b, f). Typical tight junctions and desmosomes of epithelial junction complex and a well-organized basal lamina could also be detected (Fig. 3b, c, f).
The connective tissue showed characteristic collagen Wbers, Wbroblasts (Fig. 3i, j) and smooth muscle cells (Fig. 3k). Immunohistochemistry for smooth muscle actin demonstrated a deWnite orientation of muscle cells mainly in the lower part of the tissue culture ( Fig. 2g-i). In addition, ß-tubulin III-positive enteric neurons (Fig. 2m-o) and GFAP-expressing glial cells (Fig. 2j-l) were visualized. Interestingly, some mast cell-like cells and lymphatic-like vessel structures could be detected by transmission electron microscopy ( Fig. 3g-i).
In addition to the small crypt-like structures, large cystlike formations could also be observed. These cysts with an introverted epithelium developed inside the three-dimensional culture (Fig. 3l-n). In previous studies, we demonstrated that the treatment of mice with dexamethasone strongly increased the mRNA expression of SGK1 in the mucosal epithelium of the small intestine (Grahammer et al. 2006).
We evaluated the proposed in vitro model by testing whether the in vivo eVect of dexamethasone was reproduced in our intestinal cultures. To do this, we analyzed the SGK1 mRNA expression by in situ hybridization on cryostat sections from adult murine colon and intestinal tissue cultures. Tissue cultures were incubated with 100 nM dexamethasone for 8 h. Animals were injected with dexamethasone phosphate disodium salt at a dose of 10 g/g body weight for 8 h. In the epithelium of both the adult mouse colon in vivo (Fig. 4b, e) and colonic tissue cultures in vitro (Fig. 4g, h), the application of dexamethasone strongly enhanced SGK1 mRNA expression compared to the untreated group (Fig. 4a, d, f). The sense control did not show any signals (Fig. 4c).

Infection with Candida albicans
Candida albicans occurs naturally in the gastrointestinal tract and can cause superWcial infections. We used the organotypical tissue culture for the infection assay with two Candida strains. After 10 days in vitro, colonic tissue cultures were infected with the wildtype (Candida 14) or the knockout strain (Candida 34, cph1/ efg1) (Dieterich et al. 2002). The infection of the tissue cultures with wildtype Candida cells resulted in adhesion of the Candida cells after 8 h (Fig. 5e) with subsequent penetration and inWltration after 24 h of incubation (Fig. 5a-d). Candida strain Can 34 was not capable of adhering to the culture surface (Fig. 5i) or penetrating the epithelial barrier ( Fig. 5f-h) of any of the analyzed explant cultures. These experiments indicate that this in vitro model could be of use

Discussion
In the present study, we introduced an organotypical explant culture model from the adult mouse colon for the investigation of cell-cell interactions in a three-dimensional cell arrangement.
The tissue of our in vitro model was cultivated on membrane inserts at the interface between air and culture medium to ensure adequate in vitro nutrition and oxygen supply (Metzger et al. 2007). After cellular reorganization during the Wrst week, the cultures were covered by a highprismatic epithelium similar to that found in vivo. After 6 days in vitro, these epithelial cells also possessed typical junctional complex and microvilli at their apical surface. At this point, BrdU-labeling experiments demonstrated prolif-erating epithelial cells at the surface of culture model. DiVerentiated goblet cells were identiWed by electron microscopy and Alcian blue staining. All the characteristic compounds of the epithelial barrier, such as epithelial cells, laminin-positive basal lamina and Wbrocyte-like cells underneath the basal lamina, could be detected by immunohistochemistry and electron microscopy.
However, the regeneration capacity of epithelium declined within long-term culturing. This indicates that epithelial progenitor and stem cells diVerentiated and the stem cell compartment could not be maintained in vitro. For this reason, the cultures should be used between the Wrst and second week in vitro to ensure a reliable quality of this tissue culture model.
