In Vitro Cell. Dev. Biol.--AnimaI 40:27~284, September and October 2004 �9 2004 Society for In Vitro Biolog3, 1071-2690/04 $18.00+0.00

ESTABLISHMENT OF A LONG-TERM CULTURE SYSTEM FOR RAT COLON EPITHELIAL CELLS

INGRID BARTSCH, INGRID ZSCHALER, MONIKA HASELOFF, ANO PABLO STEINBERG 1

Chair of Nutritional Toxicology, lr~stitute of Nutritional Science, University of Potsdam, Arthur-Scheunert-Allee 114-116, 14558 Nuthetal, Germany

(Received 27 April 2004; accepted 10 June 2004)

SUMMARY

The aim of this study was to establish a long-term culture system for rat colon epithelial ceils. Colonic crypts were isolated by incubating a 4-cm-long rat colon segment cut longitudinally with an ethylenediaminetetraacetic acid [disodium salt]--containing buffer, taken up in conditioned medium from the normal rat kidney fibroblast cell line NRK (i.e., the supernatant of pure NRK cultures), directly plated on mitomycin C-treated NRK cells and subcultured with conditioned medium from NRK cells. Cells started to migrate out of the crypts shortly after plating them on NRK feeder layers. Some of the crypts fell apart during the isolation procedure, whereas the vast majority of them did it within 1 to 2 h after plating. The cells proliferated extremely slowly but continuously over a period of 4 mo and were epithelial because they expressed cytokeratin 19 and were stained by crystal violet at pH 2.8. In conclusion, the experimental system described in this study allows to maintain rat colon epithelial cells for up to 4 mo in culture and can be used to study the effects of a variety of tumor-modulating factors on growth and gene expression of normal colon epithelial cells in vitro.

Key words: colonic crypt; crystal violet; eytokeratin 19; feeder layer; mitomyein C; NRK cells.

INTRODUCTION

Up to the present time, for colon epithelial cell culture systems with which one could study over a long period of time (3-4 mo) a nmnber of physiological and pathophysiological processes known to occur in the large intestine in vivo are still lacking. To circumvent this problem, various experimental approaches have been undertak- en. Most frequently, primary colon epithelial cell cultures from ,nice (Booth et al., 1995b; Tabuchi et al., 2000), rats (Ahnen et al., 1988; Schwartz et al., 1991; Traber et al., 1991; SchiSrkhuber et al., 1998), and hmnans (Whitehead et al., 1987; Baten et al., 1992; Stauffer et al., 1995; Pedersen et al., 2000) have been established. The main limitation of primary cell cultures is their short lifespan (in most eases 7-14 d), so they are not suited for cell transformation studies. Alternatively, a number of cell lines derived from colon adenomas (Paraskeva et al., 1984; Willson et al., 1987) and carcinomas (Lei- bovitz et at., 1976; Fogh et al., 1977) have become available. How- ever, because they are tumor derived and because they per se carry many genetic alterations, they are of limited value for in vitro ma- lignant cell transfomiation studies. Several research groups have made use of rat small intestinal epithelial cell lines such as IEC- 6, IEC-I8, and RIE-1 (Quaroni et al., 1979; Blay and Brown, 1994). Cells from the small and large intestine differ in a variety of aspects, e.g., the apoptosis rate is much higher in small intestinal epithelial cells than in cells from the colonic epithelium (Potten and Grant, 1998), so that one cannot infer that observations made in small

