In Vitro Cell. Dev. Biol.--Animal 32:107-115, February 1996 �9 1996 Society for In Vitro Biology 1071-2690/96 $05.00+0.00

GROWTH AND PROPAGATION OF NORMAL RAT INTESTINAL EPITHELIAL CELLS

R. M. ODEDRA, C. A. HART, J. R. SAUNDERS, B. GETTY, S. van de WALL, S. H. SORENSEN, H. EMBAYE, AND R. M. BATI"

Department of Small Animal Medicine and Surgery, Royal Veterinary College, University of London (R. M. 0., S. v.d.W., S. H. S., H. E., R. M. B.), and Departments of Medical Microbiology (C. A. H., B. G.)

and Genetics and Microbiology (J. R. & ), University of Liverpool, United Kingdom

(Received 9 June 1995; accepted 6 July 1995)

SUMMARY

A combination of mild proteolytic digestion and selective growth stimulation has been used to isolate and propagate adult rat intestinal epithelial cells with a finite life span. Growth of these cells on a variety of matrices and on mesenchymal cells has resulted in the expression of brush border enzymes including sucrase-isomahase, aminopeptidase N, and alkaline phosphatase. Examination of the cells at the electron microscopic level has revealed that although these ceils express key brush border enzymes, they do not have a fully formed brush border. These findings suggest that the expression of brush border enzymes and structural proteins represent distinct stages of enterocyte differentiation that are under separate tran- scriptional and temporal control.

Key words: differentiation; intestinal epithelium; extracellular matrix; fibroblast growth factor; heparin.

INTRODUCTION

Turnover and differentiation of intestinal epithelial cells is a tightly regulated process that allows the entire mucosal lining to be renewed every 2-3 d (Ahman and Enesco, 1967; Potten and Morris, 1988; Gordon, 1989). The renewal process appears to be driven by a population of stem cells within each crypt (Potten and Morris, 1988; Gordon, 1989). The migration of committed and initially undiffer- entiated cells along the crypt-villus axis results in controlled differ- entiation into mature, terminally differentiated enterocytes. The abil- ity of the intestinal mucosa to regenerate after damage is well documented (Altman and Enesco, 1967; Potten and Morris, 1988). However, despite this regenerative capacity in vitro, harnessing this potential for proliferation in cell culture has proved difficult. This difficulty in propagating small intestinal epithelial cells in vitro has resulted in the use of colonic tumor cells, particularly Caco-2 and HT29 (Pinto et al., 1983; Huet et al., 1987; LeBivic et al., 1988), which undergo spontaneous differentiation or can be induced to dif- ferentiate through glucose deprivation (Zweibaum et al., 1985). Al- though these cell lines have been established from colonic tumors, they do have ultrastructural and functional features of enterocytes, expressing a brush border and key digestive enzymes.

The ability of these cells to undergo enterocytic differentiation has allowed investigators to establish models to study enterocyte function and differentiation (Pinto et al., 1983; Grasset et al., 1984; Rousset, 1986; Blais et al., 1987; Huet et al., 1987; Hidalgo et at., 1989; Matsumoto et al, 1990; LeBivic et al., 1988; Vachon and Beaulieu, 1992). However, this approach has a number of drawbacks, including the transformed nature of the cells and expression of an incomplete repertoire of enterocyte functions. These cells also express a phe- notype that is closer to fetal colonic than mature small intestinal epithelium (Beaulieu et al., 1990; Wiltz et al., 1991). Similar prob- lems may arise from the use of epithelial cells from normal fetal

tissue. Fetal and also neonatal tissue have been cultured extensively but have generally been restricted to primary culture (Kondo et al., 1985; Kedinger et al., 1987a, 1987b; Whitehead and Gardner, 1987; Evans et al., 1991; Fukamachi, 1992). Intestinal epithelial cell lines that have been successfully propagated from fetal tissues do not ex- press a brush border or significant activities of brush border enzymes (Quaroni and May, 1980; Negrel et al., 1983; Louvard et al., 1992). This report describes a protocol for isolating and propagating normal adult intestinal epithelial cells that express brush border enzymes, ahhough morphologically, they do not appear to have a distinct brush border.

MATERIALS AND METHODS

Cell culture reagents were obtained from GIBCO BRL, Paisley, Scotland. General chemicals were obtained from Sigma Chemical Co., Poole, Dorset, UK. Growth factor and Matrigel were obtained from Collaborative Research, Cambridge Bioscience, Cambridge, UK. Tissue culture plastics were obtained from Falcon, Becton & Dickinson, Oxford, UK.

