Culturing explanted colon crypts highly improves viability of primary non-transformed human colon epithelial cells

Toxicology in Vitro 26 (2012) 133–141

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Culturing explanted colon crypts highly improves viability of primary non-transformed human colon epithelial cells A. Wilhelm a,c,⇑, F. Jahns a,c, S. Böcker a, H. Mothes b, K.O. Greulich c, M. Glei a a

Department of Nutritional Toxicology, Institute of Nutrition, Friedrich-Schiller-University Jena, Dornburger Strasse 24, 07743 Jena, Germany Department of General, Visceral and Vascular Surgery, University Hospital, Friedrich-Schiller-University Jena, Erlanger Allee 101, 07745 Jena, Germany c Department of Single Cell and Single Molecule Techniques, Leibniz Institute of Age Research/Fritz Lipmann Institute, Beutenbergstrasse 11, 07745 Jena, Germany b

a r t i c l e

i n f o

Article history: Received 2 July 2011 Accepted 11 October 2011 Available online 21 October 2011 Keywords: Human colon epithelial cells Cell isolation Primary cell culture Intestinal stem cells

a b s t r a c t Chemoprotective effects of nutritional compounds are usually studied in cell lines. Studies using primary human colon cells have been limited due to the lack of established methods regarding their culture. We therefore optimized isolation and culture of non-transformed human epithelial cells from individual donors to enrich viable cells and sufficient amounts of intact RNA. Isolated epithelial cells were seeded in different coated cell culture dishes combined with several media (2–24 h). To avoid cells from anoikis, also intact colon crypts were isolated to maintain cell interactions. These crypts were incubated with gut fermentation products (24 h) derived from indigestible carbohydrates. In none of the coated (fibronectin, laminin) cell culture dishes isolated epithelial cells did attach. The number of these cells remaining in suspension, decreased already after 2 h to 20%. Intact colon crypts cultured as pellets showed a stable viability up to 24 h (91 ± 4%) and were suitable to gain a sufficient quantity of RNA. The use of colon crypts with an appropriate cell culture medium could double the lifespan of intestinal epithelial cells from 12 up to 24 h and represents a promising approach to study early events in carcinogenesis and chemoprevention as well as other diseases of the colon. Ó 2011 Elsevier Ltd. All rights reserved.

1. Introduction Epithelial cells of the large intestine are permanently exposed to compounds derived from diet and digestive processes including genotoxic carcinogens that are putative risk factors for colorectal cancer (Kaeffer, 2002; Wakabayashi et al., 1992; World Cancer Research Fund and American Institute for Cancer Research, 2007). In the last decade chemoprevention using protective nutritional factors like dietary fiber or polyphenols (Bingham, 2006; Mahmoud et al., 2000) has reached great importance to fight against colorectal cancer (Das et al., 2007). But, especially studies to the underlying mechanisms have been limited due to the lack of suitable cell models of colonic epithelial cells. Many investigations therefore used tumor cell lines such as HT29 and CaCo-2 or adenoma cells such as LT97 (Borowicki et al., 2011; Miene et al., 2011; Rousset, 1986). Those cells are well suited for research of

Abbreviations: DAB, diaminobenzidine; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EGF, epidermal growth factor; FCS, fetal calf serum; HBSS, Hank‘s balanced salt solution; MEM, minimal essential medium; SFS, Synergy1Ò fermentation supernatant; RIN, RNA integrity number. ⇑ Corresponding author at: Department of Nutritional Toxicology, Institute of Nutrition, Friedrich-Schiller-University Jena, Dornburger Strasse 24, 07743 Jena, Germany. Tel.: + 49 3641 949669; fax: + 49 3641 949672. E-mail address: [email protected] (A. Wilhelm). 0887-2333/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tiv.2011.10.008

chemopreventive activities in existing pre-neoplastic lesions but less useful for prediction of chemoprotective effects in healthy, non-transformed colon cells (Fonti et al., 1994). Other groups used non-tumorigenic colon epithelial cell lines like NCM460 (human normal colon mucosa) (Moyer et al., 1996) or FHC (human fetal colon) (Hofmanova et al., 2005) cells for their studies. However, they also do not represent a real alternative, since these cells are conditionally immortal (Fenton and Hord, 2006) and have lost several organ-specific functions because of their dedifferentiated phenotype (Hidalgo, 1996). For these reasons the isolation and culture of primary colonocytes is preferred. Many investigators have already developed culture methods for intestinal cells derived from a variety of animal species due to the better availability of such colon tissue. Most of these studies were performed by using tissue of mice (Booth et al., 1995), rats (Evans et al., 1992; Kaeffer, 2002), rabbits (Benya et al., 1991) or bovines (Föllmann et al., 2000). So far, studies which compare the interspecies variation between cultured animal and human colonocytes do not exist. Hence, the comparability of these results is rather problematic. Thus, specific investigations using human primary cells are required. Such studies are able to generate the most relevant results. So far, studies have been limited due to the lack of long-term cultures of colon epithelial cells. The main reason is that in vivo half of the mucosa cells are in a terminally differentiated, resting state and already predetermined to die (Garrison et al., 2009). The human colonic