~To whom correspondence should be addressed at E-mail: steinber@rz. uni-potsdam.de

intestinal epithelial cells will also apply to cells from the colonic

epithelium. Because of the obvious difficulties in maintaining colonic epi-

thelial cells in culture for long periods of time, attempts to immor- talize colon epithelial cells have been undertaken. Whitehead et al. (1993) established conditionally immortalized colon epithelial cell cultures from a transgenic mouse bearing a temperature-sensitive mutation of the SV40 large T antigen gene. These cell cultures proliferale continuously at the pelraissive temperature (33 ~ C), but proliferation ceases at the nonpermissive temperature (39.5 ~ C). In a further development, Blum et al. (2001) used a retroviral vector to transfer the SV40 large T antigen complementary deoxyribonu- cleic acid into primary human colon epithelial cells. In both cases, cell lines were successfully established. However, one should bear in mind that SV40 large T antigen binds to p53 and Rb, so that these cells cannot be used in malignant cell transformation studies in which one would expect the two abovementioned tumor-suppres- sor genes to play an important role.

In an earlier study by Sch~srkhuber et al, (1998), it had been reported that primary cultures of normal colonic epithelial cells from both humans and rats cocultured with colon fibroblasts isolated from rat term embryos can be held for up to 2 wk in culture. On the basis of this observation, we have established a cell cultm'e system in which rat colon epithelial ceils are cultured on top of a mitomycin C-treated monolayer of NRK cells (a normal rat kidney fibroblast cell line). By doing so, the colon epithelial cells can be held for up to 4 mo in culture. In this study the estal)lishment of the long-term cell culture system as well as the characteristics of the colon epithelial cells held under the above mentioned condi- tions are described.

278

CULTURE OF RAT COLON EPITHELIAL CELLS 279

FIG. 1. Freshly isolated rat colon crypts. Rat colon crypts were isolated by incubating a rat colon segment with an ethylenediamine- tetraacetic acid (EDTA)-containing buffel, taken up in conditioned medium from nm'mal rat kidney (NRK) cells and directly plated on mitomycin C-treated NRK cells.

MATERIALS AND METHODS

Rat colon crypt isolation. Male Wistar rats from the Tierzucht Sch0nwalde (Schtinwalde, Germany) with a body weight of 250-300 g were killed by decapitation. The colon was quickly removed and rinsed in cold isotonic NaC1 solution (Ruth, Karlsruhe, Germany). A 4-cm-long middle piece cut longi- tudinally was incubated in a buffer containing 1.5 mM ethylenediaminetet- raacetic acid (EDTA) [disodium salt] (EDTA; Ruth), 109 mM NaC1 (Ruth), 2.4 mM KC1 (Ruth), 1.5 mM KHzPO 4 (Ruth), 4.3 mM NazHPO 4 (Ruth), 10 mM glucose (E. Merck, Darmstadt, Germany), and 5 mM glutamine (pH 7.4; Ruth) for 50 min at 37 ~ C in a shaking water bath set to agitate at 100 rpm. ThereMtm, the colon epithelium was scraped from the underlying tissue with a scalpel, and the scraped tissue was taken up in conditioned medium from NRK cells (American Type Cnlture Collection, Manassas, }~).

NRK culturing. To obtain conditioned medium for the long-term culture of colonic epithelial cells, NRK cells wel~ continuously subcuhured in Dul- becco's minimum essential medium (Sigma-Aldrich Chemie, Taufkirchen, Germany) supplemented with 5% v/v fetal bovine serum (Biochrom, Berlin), 0.25 U/ml insulin (Signm-Aldrich Chemie), 7.5% w/v NaHC03 (Rott~), 3% w/v glutamine (Ruth), 50 ng/ml epidermal growth factor (Signm-Aldrich Chemie), 10 p~g/ml transferrin (Sigma-Aldrich Chemic), and 5 ~ hydrocor- tisone (Sigma-Aldrich Chemie). Antibiotics included in the culture medium were penicillin (100 txg/ml; Sigma-Aldrich Chemic), streptomycin (100 txg/

ml; Sigma-Aldrich Chemie), and gentamycin (25 txg/ml; Sigma-Aldrich Chemie).