Cell culture. The jejunum was excised from freshly killed Wistar rats and flushed with phosphate-buffered saline (PBS) containing 200 ;lg/ml genta- mycin and 5 ~g/ml amphotericin. Segments of gut were opened longitudinally and scraped. The scrapings were washed twice with fresh PBS and digested in clostridial peptidase (3 mg/ml) and DNAse 1 (10 units/ml) in serum-free minimum essential medium (MEM) for up to 45 min. Tissue fragments were washed twice in MEM containing 15% fetal calf serum (FCS) and plated onto plastic culture dishes in the same medium. The fragments and cells were left to attach for 72 h before being washed and incubated in MEM containing 25% bovine plasma supplemented with heparin (10 ~tg/ml) and acidic fibro- blast growth factor (20 ng/ml). We removed contaminating endothelial cells manually by scraping the culture surface.

Colonies originating from crypts, and crypt and villus fragments, were al- lowed to reach a diameter of 2-3 mm before they were selectively transferred to a collagen or gelatin-coated well of a 24-well plate according to the method of Freshney (1987). Media for these cells were initially supplemented with mesenchymal cell-conditioned ntedium (25% vol/vol). The isolated cells were

107

108 ODEDRA ET AL.

FIG. 1. Cultures of adult rat intestinal epithelial cells. Cell fragments (A) were allowed to attach and spread to form colonies of epithelioid cells (B). These cells were ring-cloned and tested for acetylated low density lipoprotein uptake. Clones that tested negative were pooled and allowed to grow to confluence.

RAT INTESTINAL EPITHELIAL CELL CULTURE 109

O ~

5 0 0

400

300

2 0 0

100

t I I I I

0 2 4 6 8 10

D a y s

FIG. 2. Effect of heparin and acidic fibroblast growth factor (aFGF) on mesenchymal cells. Cells were incubated alone ( I ) or with heparin (O), aFGF ([]) , or both (O) as described in methods. The experiment was started 24 h after cells were seeded (Day 0). The results are expressed as a per- centage of cell numbers at Day 0. Each data point was calculated from a mean of six counts, and standard errors were less than 5% of the mean.

tested for acetylated low density lipoprotein (LDL) uptake as described later. Once in secondary culture, the cells were passaged 1:2 when confluent. Stock ceils were preserved frozen in liquid nitrogen in MEM containing 60% FCS and 9% DMSO.

Intestinal mesenchymal cells were derived from contaminating nonepithe- lial cells in primary cultures just described. Cells were classified as mes- enchymal on the basis of cell morphology in secondary culture.

Cell viability. Trypan blue was added to a suspension of cells to a final concentration of 0.1%, and the percentage of clear cells was determined with a hemocytometer.

Acetylated LDL uptake. Acetylated LDL was prepared as described by Sanan et al. (1985). Cells were grown on coverslips and incubated with 40 I.tg/ml acetylated LDL for 4 h. After the incubation period, cells were washed six times with PBS containing 5% FCS, then observed under a fluorescent microscope.

Production of extracellular matrix (ECM)-coated surfaces. Corneal endo- thelial cells, derived from bovine corneas by the method of Yue et al. (1989), were grown on the surface to be coated. The cells were allowed to reach confluence and were incubated for a further 14 d in the presence of 10 lag/ ml ascorbate. After the incubation period, we lifted cells from the deposited matrix by adding 30 mM ammonium hydroxide solution. The deposited matrix was washed three times with PBS before the surface was seeded with test cells.

Collagen was extracted from rat tails with 1 M acetic acid according to the method of Bornstein and Murray (1958). Purified collagen was redissolved in 10 mM HC1 at a concentration of 1 mg/ml. We generated a collagen film by adding 25 lal of the collagen solution per square centimeter of surface and allowing it to dry. The dried surface was washed three times with PBS before test cells were seeded.

Sufficient autoclaved 1% (wt/vol) bovine gelatin was added to cover the plastic growth surface. The flasks and plates were left a 4 ~ C for 1 h and then washed twice with PBS before being seeded.

Conditioned media. Rat intestinal mesenchymal cells were allowed to grow to confluence in 75-cm z flasks. Then 30 ml of medium was added to confluent cultures, and the cells were incubated for 48 h before the medium

was removed and fiher-sterilized. The medium was kept at 4 ~ C and used within 7 d.

Cell proliferation assays. Changes in rates of cell proliferation were deter- mined directly by cell counts. Test cells were seeded in 6 well plates at an initial density of 20 000 cells/well. The following day the plates were washed twice with PBS, and the test material in MEM containing 1% FCS was added to duplicate wells. Cells were incubated at 37 ~ C for 9 d and the medium containing the appropriate test material was changed every third day. Cells were trypsinized and resuspended in 0.5 ml of PBS; three aliquots were counted on a hemocytometer to give a mean of six determinations of cell number.

Measurement of cell numbers for enzyme assays. Cell numbers were deter- mined indirectly with the Hoechst 33258 dye binding assay (Labarca and Paigen, 1980). Briefly cells were disrupted by the addition of 0.1 ml 0.1 N NaOH, and the resulting solution was neutralized with an equal volume of 0.1 N HC1. The neutralized solution was transferred to a tube containing 0.1 ml of 50 mM Tris (pH 7.5) and 0.05 ~g/ml Hoechst dye, and fluorescence was measured in a fluorescence spectrophotometer (excitation 360 nm, emis- sion 495 nm). Cell numbers were determined from separate standard curves of DNA derived from Caco-2 and the rat intestinal epithelial cells (RJEC), respectively.