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mucosa is characterized by the presence of crypts (14,000 crypts/ cm2) (Ricci-Vitiani et al., 2009) forming finger-like invaginations into the underlying tissue of the lamina propria (Cheng et al., 1984). Each of them contains from 250 (Booth and Potten, 2000) up to 2000 cells (Nicolas et al., 2007) in total, consisting of a mixture of undifferentiated stem cells, proliferating and differentiated cells. Stem cells are located at the bottom of each crypt. The real number of these cells is still in discussion and data point from probably 1 cell to 20 cells per crypt or approximately 1% of all crypt cells (Potten and Loeffler, 1990). Until now, most investigations on defining location, number and function of intestinal stem cells were performed in murines. The most important step was the discovery of well validated stem cell markers like Lgr5 (Barker et al., 2007). In recent years, especially Clevers and his colleagues carried out intensive research in the field of intestinal stem cells. They estimated the number of stem cells in the small intestine between four and six (Sato et al., 2009). As each stem cell produces a large number of transit and differentiated cells, slight changes in the number of stem cells have important implications for maintenance of the integrity of the crypt. Therefore, it is an important aim to cultivate epithelial cells including stem cells. Intestinal stem cells represent the actual target cells of carcinogenesis since they are defined as long-lived and therefore at risk of incurring the series of somatic mutations leading to carcinoma. All other intestinal epithelial cell types are short-lived (Cammareri et al., 2008). Intestinal stem cells are highly sensitive to anoikis, a special form of apoptosis (Frisch and Francis, 1994). Thereby, it has not been possible to cultivate isolated colon stem cells which could be used as indicator cells for in vitro toxicological investigations (Berlau et al., 2004). Until now, primary colon cells in suspension culture remained viable for only short time periods of about 12 h (Sauer et al., 2007). Attachment of epithelial cells to the extracellular matrix plays an important role in the regulation of cell growth and differentiation. Particularly fibroblasts are a source of matrix proteins, especially collagens and fibronectin. The gut basement membrane, a specialized extracellular matrix, is produced by epithelial cells as well as mesenchymal cells in the intestine (Basson, 2003; Kalabis et al., 2003). Loss of cell–cell contact and detachment induces anoikis (Frisch and Francis, 1994; Strater et al., 1996). Therefore, an adequate in vitro microenvironment is necessary for optimal culture of colonic epithelial cells. The aim of the present study was to investigate different culture conditions to prolong the colon epithelial cells‘ life, including that of stem cells for at least 24 h to enable further examinations of potential harmful and protective compounds for incubation periods longer than the current 12 h with adequate cell viability and sufficient amounts of intact RNA for gene expression analyses. Thus, we analyzed relevant factors such as various cell isolation procedures and culture conditions that might influence cell attachment and survival of primary colon cells.

2. Materials and methods 2.1. Primary colon tissue preparation Normal human colonic mucosa was obtained from 24 patients undergoing surgery of colorectal tumors or other gastrointestinal diseases like colon polyps and diverticulitis. Mean age of the donors was 67.5 ± 9.2 years; 13 of the donors were male and 11 were female. The study was approved by the ethic committee (approval no. 1601-08/05) of the Friedrich-Schiller-University Jena and all patients gave their informed consent. Normal colon tissue (about 3–7 cm2), which was macroscopically and microscopically determined as non-malignant, was taken at least 10 cm away from the side of tumor or inflammation. The