NRK-colon crypt coculturing. In a first step, the feeder layel~ were pre- pared: confluent NRK cultures were mitotically arrested by treating the cells with medium containing mitomycin C (10 tzg/ml; Calbiochem-Novabiochem, Schwalbach, Germany) fur 90 rain at 37 ~ C in a CO~ incubator. Then the superuatant was removed, the NRK cells were washed with fresh medium and allowed to recover for 24 h before being used as feeder layers. Then, the freshly isolated colonic mTpts were plated onto the growth-arrested NRK cells in conditioned medium from NRK cells. One half of the cell culture medimn was changed every 3-4 d.

If the eocuhures need to be splitted, one can apply a technique described by Danes and Sutanto (1982) to get rid of the NRK cells being part of the "old" feeder layer. Tbe cocultured cells are dispersed by trypsinization and allowed to attach fi~r 15 rain at room temperature (only the NRK cells will adhere). Thereafter, the supernatant containing predonfinantly the colonic epithelial cells is treated with collagenase (Sigma-Aldrich Chemic) to inhibit the growth of any remaining NRK cells, and the epithelial cells are seeded on top of a new feeder layer.

Cyzochemistry. Cell culture medium was removed and the cells were washed twice with phosphate-buffered saline solution. A 0.1% w/v solution of crystal violet (Ruth) in distilled water (pH 2.8) was added, and after 5 min the cells were washed with distilled water (pH 2.8).

280 BARTSCH ET AL,

F~G. 2. Primary cuhure of rat colon epithelial cells. Rat colon epithelial cells held for 1 wk in culture on a mitomycin C-treated normal rat kidney (NRK) cell feeder layer. No colonic fibroblasts proliferate in between.

lmmtulocytochemistr% Staining with antibodies was performed on cells fixed with methanol for 10 min. The fixed cells were first incubated with an antibody against cytokeratin 19 (Dako, Hamburg, Germany) and then treated with the con~spnnding horseradish peroxidase-coujugated secondary anti- bodies (Dako). The site of peroxidase binding was revealed hy using 3,3'- diaminobenzidine (Wisse, 1974).

RESULTS

Rat colon epithelial cells had to be taken up and subcuhured in conditioned medimn from NRK ceils: the use of a number of alter- native cell culture media led to the death of the cells within the first 4 d after plating. Howevel; rat colon epithelial cells cultured either in conventional or collagenized cell culture flasks (without NRK feeder layers) in conditioned medium from NRK cells alone also died within the first 4 d after plating the colonic crypts. Thus, the use of the conditioned medium fl'om NRK cells as well as the direct contact with the growth-an'ested NRK cells are absolutely necessary for the successful long-term culture of rat colon epithelial cells. Furthermore, NRK cells had to be treated with mitomycin C. If mitomycin C was omitted, NRK cells overgrew the epithelial cells and in the end the epithelial cells (tied. As shown in Fig. 1, the

isolation procedure allows us to obtain intact colonic crypts, and

cells started to migrate out of the crypts shollly after plating them on NRK feeder layers. Some of the crypts fell apart during the isolation procedure, whereas the vast majority of them did it within 1 to 2 h after plating. The viability of the adherent cells was higher than 90% as determined by trypan blue exclusion.

After 1 wk in culture, colonic epithelial cells but no colonic

fibroblasts, endothelial cells, or lymphoeytes were observed on top of the feeder layers (Fig. 2), as checked by light microscopy and staining with antibodies against the epithelial cell marker cytoker- atin 19. The cells proliferated extremely slowly but continuously over a period of 4 mo and were not overgrown by the underlying

NRK cells. Figure 3 shows exemplarily colonic epithelial cells held

in culture for 4 mo and stained with crystal violet at pH 2.8. At this pH, crystal violet has proved to be epithelial cell-specific stain- ing (Booth et al., 1995a): as expected, rat colonic epithelial cells

but not the underlying NRK cells were stained by c~ystal violet (Fig. 3). In accordance with previous publications (Vidrich et al.,