Enzyme assays. Epithelial cells were seeded into 96-well plates coated with the appropriate matrix at a density of 10 000 cells/well and were incubated for up to 16 d. At 1, 4, 8, 12, and 16 d postconfluence, cells were washed with Tris-buffered saline (TBS: 0.13 M NaCI, 30 mM Tris pH 7.4) before brush border enzymes were assayed by the addition of the appropriate buf- fered substrate.

Aminopeptidase N was assayed with 1-1eucine-p-nitroanilide in TBS con- taining 2 n~/CaC12, and 1 mM ZnCI 2. Aminopeptidase A (ApA) was assayed with 7-glutatnyl-p-nitroanilide in TBS, pH 8, 1 mM ZnC1 z. Dipeptidylpepti- dase IV (DPP) was assayed with gly-pro-p-nitroanilide in TBS, pH 8. Alkaline phosphatase was assayed with p-nitrophenylphosphate in TBS, pH 9.0.

A 100 ~tl aliquot of one of the substrates just described was added to each well of a column on a 96-well plate and incubated at 37 ~ C for 90 rain. Absorbance was measured on a Titertek MCCII plate reader at 414 nm for the peptidases and 405 nm for alkaline phosphatase.

600

500-

400-

e.,

o

-~ 30o

200

100

[ [ I I

0 2 4 6 8 lO

D a y s

FIG. 3. Effect of heparin and acidic fibroblast growth factor (aFGF) on secondary cultures of rat intestinal epithelial cells. The experiment was started 24 h after seeding (Day 0). The results are expressed as a percentage of cell numbers at Day 0. Cells were incubated alone ( I ) or with heparin (O), aFGF (D), or both (C)). Each data point was calculated from a mean of six counts, and standard errors were less than 5% of the mean.

110 ODEDRA ET AL.

FIG. 4. Cells grown to confluence maintained a characteristic epithelial morphology for up to 12 population doublings (A). Excessive subcuhuring resulted in progressive cell senescence and loss of coherent polygonal mor- phology (B).

Sucrase was assayed by the modification of the method of Dalquist (1970). Sucrose (50 mM in 0.1 M maleate buffer, pH 6) was added to wells and incubated at 37 ~ C. After 1 h, the reaction was stopped by the addition of glucose oxidase reagent (0.02% peroxidase, 0.02% glucose oxidase, and 20 mM potassium hexacyanoferrate in 0.7 M Tris, pH 7). The color reaction was allowed to go to completion, and absorbance of the product was measured at 490 nm on a Titertek MCC II plate reader.

Electron microscopy. Cells were washed three times with PBS and fixed for 15 min in 2.5% gtutaraldehyde in caeodytate buffer, pH 7.4. The cells were washed in cacodylate buffer and postfixed in osmium tetroxide for 15 min before being embedded and sectioned.

RESULTS

Culture conditions. Optimal conditions for culturing intestinal ep- ithelial cells were established. The initial digestion of the tissue was found to be critically important in determining percent viability and attachment efficiency. Prolonged digestion resulted in dramatic de- terioration in attachment efficiency of the extracted cells. Batch test- ing of collagenases suggested that a high clostrapain activity was also detrimental. Low tryptic collagenase yielded cells of greater percent viability, but these cells failed to attach to the substratum and rapidly deteriorated. A short partial digestion step that predominantly gen- erated clumps of cells rather than single cells resulted in optimal survival and attachment.

Putative epithelial cells isolated as described earlier were allowed to attach and spread to form colonies (Fig. 1). We removed contam- inating nonepithelial cells within a 5-mm perimeter of growing col- onies by manual scraping every third day. Once the colonies had reached a diameter of 3-4 mm, they were selectively removed from the culture surface by collagenase digestion, passaged into gelatin or collagen-coated 24-wetl plates, and allowed to reach confluence. At this stage, the cells were grown in medium supplemented with mesenchymal cell-conditioned medium (25% vol/vol). Confluent cul- tures were subcuhured into six-well plates (well diameter 35 mm); a few cells were plated onto microscope slides and evaluated for rate of acetylated LDL uptake.

Although both epithelial and endothelial cells were able to take up acetylated LDL (ac.LDL), the rate of uptake by endothelial ceils was considerably more rapid, allowing the two cell types to be easily distinguishable. Cell cultures that tested negative with ac.LDL were pooled so that a heterogeneous population of epithelial cells was generated. Data from three separate extractions showed that micro- vascular endothelial cell colonies represented 35.3% ( + 17.6; n = 17) of the isolated colonies in primary culture.