colon tissue samples were directly stored in Hank’s balanced salt solution (HBSS; 8.0 g/l NaCl; 0.4 g/l KCl; 0.06 g/l Na2HPO4
2H2O; 0.06 g/l K2HPO4; 1 g/l glucose; 0.35 g/l NaHCO3; 4.8 g/l Hepes; pH 7.2) after removal and transported on ice from the hospital to the laboratory within 1 h. After extensive washing in HBSS epithelial strips (0.3–0.5 cm2) were stripped from the submucosa by perfusion supported mechanical disaggregation as described previously (Schaeferhenrich et al., 2003) and rinsed with HBSS several times. The resulting epithelial strips were used for isolation of single cells and colon crypts. 2.2. Isolation of single cells Single cells were isolated from epithelial strips by mincing followed by enzymatic digestion with 6 mg proteinase K (Sigma, Steinheim, Germany) and 6 mg collagenase P (Boehringer, Mannheim, Germany) dissolved in 5 ml HBSS (90 min, 37 °C). Undigested tissue pieces were removed by passing the solution over a common household sieve. The obtained suspension containing single cells was washed with HBSS, centrifuged (5 min, 4 °C, 100g) and resuspended in HBSS. Number and viability of the cells were determined by trypan blue exclusion test. 2.3. Cultivation of single cells in laminin- and fibronectin-coated cell culture dishes A low attachment rate is a strong limitation factor for culturing of intestinal cells. Laminin and fibronectin are extracellular matrix proteins which should enhance the attachment of the cells (Mahida et al., 1997). Commercially available laminin- and fibronectincoated cell culture dishes (60  15 mm, Greiner bio-one, Frickenhausen, Germany) were used to improve the attachment of the intestinal cells in culture. After centrifugation of single cells, the pellet was resuspended in a primary cell culture medium according to Rogler. This medium consisted of minimal essential medium (MEM) with Earle’s salts enriched with 20% fetal calf serum (FCS), 2 mM glutamine, 100 lg/ml gentamycin, 2.5 lg/ml fungizone, 10 ng/ml epidermal growth factor (EGF), 5 lg/ml insulin, 5 lg/ml transferrin and 5 ng/ml sodium selenite (Rogler et al., 1998). The suspension of epithelial cells was seeded at a density of 2  106 cells in uncoated, laminin- and fibronectin-coated cell culture dishes and was cultivated for 24 h (37 °C, 5% CO2, 95% humidity). 2.4. Suitability of several cell culture media Single epithelial cells were isolated as described before. The cell suspension was seeded in 6-well plates (culture area 9.6 cm2/well, nunc, Roskilde, Denmark) at a density of 2  106 epithelial cells per well. We compared viability, number and attachment of cells cultured in medium according to Rogler and MCDB (molecular, cellular and developmental biology) 302 medium (Biochrom, Berlin, Germany) containing 20% L15 Leibovitz medium, 2% FCS, 0.2 nM triiodo-L-thyronine, 1 mg/ml hydrocortisone supplemented with 10 mg/ml insulin, 2 mg/ml transferrin, 5 nM sodium selenite, 30 ng/ml EGF and 50 mg/ml gentamycin. M3:10 culture medium (Incell, San Antonio, Texas) was used as a third medium. Viability and cell number were determined after 2, 12 and 24 h by trypan blue exclusion test. Attachment was assessed by microscopical estimation of the cell confluence using an Axiovert 25 (Carl Zeiss AG, Jena, Germany). 2.5. Suitability of laminin-coated cell culture dishes in combination with several cell culture media In a further series of experiments we tested the addition of several media to laminin-coated cell culture dishes. Single epithelial

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cells were isolated as described before. The obtained cells were plated in laminin-coated cell culture dishes at a density of 2  106 epithelial cells using the above mentioned cell culture media. Viability and number of cells in suspension were determined after 12 and 24 h by trypan blue exclusion test. 2.6. Isolation and cultivation of colon crypts Colon crypts were isolated as previously described (Grossmann et al., 2003) with minor modifications. Briefly, epithelial strips were used for the isolation of intestinal colon crypts. The obtained epithelial strips were incubated for 15 min in 1 mM dithiothreitol (DTT) solution at 37 °C to remove the mucus. Afterwards the strips were washed in HBSS to completely remove DTT. Next, the epithelial strips were digested with 1 mM Ethylenediaminetetraacetic acid (EDTA, pH 8) for 10 min at 37 °C. The supernatant was discarded and the EDTA digestion step was repeated. Crypts were liberated from the digested epithelial strips by vigorous repeated shaking in 10 ml HBSS until a clear supernatant was obtained. The resulting supernatant containing the crypts was collected in a 50 ml Falcon tube and passed over a mesh filter (70 lm, BD Bioscience, Franklin Lakes, USA). The crypts were eluted by washing the filter with medium. Subsequently, they were seeded in collagen-coated cell culture dishes and collagen-coated 6-well plates and cultured in a humidified incubator at 37 °C up to 48 h. The colon crypts were maintained in DMEM (Dulbecco´s modified Eagle´s medium) supplemented with 100 U/min penicillin, 100 lg/ml streptomycin, 2.5 lg/ml gentamycin, 2.5 lg/ml amphotericin, 5 lg/ml transferrin, 10 lg/ml insulin, 0.15 mM non-essential amino acids, 1 lg/ml hydrocortisone, 30 ng/ml EGF, 4 mM L-glutamine, 2.7 mg/ml glucose and 10% FCS according to Föllmann et al. (2000). 2.7. Evaluation of cell‘s epithelial origin and detection of stem cells using immunohistochemistry Intact crypts were seeded on collagen-coated coverslips and incubated at 37 °C for 24 h. The epithelial nature of the cell layer was examined by immunohistochemistry using anti-pan-cytokeratin antibody (Dako, Glostrup, Denmark). Antibodies against vimentin (Dako, Glostrup, Denmark) were used as a negative control, due to the fact that vimentin is specific for fibroblasts and other mesenchymal cells. The existing stem cells of the cell layer were identified by anti-Lgr5 antibody (LifeSpanBioScience, Seattle, USA). Cells originating from the crypts were fixed in 4% paraformaldehyde for 20 min. Afterwards fixed cells were washed and incubated with 3% hydrogen peroxide for 7 min to inhibit endogenous peroxidase activity followed by incubation with non-immune goat serum to block non-specific binding of secondary antibodies. Sections were further incubated overnight at 4 °C in a humidity chamber with the following primary antibodies: anti-pan-cytokeratin (1:500), vimentin (1:500) and Lgr5 (1:100). Afterwards the tissue sections were washed and incubated with a goat secondary antibody (Dako, Glostrup, Denmark) for 30 min. Immunoreactivity of cells was visualized with diaminobenzidine (DAB). Sections were counterstained with hematoxylin providing a clear blue nuclear staining. In the negative controls normal serum was added instead of the primary antibody. Subsequently, the stained tissue sections were covered permanently by using gelatin for light microscopy (Axioplan 2, Carl Zeiss MicroImaging GmbH, Jena, Germany). The DAB containing substrate gives a brown color at the site of the target antigen recognized by the primary antibody. Three independent replicates were performed with tissue from different donors. Qualitative evaluation of the immunohistochemical staining was performed independently by two experienced scientists.