1988; SchiJrkhuber et al., 1998), in which colon epithelial cells stained positive when incubated with anti-pancytokeratin antibod-

CULTURE OF RAT COLON EPITHELIAL CELLS 281

FIG. 3. Crystal violet staining of rat colon epithelial cells in long-term culture. Rat colon epithelial cells were held for 4 wk in culture on mitomycin C-treated normal rat kidney (NRK) eells and stained with crystal violet at pH 2.8. Rat colon epithelial cells were stained, whereas the underlying NRK cells were not.

ies, rat colon epithelial cells cultured on NRK cell feeder layers expressed eytokeratin 19, whereas, as expected, the fibroblasts building the feeder layers did not express eytokeratin 19 (Fig. 4).

]DIscussION

The aim of this study was to establish a long-term culture system for rat colon epithelial cells. In the past, it has been shown that primary isolated colon epithelial cells survive for about 4 d when held as pure cultures (Buset et al., 1986; Seh6rkhuber et al., 1998). It has been suggested (Marian, 2002) that this extremely short life- span is most probably due to the fact that: (1) a certain number of cells within the colon cl3,pts are tem~inally differentiated and com- mitted to go into apoptosis (Lipkin et al., 1963; Ltiltler et al., 1993); and (2) the isolation procedure leads to an interruption of interac- tions between colon epithelial cells and neighboring cells such as colonic fibrohlasts, which are needed to maintain colon epithelial cells in a pmlii~rating state (Whitehead et al., 1987; Vidrich et al., 1988; Seh~rkhuber et al., 1998). Only those groups coping with the second point have been successful in culturing colon epithelial cells

for longer periods of time (Whitehead et al., 1987; Vidrich et al., 1988; Schbrkhuber et al., 1998). Sch/irkhuber et al. (1998) were able to maintain rat colon epithelial cells for about 14 d in culture by coculturing them with rat embryo colonic fihroblasts. Besides the limited survival time of rat colon epithelial cells being cultured this way (up to 14 d), a further disadvamage of this method is that rat

embryo colon fibroblasts have to be repeatedly isolated because they are not immortalized, are not commercially available, and lose their "supporting" activity as feeder layers beyond the fifth passage. Whitehead et al. (1987) reported that human colon epithelial cells could be held in culture tor up to 16 d if cultured on a feeder layer consisting of a bovine aortic endothelial cell line (Mendelsohn et al., 1982). To our knowledge, up to the present time this culture system has not been applied to rat colon epithelial cells. Vidrich et al. (1988) were successful in euhm'ing normal adult rabbit colon epithelial cells by plating them on mitomyein C-treated 3T3 Swiss albino mouse embryo fibroblasts, However, if isolated rat colonic epithelial cells were plated on mitomyein C-treated 3T3 Swiss al- bino mouse embryo fibroblasts, they only survived 3 d in culture

282 BARTSCH ET AL.

FIG. 4. Cytokeratin 19 expression in a long-term euhure of rat colon epithelial cells. Rat colon epithelial cells were held for 3 mo in culture on mitomycin C-treated normal rat kidney (NttK) cells and stained with anti-cytokeratin 19 antibodies. Rat colon epithelial ceils were stained, whereas the underlying NRK cells were not.

(SchSrkhuber et al., 1998) (i.e., the cocuhure system was not able to significantly increase the survival time of rat colon epithelial cells in culture).