Cells were initially cultured in MEM containing 15% bovine plasma, 20 p.g/ml heparin, and 10 ng/ml acidic fibroblast growth factor (aFGF). In subsequent experiments, heparinized plasma was used instead of oxalated plasma, and 10 ttg heparin was added to the culture medium. Once the cells had been passaged into second- ary culture, media were supplemented with 10% FBS.

Heparin was added to the primary cuhures to inhibit the growth of smooth muscle ceils and to potentiate the action of acidic fibroblast growth factor to selectively stimulate proliferation of the epithelial cells. The inhibitory effect of heparin on intestinal mesenchymal cells was confirmed by the inhibition of a secondary culture of rat intestinal mesenchymal cells (Fig. 2). Although aFGF mildly stim- ulated mesenchymal cells, this effect was negated in the presence of heparin (Fig. 2). In contrast, heparin alone had no effect on the proliferation rate of the secondary culture epithelial cells. However, in the presence of aFGF, the cell proliferation was significantly stim- ulated (Fig. 3).

Epithelial cell phenotype. Cells isolated as described earlier showed uniform epithelial morphology by light microscopy (Fig. 4 A). We investigated the potential of RJEC to differentiate by mea- suring the activity of key brush border enzymes under different growth conditions. Cells were grown on a number of different matri- ces and allowed to reach confluence. The activity of brush border enzymes was determined in the postconfluent cultures during a 16- d period.

The cells could be subcultured for up to 12 population doublings in the absence of exogenous growth factors other than FCS before onset of senescence; the morphology of the cells became progres- sively less uniform, indicating an irreversible change in cell phe- notype (Fig. 4 B).

Enzyme activities. Activities of all the brush border enzymes as- sayed increased significantly in the RJEC. The pattern of enzyme expression depended on the matrix, and differences between RJEC and Caco-2 were also observed. Aminopeptidase N (ApN) (Fig. 5 A) and sucrase-isomaltase (SI) (Fig. 5 B) activities steadily increased in all the cultures tested throughout the 16-d period. However, much greater increases were observed for cells grown on Matrigel or ECM than plastic or gelatin. Increased ApA activity was also observed in RJEC and Caco-2 cells grown on all matrices (Fig. 5 C). In contrast

RAT INTESTINAL EPITHELIAL CELL CULTURE 11 ]

0.8

0.6

0.4

0.21

0.2

0.1

<

A

rn- L I~.dl,, ~L .~L 1 4 8 12 16

Days post-confluence

,

3,

1 4 8 12 16 Days post-confluence

B

1 4 8 12 16 Days post-confluence

0.3

0.2

0.1

D

1 4 8 12 16

Days post-confluence

ECM

Matrigel [ ]

Plastic [ ]

Gelatin �9

RJEC

[] Caco-2

[ ] < 1,

s t 4

E

1 4 8 12 16

Days post-confluence

FIG. 5. Expression of (A) aminopeptidase N, (B) sucrase-isomahase, (C) aminopeptisase A, (D) alkaline phosphatase, (E) and dipep- tidylpeptidase IV, in Caco-2 cells and rat intestinal epithelial cells (RJEC). Cells were assayed for activity as described in methods for cells grown for up to 16 d postconfluence.

112 ODEDRA ET AL.

to ApN and SI, Caco-2 ceils grown on gelatin expressed more ApA than those growing on other matrices, whereas for RJEC there was a considerably greater expression of ApA on gelatin and Matrigel than on plastic. These observations suggest a differential effect of matrix on enzyme expression, with gelatin inducing greater expression of ApA than ApN and SI. The RJEC expressed a higher initial activity of AP than Caco-2 (Fig. 5 D). Although the overall AP activity was greater for RJEC, the increase in stimulation was greater for Caco- 2. The latter suggested a difference between the two cell types, both in their response to the growth matrix and basal expression. The RJEC showed higher DPP activity than Caco-2 with each matrix (Fig. 5 E). This further reinforces observations with AP, where the two cell types differed in their response to growth on different matrices. In common with the other enzymes, a significant increase in DPP activity was observed throughout the experiment for all the matrices tested, suggesting an expression of a progressively more differenti- ated phenotype. However, differences between matrices were less pronounced for DPP than for other enzymes tested. These data from enzyme activity studies clearly demonstrated the ability of RJEC to express active brush border enzymes, pointing strongly to the ability of these cells to undergo differentiation.

Electron microscopy. Cells grown under the conditions described for brush border enzyme expression were examined by transmission electron microscopy. In contrast to the enzyme activity studies, there was a dramatic difference between Caco-2 and RJEC morphology. Whereas Caco-2 cells expressed a fully differentiated brush border (Fig. 6 F), RJEC showed nondifferentiated epithelial morphology (Fig. 6 A-E). The nature of the matrix did not significantly influence the morphological appearance of the brush border of RJEC or Caco- 2. Growing RJEC on intestinal mesenchymal cells (Fig. 6 D) also failed to induce brush border formation. However, when RJEC were cocultured with IL3 (Interleukin-3) producing feeder cells (WEHI- 3b) separated a by microporous filter, there were distinct changes in morphology with longer projections and increased intracellular or- ganelles (Fig. 6 E).