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2.8. Generation of colon crypt pellets Intact colon crypts were isolated as described before. After eluting with medium according to Föllmann et al. (2000) the crypts were collected in a 50 ml Falcon tube and centrifuged (5 min, 4 °C, 100g). The resulting pellets were seeded in collagen-coated cell culture dishes and collagen-coated 6-well plates and cultured up to 48 h. 2.9. Determination of cell viability after incubation of colon crypt pellets with gut fermentation products Fresh isolated colon crypts were treated as pellets with non-toxic concentrations of butyrate (10 mM) and Synergy1Ò fermentation supernatant (SFS) for 24 h. A faeces control (blank, fermentation buffer only) and the medium (according to Föllmann, solvent for butyrate) served as negative controls. Synergy1Ò is a commercially available mixture of inulin enriched with oligofructose. The fermentation of Synergy1Ò was conducted in vitro under anaerobic conditions in a batch-culture system with faecal incolum of different donors with minor modifications according to the procedure described by Stein et al. (2011). After 24 h treatment single cells were isolated by incubating the colon crypt pellets (60 min, 37 °C) in 5 ml HBSS supplemented with 6 mg collagenase P (Roche Diagnostics GmbH, Mannheim, Germany) and 6 mg proteinase K (Sigma Aldrich, Deisenhofen, Germany). Viability was determined by trypan blue exclusion test. 2.10. Isolation of RNA Total RNA was isolated from the crypt pellets by using the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer‘s instructions. Total RNA was eluted in 25 ll RNase-free water and stored at 80 °C. Yield and purity were determined with the NanoDrop™ND-1000 spectrophotometer (Peqlab, Erlangen, Germany). RNA quality indicated as RNA integrity number (RIN) was assessed by using the Agilent Bioanalyzer (Agilent Technologies, Böblingen, Germany). 2.11. Statistical analyses Results of viability and cell number were statistically evaluated. Mean values and standard deviations (SD) were calculated from at least three independent experiments representing individual donors. Differences were calculated by two-way ANOVA, including Bonferroni post-test with selected pairs using GraphPad Prism software, version 5.02 for Windows (GraphPad Software, San Diego, CA, USA). The parametric Pearson correlation analysis and linear regression were used for the RNA measurement experiments. Values were shown with 95% confidence intervals. All results reached significance when p < 0.05. 3. Results 3.1. Influence of laminin- and fibronectin-coated cell culture dishes on viability and cell number of colonic epithelial cells in culture To investigate the effect of the extracellular matrix proteins laminin and fibronectin, freshly isolated single epithelial cells from human non-transformed colon tissue were seeded in cell culture dishes coated with one of these proteins. Uncoated cell culture dishes served as control. In none of the tested cell culture dishes epithelial cells did attach. The number of these cells, which stayed in suspension was already dramatically decreased after 2 h (p < 0.05). Approximately 20% of the seeded cells could be recovered

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at this time. During further cultivation of the cells (2–24 h) the cell number remained relatively stable (Fig. 1A). When calculating the viability of the cells, a comparably high number of vital cells (80%) was observed in the uncoated and laminin-coated cell culture dishes at all investigated time points. Only cells cultured in fibronectincoated dishes showed a significantly reduced viability after 24 h.

3.2. Influence of different cell culture media on attachment, viability and cell number during culturing Moreover, we investigated the influence of several media on single colonic epithelial cells in uncoated cell culture dishes. Microscopic analysis showed that the primary non-transformed

Fig. 1. (A) Viability and relative cell number (rcn) of human primary colon epithelium cells in laminin- and fibronectin-coated cell culture dishes, (B) in several cell culture media, (C) in laminin-coated cell culture dishes in combination with several media after 0 h (control = 100%), 2 h (except (C)), 12 h and 24 h cultivation determined by trypan blue exclusion test. Shown are means + SD of three independent experiments representing individual donors (Two-way ANOVA, Bonferroni post-test viability uncoated vs. viability fibronectin 24 h: p < 0.05 (A), rcn Rogler vs. rcn MCDB 12 h: p < 0.05 (C), significant differences between the various media did not exist (B)).

