Co-cultures of human intestinal goblet (HT29-H) and absorptive (Caco-2) cells for studies of drug and peptide absorption

EUNOPIEAN

ELSEVIER

European Journal of Pharmaceutical Sciences 3 (1995) 171-183

JQUIINAL

OF

PHARMACEITIfAL SCINES

Co-cultures of human intestinal goblet (HT29-H) and absorptive (Caco-2) cells for studies of drug and peptide absorption A. Wikman-Larhed, P. Artursson* Department of Pharmacy, Biomedical Centre, Uppsala University, Box 580, S-75I 23 Uppsala, Sweden Received 6 October 1994; revised 30 January 1995

Abstract

A co-culture model of the two most abundant intestinal epithelial cell types (absorptive and goblet cells) was developed on permeable supports using the human Caco-2 and HT29-H cell lines. Seeding conditions were selected to give a proportion of goblet cells comparable to the 25-55% found in human colon. The co-cultures comprised small clusters of HT29-H goblet cells embedded in Caco-2 absorptive cells. Electron microscopy showed that tight junctions formed between the two cell types in co-culture. Mono-cultures of HT29-H goblet cells were more permeable to ions and hydrophilic marker molecules and peptides and had a lower Isc than mono-cultures of Caco-2 absorptive cells. The permeability of the co-cultures was intermediate between those of the two mono-cultures. The co-cultures provide a model where absorptive and goblet cells can be studied simultaneously in studies on e.g. drug binding and transport, as well as in studies on absorption enhancement. Keywords: Caco-2; HT29; Intestinal epithelium; Tight junction; Drug transport; Peptide transport; Cell culture

1. Introduction

The human intestinal epithelial cell line Caco-2 forms monolayers of absorptive enterocytes with brush border and tight junctions under standard cell culture conditions (Pinto et al., 1983). Caco2 cells express carriers/receptors for nutrients, macromolecules and drugs (Artursson et al., 1991; Blais et al., 1987.; Dix et al., 1990; Karlsson et al., 1993a). It is therefore widely used as an in vitro model of the absorptive intestinal epithelium, for example in studies of epithelial polarity and drug transport (Matter et al., 1990; Mostov et al., 1992; Neutra et al., 1989). The permeability of Caco-2 monolayers to paracellular marker molecules is at least 20 times lower than that in the human colon and up to 100 times lower than that in the human small intestine in * Corresponding author. 0928-0987/95/$0V.50 © 1995 Elsevier Science BN. All rights reserved S S D I 0928-0987(95)00007-0

vivo (Artursson et ai., 1993). Clonal cell lines derived from Caco-2 have had lower permeability but clones with higher permeability than the parental cell population have not yet been found (Woodcock et al., 1991). There are several factors that could contribute to this difference in permeability from the intestinal epithelium in vivo. For instance, the absence of central nervous control, the absence of systemic blood flow, reduced motility and differences in the thickness of the mucus layer could influence ion and water transport. Additional factors possibly further reducing permeability involve the absence of the crypt-villus axis, a lower turnover rate (no signs of cell extrusion) and the absence of goblet cells. Goblet cells comprise the second major cell type of the intestinal epithelium, and it is suggested that they form tight junctions with an irregular structure which are more permeable than those of absorptive cells (Madara et al.,

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1982). The tight junction permeability of the goblet cells has not been investigated quantitatively, since no pure goblet cell populations have been available. However, since the original observation that HT29 cells can be made to differentiate into mature intestinal cells (Pinto et al., 1982) a number of different laboratories have isolated differentiated HT29 subpopulations and clones (Huet et al., 1987). Some are of absorptive type (Hafez et al., 1990; Huet et al., 1987; Zweibaum et al., 1985). While others are of goblet type (Augeron et al., 1984; Hafez et al., 1990; Hanski et al., 1992; Huet et al., 1987; Kreusel et al., 1991; Lesuffleur et al., 1990). The latter clones form monolayers with a large proportion of mature goblet cells and also secrete mucin molecules (Huet et al., 1987; Phillips et al., 1988; Roumagnac et al., 1987; Wikman et al., 1993). One of the clones, HT29-H, forms monolayers comprising approximately 80% mature goblet cells with tight junctions (Wikman et al., 1993). The secretion of mucin molecules results in a visible mucus layer that forms a diffusion barrier to the lipophilic marker molecule testosterone (Karlsson et al., 1993b; Wikman et al., 1993). The tight junction permeability of HT29-H monolayers to the hydrophilic marker molecule mannitol was up to 50 times higher than that of Caco-2 monolayers (Wikman et al., 1993), supporting the in vivo observation that the tight junctions of goblet cells are more permeable than those of absorptive cells (Madara and Trier, 1982). This difference suggests that the development of a co-culture of Caco-2 and HT29-H cells could give monolayers with a permeability closer to that seen in vivo. The first aim of this study was therefore to establish cell culfure conditions where co-cultures of Caco-2 and HT29-H cells formed monolayers with tight junctions. A preliminary report on the co-culture of Caco-2 and HT29-H cells under standard culture condition suggest that the coculture form 'leak-proof' cell-layers (Allen et al., 1991). The second aim was to investigate how the two cell populations mixed in the monolayers. The mixing of intestinal goblet cells with other cell populations in vivo is well documented (Bjerknes et al., 1985; Goralski et al., 1975). The third

aim was to characterise the tight junction permeability of the mono-and co-cultures. It is not known if the difference in permeability between the absorptive and goblet cell populations is a result of differences in the distribution or number of the junctional pores (i.e. the assumed routes of aqueous communication in the tight junctions). The results show that HT29-H goblet cells and Caco-2 absorptive cells can be grown as co-cultures with barrier properties that are similar to but not identical with those of the human colon.