DISCUSSION

The main obstacles to successful culture of adult intestinal epi- thelial cells were the initial digestion and the subsequent propaga- tion and stimulation of cell proliferation. The first of these problems was addressed with experiments that demonstrated the sensitivity of the intestinal cells to excessive digestion, and more specifically to raised clostrapain and other trypsin-like activities. A similar sensi- tivity of neonatal intestinal epithelium to harsh digestion has been observed previously (Evans et al., 1991). We overcame this problem by batch-testing collagenases with low clostrapain activity and by reducing digestion times to generate fragments rather than single cells. The second problem--contamination by unwanted cells--was associated with the culture of cells from tissue fragments and orga- noids. Smooth muscle cells and endothelial cells represent the main contaminants in cultures from the intestine, and the high level of contamination meant that manual removal of cells at the periphery of growing colonies was essential. The use of heparin to inhibit smooth muscle cells but not epithelial cells proved to be essential in preventing smooth muscle cells from overgrowing the cultures. However, the epithelial cells rapidly deteriorated if most of the con- taminating ceils were removed, strongly implicating a stimulatory role for mesenchymally-derived soluble factors in stimulating the

growth of the epithelial cells. A role for soluble mesenchymal cell products was further demonstrated by the survival of cloned cells in the presence of conditioned medium and by the ability of conditioned medium to stimulate epithelial cell proliferation in secondary cul- tures (Fig. 7). Once the epithelial cells had grown sufficiently using conditioned media was no longer necessary. Therefore, these ceils were probably able to produce autostimulatory factors. The require- ment for conditioned medium may therefore simply reflect the small number of cells present during the primary culture and cloning stages.

In the past, it has been possible to establish short-term primary cultures of rat intestinal epithelium (Kondo et al., 1985; Whitehead and Gardner, 1987; Evans et al., 1991), but establishing secondary cultures of these cells has proved difficult. In the present study, the growth of epithelial cells was selectively stimulated by aFGF in con- cert with heparin. This selective growth was further enhanced with plasma instead of serum, thus depriving mesenchymal cells of plate- let-derived growth factors (Castellot et al., 1981; Wright et al., 1988). This combination of added growth factor and conditioned medium allowed the propagation of adult intestinal epithelium. The condi- tions used for the isolation procedure were equally favorable to the growth of endothelial cells, with nearly 50% of the resulting colonies being of endothelial origin. However, the two cell types were easily distinguished by the difference in their rate of ac.LDL uptake. The cloning of individual colonies and testing before pooling allowed the establishment of pure epithelial cultures.

Although it has been possible to culture epithelial cells from fetal and neonatal intestine and generate cell lines, it has not proved possible to stimulate these cells to undergo differentiation (Quaroni and May, 1980; Castellot et al., 1981; Negrel et al., 1983; Wright et al., 1988). This has been achieved only for cells grown in primary mixed culture (Kedinger et al., 1987a, 1987b; Whitehead and Gard- ner, 1987; Weiser et al., 1990; Evans et al., 1991; Fukamachi, 1992). This experience has led to the notion that direct cell contact is an absolute requirement for differentiation and brush border formation. Experiments in which RJEC were grown on a confluent layer of rat mesenchymal cells also demonstrated that direct cell-to-cell contact with intestinally-derived mesenchymal cells is not sufficient to in- duce brush border formation in this culture system. However, the mesenchymal cells used in these experiments had relatively homo- geneous morphology. It is therefore possible that these mesenchymal cells have become less heterogeneous in secondary culture, with the possible loss of less vigorous cell types during each passage. Endo- thelial ceils are also absent from these cell cultures. It is not clear whether endothelial cells play any role in the differentiation of in- testinal cells in vivo. However, diffusible products for endothelial cells, albeit corneal, have been reported to stimulate intestinal epi- thelial cell growth in vitro (Evans et al., 1991). The finite lifespan and ability of RJEC to express brush border enzymes is in marked contrast to cell lines established from fetal tissue. These differences may reflect either the age of the donor or the site from which the cells were isolated.