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colon cells did not attach to the plastic surface in any of the media used. Fig. 1B displays that there were no significant differences between viability and relative number of cells in suspension after addition of the various media differing in their composition. Viability of cells was relatively stable over the time. Even after 24 h we could measure viabilities of over 60%. The relative cell number decreased insignificantly after 12 h incubation. Unlike the first set of experiments, more than half of the cells seeded could be recovered again even after 24 h in culture. 3.3. Suitability of laminin-coated cell culture dishes in combination with several media Colon cells cultured in laminin-coated dishes showed the highest viability and cell number when compared to cells in fibronectin-coated and uncoated dishes even the difference did not reach significance (Fig. 1A). Therefore, the effect of different media on viability and cell number was tested in laminin-coated cell culture dishes. Microscopic studies revealed that none of the used culture media prompted the cells to attach. All of the cells seeded could be found in the cell culture supernatant. Fig. 1C shows that both the viability of cells in suspension and the cell number decreased continuously during culturing up to 24 h. By comparing the various media, a significant difference was observed between the MCDB medium and the Rogler medium according to the performed Two-way ANOVA. Only half of the cells counted in the Rogler medium could be detected in the MCDB medium after 12 h culturing. Taken together, these studies demonstrate that special coatings with extracellular matrix proteins did not enhance the attachment of cells to cell culture dishes compared to uncoated dishes. The number of these cells, which maintained in suspension decreased already after a short time in culture. But, the viability of surviving cells remained relatively stable up to 24 h. Based on these results, we decided to preserve the cells as crypts, since cell–cell contacts play an important role for survival of primary human colon cells. 3.4. Culture of colon crypts Isolated crypts that retained a normal morphology (Fig. 2A) during the isolation process were seeded in collagen-coated plastic flasks and cultivated up to 48 h. We used collagen as the coating layer because studies by Föllmann et al. have shown successful results with cultured bovine colonic epithelial cells (Föllmann et al., 2000). A small fraction of crypts rapidly (after 2 h in culture) attached and intestinal cells grew out of the crypts by proliferation after 1 day in culture (Fig. 2B). The cells attached closely to each other and exhibited a typical epithelial morphology. The resulting monolayer clusters were stable up to 48 h after seeding. Fragments of the crypts were often visible on the monolayer. Thereafter, monolayers started to degenerate and subsequently detached from the surface (Fig. 2C).

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3.5. Evaluation of epithelial nature of cells The epithelial nature of cultured primary cells was confirmed by immunohistochemistry using an anti-pan-cytokeratin antibody. Cytokeratin is a part of the epithelial cell cytoskeletal complex present during differentiation of epithelial cells (Chandrakasan et al., 1991; Schlage et al., 1998). A specific marker antibody against vimentin was used as negative control, due to the fact that vimentin is specific for fibroblasts and other mesenchymal cells (Bernal and Stahel, 1985; Mahida et al., 1997). As demonstrated in Fig. 3A, the majority of cells reacted positive with the cytokeratin antibody (brown colored cells), suggesting that most cells were of epithelial origin. Cells at the periphery of a cell cluster were stained stronger with anti-cytokeratin antibody than those in the center. Only few cells showed a positive reaction with the vimentin antibody (brown colored cells, Fig. 3B). Hence, a contamination of the isolated colonocytes with fibroblasts could be avoided almost completely by discarding single cells during the isolation procedure of the crypts. Accordingly, overgrowth of the culture by fibroblasts was impeded. The cell nuclei were stained with hematoxylin and showed a blue color. 3.6. Detection of stem cells in colon crypts after 24 h cultivation Intestinal stem cells could be detected by immunostaining with the stem cell marker Lgr5. Primary cells, indicated by a round morphology, showed a positive reaction towards Lgr5 antibody (brown colored, Fig. 4A). The cellular staining was cytoplasmic distributed. The cell nuclei were stained with hematoxylin and exhibited a blue color. We also investigated the distribution of epithelial stem cells in paraffin-embedded, formalin-fixed, normal human colon samples. The immunostaining showed expression of Lgr5 in a few cells at the crypt base (data not shown). The cellular staining was located in the cytoplasm. A similar staining pattern was previously reported for mice (Barker et al., 2007) and for normal human colon tissue (Becker et al., 2008). 3.7. Viability of colon crypts after 24 h incubation with gut fermentation products The cultured monolayer clusters consisted predominantly of epithelial cells and were stable up to 48 h, but did not develop a confluent monolayer. Therefore, cell material was not suitable for studies on chemoprevention. We were unable to obtain a sufficient number of cells or RNA. Further studies are necessary to optimize this promising approach of epithelial cell culture. As another alternative, we cultured pellets of intact crypts and determined cell viability and quantity as well as quality of isolated RNA. For this, we investigated the viability of crypts from 3 donors directly after isolation of single cells (0 h) and after 24 h treatment

Fig. 2. (A) Freshly isolated colon crypts after EDTA digestion. (B) Proliferating epithelial colon cells were grown out of the crypts after 24 h in culture. Fragments of crypts were often visible on the monolayer clusters. (C) After 48 h in culture cells degenerated and detached from the surface. Shown are representative images of three independent experiments. Each experiment was performed with tissue from different donors.