2. Experimental procedures 2.1. Materials

The Caco-2 cell line, originally derived from a human colon carcinoma (Fogh et al., 1977), was obtained from American Type Culture Collection, Rockville, MD, USA. HT29-H cells, a mucus producing sub clone of the HT29 human colon carcinoma cell line (Wikman et al., 1993), was a gift from Dr. Daniel Louvard, Pasteur Institute, Paris, France (Huet et al., 1987). Roswell Park Memorial Institute 1640 medium (RPMI), foetal calf serum (FCS), PenicillinStreptomycin solution (10000 I U / m l and 10 mg/ ml, respectively), Dulbecco's phosphate buffered saline without calcium, magnesium and sodium bicarbonate (PBS) and Hank's Balanced Salt Solution were obtained from Gibco Laboratories through Laboratory Design AB, Liding6, Sweden. Human transferrin and N-2-hydroxyethyl-piperazine-N'-2-ethanesulfonic acid (HEPES) were from Sigma Chemical Co, St. Louis, MO, USA. Rat tail collagen (Type I), was obtained from Collaborative Research, Bedford, MA, USA. Transwell TM, 6.5 mm diameter and Snapwell TM, 12 mm cell culture chambers, polycarbonate membrane, 0.4 /xm pore size were purchased from Costar, Badhoevedorp, The Netherlands. ~4C-mannitol (specific activity: 52.0 mCi/mmol), 3H-testosterone (specific activity: 141.1 Ci/mmol), 3H-raffinose (specific activity: 5-15 Ci/mmol), 3H-TRH (thyrotropin releasing hormone, specific activity: > 100 Ci/mmol), and ~4C-carboxyl-inulin (specific activity: 1-3 mCi/g) were from New England Nuclear, Boston, MA,

A. Wikman-Larhed, P. Artursson / European Journal of Pharmaceutical Sciences 3 (1995) 171-183

USA. Mono-disperse 14C-PEGs (polyethylene glycols) with molecular weights of 238, 282, 326, 414 and 502 g/mole were gifts from Dr AnnaLena Ungell AB, Astra H/issle, M61ndal, Sweden. 125I-dDAVP (1-deamino-8-o-argininevasopressin, specific activity: 4000 cpm/1.5 fmol) was a gift from Dr Anja Broeders, Ferring AB, Malm6, Sweden. 2.2. Cell culture Caco-2 cells were maintained in RPMI supplemented with 10% heat inactivated foetal calf serum, in an atmosphere of 95% air and 5% carbon dioxide, at 37°C. The HT29-H cells were maintained under the same conditions as the Caco-2 cells except that 5 /zg/ml human transferrin was added to the cell culture medium. Polycarbonate cell culture inserts were coated with rat tail collagen type I, approximately 5 tzg/cm 2. The collagen coated filters were dried overnight in a laminar air flow hood and rinsed twice with PBS prior to seeding. Caco-2 and HT29-H cells were seeded at a density of 12 × 105 cells/cm z and co-cultures at a density of 6.0 × 105 cells/cm 2, and the cells were fed every second day with complete medium containing benzylpenicillin (100 U / m l ) and streptomycin (10 tzg/ml). Experiments were performed on 24- to 26-day-old monolayers. Caco-2 cells of passage 97-102 and HT29-H cells of passage 29-32 were used. 2.3. Co-cultures Caco-2 and HT29-H cells were detached from the cell culture flasks, suspended thoroughly with ice-cold medium and kept on ice to avoid the formation of cell aggregates. Initially, the suspensions were filtered through a 23 /xm nylon mesh (Gonzfilez-Mariscal et al., 1989), but this step was excluded since it did not reduce the number of cell clumps further. The suspensions were diluted so that equal volumes of the two cell suspensions could be mixed to give the desired proportions of each population. Experiments were performed after 24-26 days in culture. Filters with mono-cultures of HT29-H and Caco2 cells were always used as controls.