Interactions between epithelial cells and the mesenchyme in vivo are likely to be mediated by both the basement membrane synthe- sized by the mesenchyme and by the release of diffusible morpho- gens. The basement membrane is known to be a key element in the relationship between the epithelium and the mesenchyme (Weiser et al., 1990; Louvard et al., 1992). The nature of mesenchyme-derived diffusible factors in the growth and differentiation process remains

RAT INTESTINAL EPITHELIAL CELL CULTURE 113

B

D

F

j~

#

FIG. 6. Transmission electron micrographs (magnification • 30 000) of rat intestinal epithelial cells grown on (A) plastic, (B) ECM, and (C) Matrigel demonstrate the lack of a fully formed brush border when the cells were grown on different matrices. Similar results were obtained for cells grown on monolayers of mesenchymal cells (D). However, cells grown on WEHI-3b cells showed a markedly different morphology, with large vacuoles and more elongated microvilli (E). These cells also failed to express a fully formed brush border. The RJEC consistently expressed a few short, disorganized microvilli associated with nondifferentiated epithelium. In contrast, Caco-2 cells grown on plastic were able to form a well-ordi~red brush border (F).

to be determined. However, there is evidence for the presence of basic fibroblast growth factor (FGF) in mueosal basement membranes (Cordon-Cardo et al., 1990). Basic FGF is a pluripotent mitogen capable of stimulating a wide variety of cell types including cells of

endodermal origin (Gospodarowicz et al., 1986; Thomas, 1987; Fox, 1988) and is a member of a diverse group of heparin-binding growth factors that include aFGF (Thomas, 1987). The efficacy of aFGF in promoting the growth of epithelial cells and allowing the RJEC cell

114 ODEDRA ET AL.

o~ ,.Q

Z

300

200-

100-

I I I I I

20 40 60 80 tO0

C o n d i t i o n e d m e d i u m ( % )

demonstrated expression of brush border enzymes including sucrase- isomahase and aminopeptidase N when cells were grown on a variety of matrices in pure culture. However, electron microscopy demon-

J

strated that the ceils did not possess a brush border or tight junctions. Although there was little difference in the uhrastructural appearance of the cells under the different conditions tested, there were signif- icant differences in the expression of brush border enzymes, dem- onstrating the subtle influences of matrix on epithelial cell function. Moreover, the finding that altering the underlying growth matrix can alter expression of brush border enzymes but not induce complete differentiation suggests that these events are under separate tran- scriptional control. These abservations are further reinforced by the cell morphology observed with epithelial cells cocultured with WEHI-3b cells and in direct contact with intestinal mesenchymal cells. A similar heterogeneity in expression of differentiation markers has been observed to a lesser extent in Caco-2 cells, which appear to express a spectrum of phenotypes including the expression of brush border enzymes before the brush border is fully formed (Va- chon and Beaulieu, 1992). The RJEC cell line should therefore prove a valuable tool for determining the nature of interactions involved in stimulating growth and differentiation of intestinal epithelial cells.

FIG. 7. Stimulation of rat intestinal epithelial cell (RJEC) proliferation by medium conditioned by rat mesenchymal cells in secondary culture. Cul- tures of RJEC were incubated for 8 d in medium containing 0-100% con- ditioned medium that was changed every third day throughout the assay. At the end of the experiment, cells were trypsinized and counted on a hemo- cytometer. The results are expressed as means of six counts. Mesenchymal cell-conditioned medium stimulated proliferation of RJEC up to a concentra- tion of 50% (vol/vol). At higher concentrations stimulation was reduced and finally inhibited at 100% conditioned medium. The latter probably resulted from medium exhaustion.

line to be established further reinforces a role for this group of growth promoters in the intestine in vivo. The discovery of a keratinocyte- specific member of the heparin-binding growth factor family (Finch et al., 1989; Rubin et al., 1989; Aaronson et al., 1991) invites spec- ulation on the possible existence of similar factors for epithelial tis- sues other than the intestine.

The intestinal mucosa is not a homogeneous structure, and there is a progressively decreasing structural and functional gradient from the duodenum to the ileum (Ahman, 1971). In addition, grafting ileal segments closer to the duodenum increases villus size within the segments (Ahman, 1971). These observations suggest that humoral factors within the lumen of the intestine are exerting atrophic influ- ence on the mucosa. A number of substances have been implicated in the process including enteroglucagon (Gleeson et at., 1971; Sagor et al., 1981, 1983), epidermal growth factor (Gleeson et at., 197t; Sagor et al., 1981; A1-Nafussi and Wright, 1982; Malo and Menard, 1982; Olsen and Nexo, 1983; Sagor et al., 1983; Olsen et al., 1984), and bombesin (Lehy et al., 1986). The role of these factors on the differentiation of RJEC, either singly or in concert, remains to be fully evaluated, but from our results, it seems clear that the intestinal epithelium is under multifactorial control, including cell and matrix interactions as well as humoral interactions with paracrine and lu- minal substances.

The RJEC isolated from adult rat intestine in the present study appear to display anomalous expression of differentiation markers. The ceils clearly show an epithelial morphology, and enzyme assays

ACKNOWLEDGMENTS

This work was supported by a program grant from Wellcome trust.

REFERENCES

Aaronson, S. A.; Bottaro, D. P.; Miki, T., et at. Keratinocyte growth factor. A fibroblast growth factor family member with unusual target specificity. Ann. NY Acad. Sci. 638:62-77; 1991.