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Fig. 3. (A) Strong immunostaining of epithelial cells by the pan-cytokeratin marker (brown colored) in proliferating crypt cells after 24 h in culture. (B) Weak immunostaining of the mesenchymal cell marker vimentin (brown colored) in proliferating crypt cells after 24 h in culture. The cell nuclei were stained with hematoxylin (blue colored). Shown are representative images of three independent experiments. Each experiment was performed with tissue from different donors.

single crypts and those cultivated as pellets were not observed (data not shown). However, for subsequent studies investigating gene expression of several targets related to chemoprotection, we decided to use crypts as pellets, because this could protect the colonocytes from anoikis (Hofmann et al., 2007). In addition, the use of pellets was connected with a more convenient handling during cultivation and RNA isolation. A comparison of the weight of pellets and isolated RNA amounts revealed that both parameters were positively correlated. With increasing weight of the pellet an increasing RNA concentration could be determined (r = 0.55, p = 0.08, Fig. 5). In general, we could show that at least 0.1 g crypt tissue is compulsory for a RNA amount of 100 ng/ll needed for subsequent gene expression studies. RNA quality indicated as RIN was on average 6.3 ± 1.1. 4. Discussion

Fig. 4. (A) Immunostaining of stem cells by the Lgr5 marker (brown colored) in proliferating crypt cells after 24 h in culture. (B) Normal serum was added instead of Lgr5 in the negative controls. The cell nuclei were stained with hematoxylin (blue colored). Shown are representative images of three independent experiments. Each experiment was performed with tissue from different donors.

with SFS and butyrate by using the trypan blue exclusion test. The cell viability of colon crypts was still largely over 80% after 24 h treatment with butyrate (92.1% ± 1.8%) and SFS (86.2% ± 1.5%) as well as with the corresponding controls medium (91.3% ± 3.8%) and blank (87.4% ± 9.32%) (data not shown).

Human primary colon cells isolated from surgically removed tissue will provide a relevant model to study early events of carcinogenesis and chemoprevention. The cultivation of nontransformed colonic epithelial cells is a prerequisite to ensure further investigations on colon-specific effects of nutrients or toxins. Therefore, the major aim of the present investigation was to prolong the lifespan of cultivated colonocytes to an in vivo comparable time of at least 24 h with stable and high cell viability. This allows treating cells with bioactive components to measure e. g. effects on gene expression. For this, the quality of cells has to enable the isolation of sufficient amounts of intact RNA. However, establishment of a primary cell culture is difficult due to the fact that colon epithelial cells in vivo have a finite lifespan of 3–5 days (Potten and Allen, 1977). Concerning the isolation and culture of primary non-transformed human colon cells, the literature reports various technical strategies with different outcomes on cell viability. Colonic epithelial cells loose their viability very quickly during the isolation procedure (Grossmann et al., 1998). Due to this fact, it is very important to find an optimal method to minimize cell damages and to ensure the viability over an acceptable period. Therefore, several isolation and cultivation methods have been compared in our investigations to identify conditions for an optimal culture over a longer time (>12 h).

3.8. RNA quantity of cultured colon crypt pellets First, we analyzed cell number and viability after culturing of crypt pellets and single crypts. The viability of the epithelial cells was over 80% even after 24 h incubation in cell culture medium according to Föllmann et al. (2000). Thereby, differences between

Fig. 5. Correlation between RNA concentration and cell pellet weight. The figure includes data from 11 patients. The parametric Pearson correlation coefficient was calculated. 95% confidence intervals are shown as well as the linear regression line.

4.1. Laminin- and fibronectin-coated cell culture dishes as well as several media did not influence viability and cell number of colonic epithelial cells in culture Normal epithelial cells induce apoptosis when they are separated from their respective connective tissue (Hofmann et al., 2007). To inhibit this process and further approximate to the in vivo situation, different coatings of extracellular matrix proteins (laminin and fibronectin) were used. One major function of these proteins is their ability to attach epithelial cells to the extracellular matrix via a-distoglycan and cell surface receptors such as integrins (Aumailley and Krieg, 1996; Sasaki et al., 2002). However, in the present study epithelial cells did not attach despite the coating with extracellular matrix proteins. Furthermore, the number of cells which remained in suspension decreased dramatically already after 2 h, but was relatively stable from 2 to 24 h. When calculating the viability of the residual cells, a comparably high number of vital cells (80%) was observed in the uncoated and laminin-coated cell culture dishes at all investigated time points. Only cells cultured in fibronectin-coated dishes showed a significantly reduced viability after 24 h. Preliminary studies by Sauer et al. used Matrigel and Collagen (I, IV)-coated cell culture dishes for cells‘ attachment but without success (unpublished data). Number and viability of cells in suspension were also decreased. Besides the