173

2.4. Transmission electron microscopy Specimens were prepared for transmission electron microscopy essentially according to standard procedures (Robards et al., 1993). Briefly, cells grown on permeable inserts were rinsed with PBS and fixed in 1.5% glutaraldehyde in phosphate buffer. Specimens were treated consecutively with 1% osmium tetroxide and 1% uranylacetate. They were then dehydrated through a graded series of ethanol and embedded in Epon. Thin sections were cut with an ultramicrotome and stained with uranylacetate and lead citrate. The specimens were studied in a Philips 420 electron microscope (Philips, Eindhoven, The Netherlands) operated at 60 kV. 2.5. Fluorescence microscopy Monolayers used for transport and resistance studies were stained with the intercalating dye Hoechst 33258 for fluorescence microscopy. The cells were first fixed in a 50% solution of fixative (acetic acid:methanol, 1:4) in Hanks'Balanced Salt Solution containing 25 mM H E P E S (HBSS) and then in fixative alone. The monolayers were dried and the cell nuclei were stained in the dark. The filters were rinsed with deionized water, air dried, mounted with a cover glass in PBS: glycerol (1:1) and examined in a fluorescence microscope (Zeiss Axioskop, Oberkochen, Germany). HT29-H cells in suspension were stained with the fluorescent cell linker PKH-26 GL (Sigma Immunochemicals, Deisenhofen, Germany), according to the manufacturers' instructions. The number of HT29-H cells occurring as single cells or in aggregates were recorded in the fluorescence microscope, 1 h and 24 h after seeding. 2.6. Determination of the size of the cell nuclei and the surface area covered with HT29-H cells NIH Image, a public domain image analysis program for the Macintosh, was used to measure the cross-sectional area of the cell nuclei of Caco2 and HT29-H cells in the fluorescence microscope. Four monolayers of each cell type were analysed and three images were taken at random

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A. Wikman-Larhed, P. Artursson I European Journal of Pharmaceutical Sciences 3 (1995) 171-183

positions on each monolayer. A total of 240 cells of each cell type were analysed. The relative proportions of the two cell types in the co-cultures were determined from differences in the size of the cell nuclei. The cocultures were stained with Hoechst 33258 and six images were taken at 1 mm intervals along the midsection of each of four monolayers. The areas that contained HT29-H cells were measured and the mean percentage area covered with HT29-H cells was calculated.

2.7. Cell density Hoechst 33258 stained HT29-H and Caco-2 monolayers were examined in the fluorescence microscope. All cells in the field of vision were counted at three positions on each filter. Four filters of each cell type were used. The mean number of HT29-H cells/cm 2 or Caco-2 cells/cm 2 on each filter was calculated.

2.8. Transport studies All transport experiments were performed at 37°C in HBSS. The apical culture medium was removed and the inserts transferred to wells containing 600 /xl prewarmed HBSS. 300 /zl prewarmed HBSS containing the radioactive compound was added to the apical chamber of each cell culture insert. At selected times, the filters were transferred to a rlew well containing 600/zl of fresh HBSS. At the end of the experiment samples were taken from the wells for determination of the radioactivity in a Tricarb 1900 CA liquid scintillation counter (Canberra Packard Instruments, Downers Grove, IL, USA). The initial donor concentration in the apical chamber, C O, was determined from the mean value of four samples (50 /zl) taken from the radio labelled drug solutions. Apparent permeability coefficients (Papp) were calculated according to Papp = dQ/dt x 1/(ACo)

(1)

where dQ/dt is the permeability rate, A the surface area of the monolayer, and C O the initial concentration in the donor chamber. Enzymatic degradation of the peptides when incubated with

cells did not occur, as analysed previously (Lundin et al., 1990).

2.9. Extracellular mucus The surface of the mono- and co-cultures were investigated by ocular inspection or in the light microscope for the presence of a mucus layer. By this simple method a visible mucus layer can be seen as a gel like material on mono-cultures of HT29-H cells grown in DMEM. The barrier properties of the putative mucus layer was investigated using 3H-testosterone as a marker molecule. Testosterone diffusion across HT29-H monolayers grown in D M E M is significantly reduced by the mucus layer (Karlsson et al., 1993b; Wikman et al., 1993).

2.10. Electrical parameters The electrical parameters were determined with a new in house computer based automatic system (Johan Grgtsj6, unpublished). A single unit Transwell diffusion chamber was used (Precision Instrument Design, Tahoe City, CA, USA). The apical and basolateral compartments were connected to current generated by custom made platinum electrodes and to voltage sensitive Ag/AgCI electrodes (Radiometer, Copenhagen, Denmark) via salt bridges (polyethylene tubing, 2% agar in 2 M KC1). The resistance measurements were based on voltage measurements at five different currents (0, 15, - 1 5 , 30 and - 3 0 /zA). The values were fitted to a straight line in a current-voltage diagram and the resistance was determined from the slope of the line. The T E E R values were calculated after subtraction of the resistance of cell culture inserts without cells. The short circuit current and potential difference were calculated from the resistance slopes. A theoretical value for the transepithelial resistance of the co-cultures was calculated according to the following equation (Fuller et al., 1986;Gonz~ilez-Mariscal et al., 1989):

Rtheo r

=

II(fHIRH + (1 --fH)lRc)

(2)

where fH is the fraction of the filter covered with HT29-H cells, RH the resistance of the HT29-H

A. Wikman-Larhed, P. Artursson / European Journal of Pharmaceutical Sciences 3 (1995) 171-183

monolayers, and R c the resistance of the Caco-2 monolayers. Since P~pp is the reciprocal of R, then a theoretical value o f Papp can also be calculated according to Eq. 2. 2.11. Statistics

The results are generally expressed as mean--. SD. When a mean value was derived from the mean of individual monolayers (i.e. for cell densities of mono-cultures and the proportion of HT29-H cells in co-cultures), then m e a n - s.e.m. was used. Unpaired student's t test (two-tailed) was used to test the significance of the difference between two mean values. When comparisons of more than two mean values were made, the data were analysed with one way ANOVA.