A1-Nafussi, A.; Wright, N. A. The effect of epidermal growth factor EGF: on cell proliferation of the gastrointestinal mucosa of rodents. Virchows Arch. Cell. Path. 40:63-69; 1982.

Ahman, G. G. Influence of bile and pancreatic secretions on the size of intestinal villi in the rat. Am. J. Anat. 132:167-178; 1971.

Ahman, G. G.; Enesco, M. Cell number as a measure of distribution and renewal of epithelial cells in the small intestine of growing and adult rats. Am. J. Anat. 121:319-336; 1967.

Beaulieu, J.-F.; Weiser, M. M.; Herrera, L., et at. Detection and character- ization of sucrase-isomaltase in adult human colon and in colon pol- yps. Gastroenterology 98:1467-1477; 1990.

Blais, A.; Bissonette, P.; Berteloot, A. Common characteristics for Na + de- pendent sugar transport in Caco-2 cells and human fetal colon. J. Membr. Biol. 99:113-125; 1987.

Borustein, M. B.; Murray, M. R. Serial observations on patterns of growth, myelin formation, maintenance and degradation in cultures of new- born rat and chicken cerebellum. J. Biophys. Biochem. Cytol. 4:499- 507; 1958.

Castellot, J. J.; Addonizio, M. L.; Rosenberg, R., et ah Cultured endothelial cells produce a heparin like inhibitor of smooth muscle growth. J. Cell Biol. 90:372-379; 1981.

Cordon-Cardo, C.; Vlodavsky, I.; Haimovitz-Friedman, A., et at. Expression of basic fibroblast growth factor in normal human tissues. Lab. Invest. 63:832~339; 1990.

Dalquist, A. Assay of intestinal disaccharidases. Enzyme Biol. Clin. 11:52- 66; 1970.

Evans, G. S.; Flint, N.; Somers, A. S., et at. The development of a method for the preparation of rat intestinal cell primary cultures. J. Cell Sci. 101:219-231; 1991.

Finch, P. W.; Rubin, J. S.; Miki, T., et at. Human KGF is FGF-related with properties of a paracrine effector of epithelial cell growth. Science 245:752-755; 1989.

Fox, G. M. The role of growth factors in tissue repair Ill. Fibroblast growth factor. In: Clark, R. A.; Henson, P. M., eds. The molecular and cel- lular biology of wound repair. New York: Plenum; 1988:265--272.

RAT INTESTINAL EPITHELIAL CELL CULTURE 115

Freshney, R. I. Culture of animal cells. New York: Wiley-Liss; 1987:137- 153.

Fukamachi, H. Proliferation and differentiation of fetal rat intestinal epithe- lial ceils in primary serum free culture. J. Cell Sci. 103:511-519; 1992.

Gleeson, M. H.; Bloom, S. R.; Polak, J. M., et al. Endocrine tumor in kidney affecting small bowel structure, motility, and absorptive function. Gut 12:773-782; 1971.

Gordon, J. I. Intestinal epithelial differentiation: new insights from chimeric and transgenic mice. J. Cell Biol. 108:1187-1194; 1989.

Gospodarowicz, D.; Neufeld, G.; Schweigerer, L. Fibroblast growth factor. Mol. Cell. Endocrinol. 46:187-204; 1986.

Grasset, E.; Pinto, M.; Dussaulx, E., et al. Epithelial properties of human colonic carcinoma cell line Caco-2: electrical parameters. Am. J. Physiol. 247:C260-C267; 1984.

Hidalgo, I. J.; Raub, T. J.; Borchardt, R. T. Characterization of the human colon carcinoma cell line (Caco-2) as a model system for intestinal epithelial perumability. Gastroenterology 96:736-749; 1989.

Huet, C.; Sahuquilo-Merino, C.; Coudrier, E., et al. Absorptive and mucus- secreting subclones isolated from a muhipotential intestinal cell line (HT29) provide new models for cell polarity and terminal differenti- ation. J. Cell Biol. 105:345-357; 1987.

Kedinger, M.; Haffen, K.; Simon-Assmann, P. Intestinal tissue and cell cul- ture. Differentiation 36:71~35; 1987a.

Kedinger, M.; Simon-Assmann, P.; Alexandre, E., et al. Importance of fibro- blastic support for in vitro differentiation of intestinal endodermal cells and for their response to glucocorticoids. Cell Differ. 20:171- 182; 1987b.

Kondo, Y.; Young, G. P.; Rose, I., et al. Organ specificity of epithelial cells grown in tissue culture from explants obtained from various levels of the rat gut. Exp. Cell Res. 159:158-170; 1985.

Labarca, C.; Paigen, K. A simple, rapid, and sensitive DNA assay procedure. Anal. Biochem. 102:344-352; 1980.