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proteins that were aforementioned, we used collagen as a cell carrier because of its important role as a main component of the extracellular matrix. But after 2, 24 and 48 h no difference between the collagen cell carrier and the uncoated plates could be determined with regard to attachment. For isolation of single cells proteinase K was chosen because of its high activity and its ability to digest native proteins (Yu et al., 2004). Among different methods of cell isolation and several enzymes that were tested in previous studies of our workgroup, the enzymatic digestion with proteinase K and collagenase P revealed the best cell viability, which is an important condition for further gene and protein expression studies (unpublished data). Therefore, we decided to isolate single cells according to this protocol. However, it is not excludable that due to the enzymatic digestion of the tissue cell surface receptors were possibly destroyed and subsequently epithelial cells could not attach to the extracellular matrix proteins. This is an important aspect which should be considered in further experiments, prospectively. A further approach of culturing normal epithelial cells is a co-culture with fibroblasts. Studies by Schörkhuber et al. showed that a co-culture with inactivated fibroblasts could prolong survival of normal colonic epithelial cells (Schorkhuber et al., 1998). We also tried to improve attachment and viability of single epithelial cells by culturing those over a feeder layer of fibroblasts. Therefor we used 3T3-mouse-fibroblastic-cells, which were inactivated by gamma radiation. We were unable to determine the number and viability of attached cells. This was due to the fact that the epithelial layer was not separable from the fibroblastic cells, although different combinations of digestion enzymes, like trypsin, trypsin/EDTA or trypsin/versen were used. Furthermore, we tested three culture media with different supplements to reproduce the physiological environment ex vivo and to create optimal conditions. Based on the culture conditions established by Rogler et al. (1998), we first used MEM with Earle’s salts enriched with 20% FCS. But these experiments showed that attachment and cell viability were not enhanced. The MCDB medium with a reduced amount of FCS (2.5%) did also not increase viability and attachment of colonocytes nor suppress survival of mesenchymal cells. In addition, we tested M3:10-culture medium that allowed successful growth of NCM460, a normal human colon mucosal epithelial cell line (Moyer et al., 1996), but again without a positive effect. Hence, there were no significant differences between the three media detectible. Due to the limited sample material, the three series of experiments (Fig. 1) had to be performed with tissues from different patients. One reason why the second experimental series caused better results in regard to cell survival might be the age of the patients. By showing an average age of 60 years the patients were younger than those whose tissues were used in the experimental series one (78 years) and three (72 years). Studies of Nooteboom et al. (2010) showed that in aging human tissue mitochondrial DNA mutations accumulated and caused respiratory chain defects. Further, they found that these defects resulted in decreased cell proliferation and increased apoptosis leading to a decreased crypt cell population. 4.2. Culture of colon crypts doubled the lifespan of intestinal epithelial cells from 12 up to 24 h On the basis of the present results using single colonocytes and literature sources (Hofmann et al., 2007; Pedersen et al., 2000), we had to consider that cell–cell contacts are essential for the survival of primary colonic cells. Loss of cell anchorage is associated with loss of survival signals and leads to the induction of anoikis in colonic epithelial cells. Studies by Hofmann et al. (2007) showed that primary colonic cells can be rescued from anoikis which is triggered through detachment, when cell–cell adhesion is preserved. Due to this fact, we isolated and cultured intact colonic crypts

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henceforth. The colonic crypts consist of stem cells located at the base as well as proliferating and differentiating cells in the upper part which are shed into the lumen and replaced by intestinal stem cells. Stem cells have exclusively the ability to produce transit cells and new stem cells. This life cycle is important for intestinal homeostasis (Barker and Clevers, 2010). The hypothesized asymmetric cell division of stem cells to a new stem cell and a non-stem progenitor cell is still under debate (Potten et al., 2002). Current studies of the murine small intestine, which presents an attractive system to study mammalian adult stem cells (Snippert et al., 2010), show a novel view on tissue stem cell organization (Buske et al., 2011). By the identification of Lgr5 as a marker of stem cells (Barker et al., 2007), different workgroups could demonstrate that Lgr5 positive-cells do not divide asymmetrically. Rather they divide symmetrically and maintain their stemness or develop to transit cells depending on niche signals from adjacent Paneth cells (Sato et al., 2011; Schepers et al., 2011). Sato et al. showed that it is possible to establish a crypt-villus structure in vitro by a single Lgr5 stem cell in combination with a limited set of growth factors (Sato et al., 2009). There are only a few studies which describe the situation of colon stem cells. Fact is that Paneth cells hardly occur in the colon. Occasionally they can be found in the ascending colon. Therefore, further studies about the colon stem cell niche are necessary. Important in this context is that intestinal stem cells persist throughout lifetime within the tissue and mutated stem cells can expand and occupy the whole stem cell niche (Humphries and Wright, 2008; Potten and Loeffler, 1990). Working with whole crypts ensures on the one hand preservation of cell–cell contacts and on the other hand the presence of stem cells in the cell culture. Initially, several methods were tested to yield a high number of complete colon crypts. The best procedure seems to be a combination of the methods by Föllmann et al. (2000) and Grossmann et al. (2003) with minor modifications. The cultivated crypts attached already after 2 h in culture and 24 h later we could observe attached crypts with migrated cells apart from the crypts forming an attached cell cluster. Thereby, former colon crypts lost their structure. After 48 h crypts detached and cells around the crypts were getting in suspension. The epithelial nature of the attached monolayer clusters was proven by the specific epithelial antibody pan-cytokeratin (Deveney et al., 1996; Vidrich et al., 1988). The majority of attached cells reacted positive with the antibody and only some cells showed a positive staining for vimentin indicating the presence of fibroblasts or other mesenchymal cells (Mahida et al., 1997). For further classification of epithelial subpopulations we used the stem cell marker Lgr5. The evidence of intestinal stem cells has been hampered for a long time by the lack of suitable markers. But, meanwhile several studies revealed immunostaining of Lgr5 in cells at the base of crypts, the so called stem cell niche (Becker et al., 2008; Mills and Gordon, 2001; van der Flier and Clevers, 2009). We found a similar distribution of Lgr5 immunostaining in normal colon tissue embedded in paraffin. In addition, Lgr5 staining was also observed in attached monolayer clusters demonstrating the presence of epithelial cells with stem cell characteristics. For establishment of a prolonged culture (>12 h) enabling studies of chemopreventive effects, it is of particular importance to cultivate epithelial cells including stem cells. Our findings demonstrated that crypts and the outgrowing epithelial cells comprised stem cells. The arising monolayer clusters are a promising approach, but have to be optimized in future investigations to enhance the cell number. Essential for gene expression studies is a sufficient amount of high-quality RNA indicated by a RIN > 5 (Fleige et al., 2006). Up to now, the number of available cells was the limitative factor. For this reason, we tried to cultivate isolated crypts as pellets to overcome these problems. After optimizing the culture conditions it was the aim of this set of experiments