3. Results

3.1. Cell culture conditions

Initial studies showed that a high proportion of the HT29-H monolayers grown under conventional culture conditions were discontinuous and

175

more than half of the monolayers had to be discarded for this reason (data not shown). However, continuous monolayers (as determined by fluorescence microscopy and electrical parameters) where obtained when the D M E M medium was changed to RPMI. Electron microscopy of confluent RPMI-grown HT29-H and Caco-2 cells showed that both cell populations formed monolayers of differentiated epithelial cells comparable to those reported previously for D M E M grown cells (Fig 1) (Pinto et al., 1983; Wikman et al., 1993). Approximately 80% of the HT29-H cells had similar morphology to that of mature goblet cells with the apical part of the cytoplasm filled with clusters of mucin granules (Fig la). The remaining cell population was mainly composed of immature goblet cells with few mucin granules and occasional absorptive like cells. The HT29-H cells had relatively few, short microvilli and formed tight junctions (Fig. la). Caco-2 cells had similar morphology to that of normal enterocytes with tight junctions and a well developed brush border (Fig. lb). No visible mucus layer was present on the apical side of HT29-H cells grown in RPMI. The absence of a functional mucus barrier was verified by comparing the

Fig. 1. Transmission electron micrographs of cell monolayers. (a) The apical portion of HT29-H cells. (b) The apical portion of Caco-2 ceils. Note that the Caco-2 cells have larger microvilli and less dense cytoplasm than the HT29-H cells and the apically located mucin granules in the HT29-H cells. The bars indicate 0.2/.~m.

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permeability of Caco-2 and HT29-H monolayers to the lipophilic and uncharged marker molecule 3H-testosterone (Karlsson et al., 1993b; Wikman et al., 1993). Thus, the permeability to 3H-testosterone was 2.8 ± 0.1 × 10 -~ cm/s and 3.1 --+0.2 × 10 -5 cm/s in HT29-H and Caco-2 monolayers, respectively. This is in contrast to previous studies where the mucus layer produced by DMEM-grown HT29-H monolayers reduced the permeability to testosterone 3-fold (Karlsson et al., 1993b).

HT29-H cells were significantly different (Fig. 2a and b). The mean areas of the cell nuclei of Caco-2 and HT29-H cells were 126.6---33.8/.~m and 46.9---15.2 /zm ~, respectively (p <0.0001, n = 240) (Fig. 2c). The cell densities were also significantly different (0.54 --- 0.07 × 10 6 cells/cm: and 1.56-+ 0.91 × 106 cells/cm: for Caco-2 and HT29-H cells, respectively (p <0.0001, n = 4 ) (Fig. 2). These differences were used to distinguish between the two cell types in co-culture. 2

3.3. Characteristics of co-cultures 3.2. Identification of Caco-2 cells and HT29-H cells Staining with Hoechst 33258 showed that the cross-sectional areas of the nuclei of Caco-2 and

To study the mixing between the two cell populations, HT29-H cells were stained with the stable membrane marker PKH-26 GL and cocultured with Caco-2 cells for 1 h and 24 h. The mixing was good with > 8 0 % of the adherent

D wt

~oo

C

80 m m

60

°m...m,.°.J°

Caco-2 ii

t_

HT29-H

40 7

20 % " ° "too , $ | | ~ ~ o * i ' P ° e o l . . . l l

0

100 2 Area, p.m

20O

Fig. 2. 24-Day-old cell monolayers stained with the nuclear dye Hoechst 33258. Only cell nuclei in the focal plane are reproduced in the correct size (40 × magnification). (a) HT29-H cells. (b) Caco-2 cells. (c) Histogram of the cross-sectional area of the cell nuclei of HT29-H cells and Caco-2 ceils•

A. Wikman-Larhed, P. Artursson / European Journal of Pharmaceutical Sciences 3 (1995) 171-183

cells occurring as single cells or cell pairs after 24 h. However, after > 3 weeks in culture, the co-cultures had an unpredictably large proportion of HT29-H cells as detected by nucleus staining with Hoechst 33258. This was related to the more rapid growth of HT29-H cells during the first days in culture (data not shown). In subsequent cultures the proportion of HT29-H was reduced in order to more closely approach the in vivo situation. An initial seeding density of 1% HT29H cells resulted in monolayers with 24.3 ± 5.0% to 30.7---8.9% of the surface area covered with HT29-H cells, corresponding to 48-56% of the total number of cells, a proportion similar to the 25-55% reported for the colon (Kulenkampff, 1975; Lacy, 1991). The rapid growth of the HT29-H cells resulted in co-cultures comprised of small clusters of HT29-H cells embedded in Caco-2 cells (Fig. 3a). A closer examination of the borders between the two cell populations indicated that mixing between them was limited (Fig. 3b). Electron microscopy showed that the monolayers were continuous at these sites and that tight junctions were consistently formed between HT29-H and Caco-2 cells (Fig. 3c). Moreover, the two cell lines displayed morphologies comparable to those observed in the mono-cultures (Figs. 1 and 3c). Electrophysiological characterisation of the monolayers showed that Caco-2 mono-cultures had a higher TEER, resting potential and short circuit current than HT29-H mono-cultures, consistent with the characteristics of the absorptive enterocyte (Table 1) (Karlsson et al., 1993b; Madara and Trier, 1982; Wikman et al., 1993). Co-cultures comprised of 30% HT29-H and 70% Caco-2 cells had an intermediate TEER as compared to the two mono-cultures, while the short circuit current and resting potential values were more similar to the Caco-2 mono-cultures (Table 1 and Fig. 4). Transport experiments with the hydrophilic paracellular marker molecule 14C-mannitol were in agreement with the T E E R studies and showed that the paracellular permeability of the monolayers increased with the proportion of HT29-H cells (p < 0.002, n = 4) (Fig. 4). The changes in mannitol permeability were larger than those in TEER. Thus, the T E E R decreased 1.5-fold and