In this study, rat colon epithelial ceils have been held in culture for up to 4 mo by plating them on a feeder layer consisting of mitomycin C-treated NRK cells, a commercially available normal rat kidney fibroblast cell line that is extremely easy to subculture, Although EDTA has effectively been used to isolate intact crypts from hmnan (Whitehead et ah, 1987) and ra~ colon mucosa (this study), in a number of cases, dispase, a fibronectase and collagenase (Stenn et al., 1989), has successfully been used as an alternative to EDTA to release intact crypt units from rabbit (Vidrich et al., 1988), mouse (Booth et al., 1995b), rat (Sch6rkhuber et al., 1998), and human colon epithelium (Schtirkhuber et al., 1998). It has often been argued that the number of colon crypts isolated from a single animal, the viability of the ceils within the isolated colon crypts, as well as the colony-forming efficiency of the colon crypt preparations is much higher if dispase instead of EDTA is used. This is because in earlier studies (Cheng and Bjerknes, 1982; Cheng et al., 1984;

Yoshino et al., 2003) a much higher EDTA concentration (30 mM) than that chosen in this report (1.5 nrM) was used, 30 mM EDTA being extremely toxic to the cells within the colon crypts.

The isolated crypts were seeded on top of a feeder layer con- sisting of mitomycin C-treated (i.e., growth-arrested) NRK cells cul- tured in conditioned medium from NRK cells, the feeder layer be- ing, as previously observed by other research groups (Whitehead et al., 1987, Vidrich et al., 1988; Sch6rkhuber et al., 1998), unavoid- able for the long-term euhure of colon epithelial cells. According to the data presented in this study, the contribution of the NRK cell feeder layers to colon epithelial cell growth is complex, most prob- ably involving both soluble factors secreted into the medium as well as matrix or membrane constituents. It may be that components of the extracellular matrix or the plasma membrane (or both) provided by NRK cells stimulate epithelial cell proliferation or enable the cells to respond to growth factors secreted into the medium. For example, it is known that on the one hand NRK cells secrete leu- kemia inhibitory factor (LIF) (Yoshino et al., 2003; data not shown in this study) into the medium and that on the other hand colon

CULTURE OF RAT COLON EPITHELIAL CELLS 283

epithelial cells possess receptors for LIF (Godard et al., 1992) and proliferate if LIF is added to the cell cul ture medium (Guimbaud et al., 1998; Kalabis et al., 2003).

The colon epi thel ium is a continuously replacing tissue, and the ceils responsible for this replacement are the stem cells located at the very base of a colonic crypt (Potten, 1998). It is tempting to suggest that the ceils proliferating extremely slowly but continu- ously on top of the feeder layers are stern cells derived from the colon crypts. However, up to the present time, a specific marker for such a cell has not been found. As unambiguously documented in this study, the cells proliferating on top of the feeder layers are epithelial in nature because they are cytokeratin 19 positive and are s tained by crystal violet at pH 2.8 (Vidrich et al., 1988; Booth et al., 1995a; Schr rkhuber et al., 1998). Whether these cells are able to give rise to a differentiated progeny and to regenerate co- lonic tissue after injury, two functional properties of stem cells, remains to be shown.

In conclusion, the experimental system descr ibed in this study allows us to mainta in rat colon epithelial cells for up to 4 mo in culture. Although we did not succeed in obtaining large areas of confluent epithelial monolayers that could be used in transport stud- ies, the experimental model could be useful to study the effects of a variety of tumor-modulat ing factors on growth aud gene expression of normal colon epithelial cells in vitro. Antibiotics such as strep- tomycin are included in the cell cul ture medium to avoid bacterial contamination. One should bear in mind that antibiotics are able to modulate the transcript ion rate of multidrug resis tance genes (Mar- c h a l e t al., 1999). This fact has to be taken into account whenever the effects of compounds, which could interact with mnhid rug re- sistance gene products, are analyzed in the cell culture system pre- sented herein. Furthermore, the low proliferation rate of colon ep- ithelial ceils eocuhured with NRK cells might be an obstacle to study certain effects of tumor-modulat ing compounds on normal co- lon epithelial cell growth. Ongoing and future experiments are being performed to determine whether this coculture system is indeed adequate for the above ment ioned application.

ACKNOWLEDGMENT

This study was supported by the grant Ste 493/7-1 from the Deutsche Forschungsgemeinschaft.

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