LeBivic, A.; Him, M.; Reggio, H. HT-29 cells are an in vitro model for the generation of cell polarity in epithelial cells during embryonic dif- ferentiation. Proc. Natl. Acad. Sci. USA 85:136-140; 1988.

Lehy, T.; Puccio, F.; Chariot, J., et al. Stimulating effect of bombesin on the growth of gastrointestinal tract and pancreas in suckling rats. Gastro- enterology 90:1942-1949; 1986.

Louvard, D.; Kedinger, M.; Hauri, H. P. The differentiating intestinal epi- thelial cell: establishment and maintenance of functions through in- teractions between cellular structures. Annu. Rev. Cell Biol. 8:157- 195; 1992.

Malo, C.; Menard, D. Influence of epidermal growth factor on the development of suckling mouse intestinal mucosa. Gastroenterology 83:28-35; 1982.

Matsumoto, H.; Erickson, R. H.; Gum, J. R., et al. Biosynthesis of alkaline phosphatase during differentiation of the human colon cancer cell line Caco-2. Gastroenterology 98:1199-1207; 1990.

Negrel, R.; Rampal, P.; Nano, J.-P., et al. Establishment and characterization of an epithelial intestinal cell line from rat foetus. Exp. Cell Res. 143:427~136; 1983.

Olsen, P. S.; Nexo, E. Quantitation of epidermal growth factor in the rat: identification of and partial characterization of duodenal EGF. Scand. J. Gastroenterol. 18:771-776; 1983.

Olsen, P. S.; Poulsen, S. S.; Kirkegaard, P., et at. Role of submandibular saliva and epidermal growth factor in gastric cytoprotection. Gastro- enterology 87:103-108; 1984.

Pinto, M.; Robin-Eleon, S.; Appay, M.-D., et al. Enterocyte like differentia- tion and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47:323-330; 1983.

Potten, C. S.; Morris, R. J. Epithelial stem cells in vivo. J. Cell Sci. Suppl. 10:45-62; 1988.

Quaroni, A.; May, R. J. Establishment and characterization of intestinal ep- ithelial cell cultures. Methods Cell Biol. 21b:403-427; 1980.

Rousset, M. The human colon carcinoma cell lines HT-29 and Caco-2-2 in vitro models for the study of intestinal differentiation. Biochemie 68:1035-1040; 1986.

Rubin, J. S.; Osada, H.; Finch, P. W., et al. Purification and characterization of a newly identified growth factor specific for epithelial cells. Proc. Natl. Acad. Sci. USA 86:802-806; 1989.

Sagor, G. R.; Ghatei, M. A.; A1-Mukhtar, M. Y. T., et al. The effects of ahered luminal nutrition on cellular proliferation and plasma concentrations of enteroglucagon after small bowel resection in the rat. Br. J. Surg. 69:14-18; 1981.

Sagor, G. R.; Ghatei, M. A.; A1-Mukhtar, M. Y. T., et al. Evidence for a humoral mechanism after intestinal resection, exclusion of gastrin but not enteroglucagon. Gastroenterology 54:902-916; 1983.

Sanan, D. A.; Strumpfer, AEM; van der Westhuyzen, D. R., et al. Native and acetylated low density lipoprotein metabolism in proliferating and quiescent bovine endothelial cells in culture. Eur. J. Cell Biol. 36:81- 90; 1985.

Thomas, K. A. Fibroblast growth factors. FASEB J. 1:434-440; 1987. Vachon, H. V.; Beaulieu, J.-F. Transient mosaic patterns of morphological

and functional differentiation in Caco-2 cell line. Gastroenterology 103:414-423; 1992.

Weiser, M. M.; Sykes, D. E.; Killen, P. D. Rat intestinal basement membrane synthesis: epithelial versus nonepithelial contribution. Lab. Invest. 62:325-330; 1990.

Whitehead, R. H.; Gardner, J. In vitro growth of epithelial cells from mucosa derived from different regions of the gastrointestinal tract. J. Gas- troenterol. Hepatol. 2:59-66; 1987.

Wiltz, O.; O'Hara, C. J.; Steele, G. D., et al. Expression of enzymatically active sucrase-isomaltase is a ubiquitous property of colon adenocar- cinomas. Gastroenterology 100:1266-1278; 1991.

Wright, T. C.; Castellot, J. J.; Diamond, J. R., et at. Regulation of cellular proliferation by heparin and heparan sulfate. In Heparin; Land, D; Lindhard, U eds. London: Edward Arnold; 1988:295-316.

Yue, B. Y. J. T.; Sugar, J.; Gilboy, J. E., et al. Growth of human corneal endothelial cells in culture. Invest. Ophthalmol. Vis. Sci. 30:248- 253; 1989.

Zweibaum, A.; Pinto, M.; Chevalier, G., et al. Enterocytic differentiation of a subpopulation of the human colon tumour cell line HT29 selected for growth in sugar free medium and its inhibition by glucose. J. Cell. Physiol. 122:21-29; 1985.