t77

Fig. 3. 24-Day-old co-cultures of HT29-H and Caco-2 cells stained with the nuclear dye Hoechst 33258. (a) HT29-H cells form clusters embedded in Caco-2 cells ( 1 0 x magnification). (b) Border between a cluster of HT29-H cells and surrounding Caco-2 cells (40 x magnification). (c) The micrograph shows cell-cell contact between a Caco-2 cell and an HT29-H cell. A tight junction is formed between the two cells. The bar indicates 0 . 5 / z m .

2.7-fold when the content of HT29-H was increased to 30% and 100%, respectively, while the corresponding increases in mannitol permeability

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Table 1 Resistance, short-circuit current (Isc) and resting potential (pd) of the cell monolayers a Cell monolayer

Resistance (1"1cm z)

lsc (/zA/cm2)

pd (mV)

Caco-2 HT29-H Co-culture

120.1 ± 24.3** 75.8 -+ 3.7 98.8 ± 15.1"

14.2 ± 2.4*** 2.7 ± 0.5 13.8 ± 1.9"**

- 1 . 7 ± 0.1"** - 0 . 2 ± 0.04 - 1 . 1 -"- 0.8***

Experiments were performed on 25-day-old monolayers. 30% of the area was covered with HT29-H cells in co-cultures. Results are expressed as mean ± S D (n = 4). * Significantly different from HT29-H monolayers (Student's t test, *p < 0.05, **p < 0.01, ***p <0.001). a

4

~

3.4. Tightjunction permeability

~ ~

The permeabilities of the monodisperse polyethylene glycols showed a non-linear dependence on their molecular weights, with the permeabilities of the monolayers decreasing in the order HT29-H > co-culture > Caco-2 (Fig. 5).

e,i

3

~

0

0 30 100 HT29-H coverage, %

q/

10.4 -5 10

Fig. 4. Apparent permeability coefficients (Papp) of mannitol and transepithelial electrical resistances of the monoand co-cultures. The height of the bars indicates mean ± SD (n = 4).

-6

10

-7

.

10

200

were 2.6-fold and 8.0-fold, confirming previous findings that the permeability of marker molecules is a more sensitive measure of paracellular permeability than TEER (Anderberg et al., 1992). The T E E R and mannitol permeability of the co-cultures were in good agreement with predicted values when the electrical resistance (or alternatively the-inverted value of the mannitol permeability) of the mono-cultures were added in parallel, taking into account the proportion of each cell type in the mixture (Eq. 2). Thus, the measured values of TEER and mannitol permeability in the co-cultures presented in Fig. 4 were 104 - 15 ~ c m 2 and 1.2 x 10 - 6 -+" 0.2 x 10 -6 cm/s while the corresponding values predicted using calculations from the values of the monocultures were 104 Ilcm 2 and 1.7 x 10 -6 c m / s , respectively.

300

-4---A-

4'

400

500

HT29-H Coculture

3~

200

300

mw

400

500

Fig. 5. Transport of monodisperse polyethylene glycols across cell monolayers as a function of molecular weight. Values are mean ± SD (n = 3-7). The insert shows the same graph on a logarithmic scale and includes permeability values of polyethylene glycols in human colon. The permeability values for human colon were calculated from data by Chadwick et al. (1977) as described previously (Artursson et al., 1993).

A. Wikman-Larhed, P. Artursson / European Journal of Pharmaceutical Sciences'3 (1995) 171-183

179

correlation between Caco-2 monolayers (cultured in DMEM) and human colon was established (Artursson et al., 1993). The tight junction permeability of the cell monolayers was also investigated using structurally different hydrophilic marker molecules with molecular weights ranging from 362 to 5000 g/ mole (Table 2). As for the polyethylene glycols, a non-linear dependence on molecular weight was observed, with the permeabilities of the monolayers decreasing in the order HT29-H> co-culture > Caco-2. The exception was the tripeptide TRH which was transported across Caco2 monolayers at a higher rate than expected from its molecular weight (Table 2). However the difference in permeability between Caco-2 and HT29-H monolayers was generally smaller than observed for the polyethylene glycols, suggesting that the characteristics of the largest junctional pores are comparable in the two cell types.