Nevertheless, the reorganization processes during culturing make this model useful for investigating in detail the time-dependent cellular mechanisms and factors involved in epithelial regeneration and degeneration (e.g. growth factors, hormones, extracellular matrix compounds, cell-cell interactions with non-epithelial cells for a review see, de Santa Barbara et al. 2003;Marshman et al. 2002;Yen and Wright 2006). Additionally, the proposed culture model is suitable for implantation experiments to analyze the integration and diVerentiation capacity of grafted epithelial stem and tumor cells. Furthermore, re-implantation of these cultures into animal models could reveal whether epithelial stem cells of grafted tissue could be re-organized in stem cell niches in a three-dimensional environment in vivo.
In the past, several groups have used organotypical cultures of various gut regions and cultivated them either as free-Xoating cultures (Autrup 1980;DeRitis et al. 1975) or on diVerent culture substrata in vitro (Altmann and Quaroni 1990;Quinlan et al. 2006;SchiV 1975). Alternatively, the chick chorioallantoic membrane (CAM) has been used to provide an in vivo-like environment (Burns 2005;Pomeranz and Gershon 1990). However, these techniques are basically restricted to embryonic tissues and are less suitable for experimental manipulations and real-time imaging.
A cultivation principle similar to that described for our model was published by Defries and Franks (Defries and Franks 1977). To our knowledge, this is the only group that has already analyzed intestinal explant cultures from adult mice in detail. However, in contrast to their culture method we used a diVerent type of scaVold membrane and a diVerent culture medium which was supplemented with adult horse serum instead of fetal calf serum.
Defries and Franks investigated the eVect of donor age and carcinogen treatment on epithelial mitotic activity in their culture model.
Our aim was to establish a culture system of colonic mucosa for investigating the biological function of intestinal epithelium in an in vivo-like environment under normal and pathological conditions. First, we evaluated the cell culture system by testing the known in vivo eVect of dexamethasone on the gene expression of SGK1 in intestinal epithelial cells. Dexamethasone is a potent synthetic member of the glucocorticoid class of steroid hormones and acts as an anti-inXammatory and immunosuppressant drug. Glucocorticoids are involved in the regulation a variety of cellular processes in the mucosal intestinal epithelium such as cell proliferation und diVerentiation, activity of digestive enzymes, epithelial transport or intestinal Wrst-pass metabolism (Binder et al. 1989;Quaroni et al. 1999).
In an earlier report we used in situ hybridization and real-time PCR to demonstrate that dexamethasone also stimulates SGK1 gene expression in the cells of the small intestine in vivo (Grahammer et al. 2006). SGK1 plays an important role in activating certain potassium, sodium and chloride channels and seems to be involved in the regulation of cell survival (for review see Lang and Cohen 2001). In the intestine of normal mice the SGK1 mRNA expres-sion of the epithelium was mainly located in diVerentiated epithelial cells at the surface of the gut wall. As in the small intestine, the SGK1 expression of colonic epithelial cells was strongly enhanced in mice treated with dexamethasone. And like in these in vivo experiments, the dexamethasone eVect could be reproduced in epithelial cells of explant cultures.
To evaluate the suitability of our in vitro system for use as an infection model we performed infection experiments with wildtype and mutant strains of Candida albicans, a pathogen leading to systemic infections in immunocompromised patients (Cole et al. 1996;Darouiche 1998). The wild-type Candida (Candida 14) adhered to the epithelial surface and inWltrated the tissue. In contrast, the mutant strain (Candida 34) lacking the transcription factors EFG1 and CPH1 (Dieterich et al. 2002) was neither able to adhere to the epithelium, nor to penetrate the epithelial barrier in our in vitro model. The results are comparable to those from studies published by Dieterich et al. 2002. In these experiments, the same Candida strains were investigated in a three-dimensional intestinal in vitro model based on the colonic human cell line Caco-2 co-cultured with human intestinal Wbroblasts embedded in collagen I matrix. Threedimensional culture techniques with immortalized epithelial cell lines have also been used in infection experiments with Salmonella typhimurium (Honer zu et al. 2006;Nickerson et al. 2001). The advantage of using immortalized cells is a high level of standardization and reproducibility. However, because of the speciWc physiology and the more complex cell organization in primary tissues, explant cultures may bear a closer resemblance to the in vivo situation.
We conclude that the proposed three-dimensional cultures from adult murine colon may provide a suitable in vitro model of the gut.