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to reach adequate cell viability and a huge amount of RNA for subsequent gene expression analyses of individual human donors. Our results demonstrated, for the first time, that even after 24 h treatment with gut fermentation products like butyrate and SFS number and viabilities of cells were sufficient to perform studies on gene expression. Therefore, it is now possible to better investigate how nutrition-related factors contribute to chemoprevention in healthy, non-transformed colon cells. 5. Conclusions The present study disclosed that using colon crypts instead of single epithelial cells in combination with collagen-coating and cell culture medium according to Föllmann et al. (2000) prolongs the lifespan of human intestinal epithelial cells ex vivo from 12 h up to 24 h. Furthermore, this culture method ensures a sufficient number of viable colon crypts as well as enough RNA of high quality suited for gene expression studies using microarray and real time PCR. The presented approach represents an advancement of studying early events in carcinogenesis and chemoprevention as well as other diseases of the colon. Conflict of Interest This article represents original work and has not been submitted concurrently to any other journal and if accepted for Toxicology In Vitro it will not be published elsewhere in the same form in English or in any other language without the written consent of Nutrition society. All the authors have approved the submitted version and have no conflict of interest. Acknowledgements The present study was supported by the German Research Foundation (DFG, PO 284/8-3). We would like to thank Kornelia Haus and Dr. Claudia Miene for carefully reading the manuscript and Stefanie Lux for helpful assistance during the in vitro fermentation. We thank all donors of colon tissue samples for giving their informed consent. References Aumailley, M., Krieg, T., 1996. Laminins: a family of diverse multifunctional molecules of basement membranes. J. Invest. Dermatol. 106, 209–214. Barker, N., Clevers, H., 2010. Leucine-rich repeat-containing G-protein-coupled receptors as markers of adult stem cells. Gastroenterology 138, 1681–1696. Barker, N., van Es, J.H., Kuipers, J., Kujala, P., van den, B.M., Cozijnsen, M., Haegebarth, A., Korving, J., Begthel, H., Peters, P.J., Clevers, H., 2007. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007. Basson, M.D., 2003. Invited research review: cell–matrix interactions in the gut epithelium. Surgery 133, 263–267. Becker, L., Huang, Q., Mashimo, H., 2008. Immunostaining of Lgr5, an intestinal stem cell marker, in normal and premalignant human gastrointestinal tissue. Sci. World J. 8, 1168–1176. Benya, R.V., Schmidt, L.N., Sahi, J., Layden, T.J., Rao, M.C., 1991. Isolation, characterization, and attachment of rabbit distal colon epithelial cells. Gastroenterology 101, 692–702. Berlau, J., Glei, M., Pool-Zobel, B.L., 2004. Colon cancer risk factors from nutrition. Anal. Bioanal. Chem. 378, 737–743. Bernal, S.D., Stahel, R.A., 1985. Cytoskeleton-associated proteins: their role as cellular integrators in the neoplastic process. Crit. Rev. Oncol. Hematol. 3, 191– 204. Bingham, S., 2006. The fibre-folate debate in colo-rectal cancer. Proc. Nutr. Soc. 65, 19–23. Booth, C., Patel, S., Bennion, G.R., Potten, C.S., 1995. The isolation and culture of adult mouse colonic epithelium. Epithel. Cell Biol. 4, 76–86. Booth, C., Potten, C.S., 2000. Gut instincts: thoughts on intestinal epithelial stem cells. J. Clin. Invest. 105, 1493–1499. Borowicki, A., Michelmann, A., Stein, K., Scharlau, D., Scheu, K., Obst, U., Glei, M., 2011. Fermented wheat aleurone enriched with probiotic strains LGG and Bb12

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