The HT29-H mono-cultures and the co-cultures were 3.0-13.6 and 1.6-5.0 times more permeable to the polyethylene glycols, respectively, than the Caco-2 mono-cultures. The non-linear dependence on molecular weight was generally comparable for HT29-H and Caco-2 monolayers, suggesting that the distribution of the junctional pores (the assumed routes of aqueous communication in the tight junctions) was roughly similar between the two cell populations. Thus, permeability showed a strong dependence on molecular weight for polyethylene glycols smaller than approximately 300 g/mole while in the 300-500 g/mole interval permeability was constant, consistent with the presence of a larger population of small pores and a smaller population of large pores. Comparisons with published permeability profiles of polyethylene glycols in human colon in vivo (Chadwick et al., 1977) showed that the human colon was more permeable than the cell monolayers (Fig. 5 insert). In addition, the dependence on molecular weight was different in the human colon since a clear molecular weight dependence could be observed up to a molecular weight of 400-500 g/mole (Fig. 5 insert). As a result, the differences in the permeability of polyethylene glycols between the cell cultures and human colon in vivo were relatively small at higher molecular weights but increased with decreasing molecular weight. This suggests that the aqueous pore distributions are different in the cell lines from those in human colon. This is in contrast to our previous studies, where the permeability profiles were comparable and a good

4. Discussion

Clones derived from the parental HT29 cell line, including the well characterised HT29-18N 2 goblet cell clone, generally grow in normal glucose-containing cell culture medium as undifferentiated multilayers (Phillips et al., 1988). However, the HT29-H goblet cell clone forms well differentiated monolayers with tight junctions consisting of about 80% mature goblet cells in glucose-containing DMEM (Wikman et al., 1993). Of these monolayers, only a fraction form a leak proof barrier with a measurable trans-

Table 2 Permeability coefficients of hydrophilic molecules in m o n o - and co-cultures a Pal'l, X 106 (cm/s) Substance

MW

Caco-2

Co-culture

HT29-H

TRH Raffinose dDAVP Inulin

362.4 594.4 1069.2 ~5000

1.07 0.34 0.i0 0.44

0.98 0.56 0.23 0.62

2.36 1.02 0.61 0.65

--- 0.38 ± 0.03 --- 0.05 - 0.04

-4- 0.13 ± 0.12 +- 0.06 ... 0.02

--- 0.92 --- 0.15 - 0.18 --- 0.09

~ T h e results are expressed as m e a n ---SD (n = 4). E x p e r i m e n t s were p e r f o r m e d on 25- to 26-day-old monolayers, with 28.1 +- 4.2% of the area covered with H T 2 9 - H cells. The permeability coefficients of the molecules d e p e n d e d significantly on the proportion of H T 2 9 - H cells in the monolayers (one-way A N O V A , p < 0.015).

180

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epithelial electrical resistance. Since parental HT29 cells have recently been shown to differentiate rapidly in RPMI medium without any signs of cell death (Polak-Charcon et al., 1989), we investigated if this simple approach could be used to obtain monolayers of HT29-H cells with acceptable barrier properties. Adaptation and cultivation of HT29-H in RPMI consistently gave confluent monolayers with a lower permeability than that reported for cells grown in DMEM. However, the morphologies of the monolayers cultivated in DMEM and RPMI were comparable. This result is in agreement with previous studies in the renal epithelial cell line MDCK, which suggested that morphological differentiation is unrelated to the regulation of tight junction permeability (Stevenson et al., 1989). The components of the RPMI that induced the formation of confluent monolayers are unknown but could involve inositol (Polak-Charcon et al., 1989). RPMI may also affect cell differentiation by regulating the rate of glucose consumption, as suggested previously (Zweibaum et al., 1985). We conclude that the problem of obtaining confluent monolayers of HT29-H under standard cell culture conditions can be solved by changing the medium from DMEM to RPMI. HT29-H monolayers grown in DMEM secrete mucin molecules and form a visible mucus layer on the apical side (Wikman et al., 1993). This mucus layer functions as a diffusion barrier to the lipophilic and uncharged drug testosterone (Karlsson et al., 1993b). However, HT29-H monolayers grown in RPMI did not form a visible mucus layer and had a permeability to testosterone that was comparable to that of Caco-2 cells (which do not form a mucus layer), indicating that RPMI-grown HT29-H monolayers do not form a functional" mucus layer. The reasons for this difference are not clear but could possibly be related to an increased differentiation of HT29-H in RPMI (Polak-Charcon et al., 1989), since recent studies on human colon cancer cell lines suggest that mucin synthesis decreases during differentiation in post confluent cultures (Niv et al., 1992). Intestinal goblet cells mix with other cell types as they migrate from the crypt to the villus tips (Bjerknes and Cheng, 1985; Goralski et al.,

1975). This is in contrast to the limited mixing between HT29-H and Caco-2 cells in the cocultures. However, goblet cell clusters may form in vivo as a result of that goblet cells are more resistant to injury than other epithelial cells (Bryan et al., 1980; Matsuyama et al., 1970). Thus, differential loss of epithelial cells and apposition of retained goblet cells as a result of migration pressure from the crypts reconstitute the integrity of the epithelium. Since epithelia generally resurface by sheet migration, the mixing of the retained goblet cells with other cells will be limited, resulting in goblet cell clusters comparable to those found in the co-cultures in the present study (Basson et al., 1992; Bryan et al., 1980). Recent studies show that Caco-2 cells migrate rapidly as sheets on collagen and that the migration is affected by EGF (Basson et al., 1992). It is therefore possible that the formation of HT29-H clusters in the co-cultures was caused in part by migration pressure generated by sheet migration of Caco-2 cells on collagen, as well as by the more rapid cell division of the HT29-H cells themselves. Studies where the migration and cell division of the two cell types are followed with time will resolve this issue. The number of HT29-H cells per unit surface area was higher than that for the Caco-2 cells, indicating that HT29-H monolayers had a longer junctional path length. Since tight junction permeability is dependent not only on the specific permeability of the tight junction but also on the junctional path length (Claude, 1978), it was possible that the difference in permeability between the two cell lines was related to the different junctional path lengths rather than a difference in the specific permeability of the tight junctions (Marcial et al., 1984). If we assume that the cells are hexagonal (Claude, 1978), the path lengths for HT29-H and Caco-2 cells become 23.2 m/cm 2 and 13.7 m/cm 2, respectively. These values are in the vicinity of the 21.8 m/cm 2 calculated for villus enterocytes in guinea pig ileum (Marcial et al., 1984). The specific tight junction permeability, adjusted for junctional path length, was calculated for each cell line. The length specific permeability in HT29-H cells was 1.6 times higher for ions and 4.7 times higher for mannitol compared to that in Caco-2 cells, in-

A. Wikman-Larhed, P. Artursson I European Journal of Pharmaceutical Sciences'3 (1995) 171-183

dicating that there is a real difference in the tight junction permeability between the absorptive and goblet cell lines. The formation of tight junctions between Caco-2 and HT29-H cells was verified by electron microscopy. To our knowledge this is the first demonstration that tight junctions can form between different intestinal epithelial cell types from different cell lines. Since HT29-H and Caco-2 are derived from different human subjects this finding opens up the possibility of developing co-cultures for drug transport studies with other well-differentiated human intestinal epithelial cells. The change of culture medium reduced the difference in permeability between the two cell lines. As a result, the permeability of the cocultures could not be increased to that of the human colon in vivo. The main advantage of the co-cultures for studies of epithelial transport is therefore that they comprise the cellular barriers of the two major epithelial cell populations, as well as carriers and receptors for nutrients, macro-molecules and drugs (expressed by Caco2) in the same monolayer. However, further optimisation of the cell culture conditions are needed in order to stimulate the production of an extra cellular mucus layer. Alternatively, a supplemental apical mucus layer from an external source may be applied [if insufficient quantities of mucus is produced by RPMI-grown HT29-H cells also after further optimisation of the cell culture conditions (Cepinskas et al., 1993)]. In conclusion, the co-cultures offers a model where absorption and goblet cells can be studied simultaneously in studies on drug transport and absorption enhancement.

Acknowledgements We thank Mr. Tapio Nikkil/i for preparing the electron micrographs, Ms. Lena Lundberg for technical assistance, Dr. Daniel Louvard, Institut Pasteur, Paris for providing the HT29-H cell line and Dr. John Allen for valuable advice regarding the identification of HT29-H and Caco-2 cells in co-culture. This work was supported by grants from The Swedish Medical Research Council (94-

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78). The Swedish Fund for Research without Animal Experiments, Centrala F6rs6ksdjursn/imnden (93-11) and The Swedish Academy of Pharmaceutical Sciences.

References Allen, J.D., Martin, G.P., Marriott, C., Hassan, I. and Williamson, I. (1991) Drug transport across a novel mucin secreting cell model: comparison with the Caco-2 cell system. J. Pharm. Pharmacol. 43, 63P. Anderberg, E.K., Nystr6m, C. and Artursson, P. (1992) Epithelial transport of drugs in cell culture. VII. Effects of pharmaceutical surfactant excipients and bile acids on transepithelial permeability in monolayers of human intestinal epithelial (Caco-2) cells. J. Pharm. Sci. 81, 879887. Artursson, P. and Karlsson, J. (1991) Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco2) cells. Biochem. Biophys. Res. Commun. 175,880-885. Artursson, P., Ungell, A.-L. and L6froth, J.-E. (1993) Selective paracellular permeability in two models of intestinal absorption: cultured monolayers of human intestinal epithelial cells and rat intestinal segments. Pharm. Res. 10, 1123-1129. Augeron, C. and Laboisse, C.L. (1984) Emergence of permanently differentiated cell clones in a human colonic cancer cell line in culture after treatment with sodium butyrate. Cancer Res. 44, 3961-3969. Basson, M.D., Modlin, I.M. and Madri, J.A. (1992) Human enterocyte (Caco-2) migration is modulated in vitro by extracellular matrix composition and epidermal growth factor. J. Clin. Inv. 90, 15-23. Bjerknes, M. and Cheng, H. (1985) Mucous cells and cell migration in the mouse duodenal epithelium. Anat. Rec. 212, 69-73. Blais, A., Bissonnette, P. and Berteloot, A. (1987) Common characteristics for Na÷-dependent sugar transport in Caco-2 cells and human fetal colon. J. Membrane Biol. 99, 113-125. Bryan, A.J., Kaur, R., Robinson, G., Thomas, N.W. and Wilson, C.G. (1980) Histological and physiological studies on the intestine of the rat exposed to solutions of Myrj 52 and PEG 2000. Int. J. Pharm. 7, 145-156. Cepinskas, G., Specian, R.D. and Kvietys, P.R. (1993) Adaptive cytoprotection in the small intestine: role of mucus. Am. J. Physiol. 264, G921-G927. Chadwick, V.S., Phillips, S.F. and Hofmann, A.F. (1977) Measurements of intestinal permeability using low molecular weight polyethylene glycols (PEG 400) II. Application to normal and abnormal permeability states in man and animals. Gastroenterology 73, 247-251. Claude, P. (1978) Morphological factors influencing transepithelial permeability. J. Membrane Biol. 39, 219-232.

182

A. Wikman-Larhed, P. Artursson / European Journal of Pharmaceutical Sciences 3 (1995) 171-183

Dix, C.J., Hassan, I.F., Obray, H.Y., Shah, R. and Wilson, G. (1990) The transport of vitamin B12 through polarized monolayers of Caco-2 cells. Gastroenterology 98, 12721279. Fogh, J., Fogh, J.M. and Orfeo, T.J. (1977) One hundred and twenty seven cultured human tumor cell lines producing tumors in nude mice. J. Natl. Cancer Inst. 59, 221226. Fuller, S.D. and Simons, K. (1986) Transferrin receptor polarity and recycling accuracy in 'tight' and 'leaky' strains of Madin-Darby canine kidney cells. J. Cell Biol. 103, 1767-1779. Gonz~lez-Mariscal, L., Chfivez de Ramirez, B., Lfizaro, A. and Cereijido, M. (1989) Establishment of tight junctions between cells from different animal species and different sealing capacities. J. Membrane Biol. 107, 43-56. Goralski, A., Sawicki, W. and Blaton, O. (1975) Nonrandom distribution of goblet cells around the circumference of colonic crypt. Cell Tissue Res. 160, 551-556. Hafez, M.M., Infante, D., Winawer, S. and Friedman, E. (1990) Transforming growth factor /~1 acts as an autocrine-negative growth regulator in colon enterocytic differentiation but not in goblet cell maturation. Cell Growth Differ. 1, 617-626. Hanski, C., Stolze, B. and Riecken, E.O. (1992) Tumorigenicity, mucin production and AM-3 epitope expression in clones selected from the HT-29 colon carcinoma cell line. Int. J. Cancer 50, 924-929. Huet, C., Sahuquillo-Merino, C., Coudrier, E. and Louvard, D. (1987) Absorptive and mucus-secreting subclones isolated from a multipotent intestinal cell line (HT29) provide new models for cell polarity and terminal differentiation. J. Cell Biol. 105, 345-357. Karlsson, J., Kuo, S.-M., Ziemniak, J. and Artursson, P. (1993a) Transport of celiprolol across human intestinal epithelial (Caco-2) cells: secretion is mediated by multiple transporters including P-glycoprotein. Br. J. Pharmacol. 110, 1009-1016. Karlsson, J., Wikman, A. and Artursson, P. (1993b) The mucus layer as a barrier to drug absorption in monolayers of human intestinal epithelial HT29-H goblet cells. Int. J. Pharrn. 99, 209-218. Kreusel, K.-M., Fromm, M., Schulzke, J.D. and Hegel, U. (1991) C1- secretion in epithelial monolayers of mucus forming human colon cells. Am. J. Physiol. 261, C574C582. Kulenkampff, H. (1975) Gastrointestinal absorption of drugs. In: Forth, W. and Rumnel, W. (Eds.) Pharmacology of Intestinal Absorption. Springer, Berlin. Lacy, E.R. (1991) Functional morphology of the large intestine. In: Schultz, S., Field, M., Frizzell, M. and Rauner, B. (Eds.) The Gastrointestinal System. Oxford University Press, New York, pp. 121-194. Lesuffleur, T., Barbat, A., Dussaulx, E. and Zweibaum, A. (1990) Growth adaptation to methotrexate of HT29 human colon carcinoma cells is associated with their ability to differentiate into columnar absorptive and mucus secreting cells. Cancer Res. 50, 6334-6343.

Lundin, S. and Artursson, P. (1990) Absorption of a vasopressin analogue, 1-deamino-8-D-arginine-vasopressin (dDAVP), in a human intestinal epithelial cell line. Int. J. Pharm. 64, 181-186. Madara, J. L. and Trier, J. S. (1982) Structure and permeability of goblet cell tight junctions in rat small intestine. J. Membrane Biol. 66, 145-157. Marcial, M.A., Carlson, S.L. and Madara, J.L. (1984) Partitioning of paracellular conductance along the ileal crypt-villus axis: a hypothesis based on structural analysis with detailed consideration of tight junction structurefunction relationship. J. Membrane Biol. 80, 59-70. Matsuyama, M. and Suzuki, H. (1970) Differentiation of immature mucous cells into parietal, argyrophil, and chief cells in stomach grafts. Science 169, 385-387. Matter, K., Brauchbar, M., Bucher, K. and Hauri, H.-P. (1990) Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cell (Caco-2). Cell 60, 429-437. Mostov, K., Apodaca, G., Aroeti, B. and Okamoto, C. (1992) Plasma membrane sorting in polarized epithelial cells. J. Cell Biol. 116, 577-583. Neutra, M. and Louvard, D. (1989) Differentiation of intestinal cells in vitro. In: Matlin, K.S. and Valentich, J.D. (Eds.) Modern Cell Biology. Alan R. Liss, New York, pp. 363-398. Niv, Y., Byrd, J.C., Ho, S.B., Dahiya, R. and Kim, Y.S. (1992) Mucin synthesis and secretion in relation to spontaneous differentiation of colon cancer cells in vitro. Int. J. Cancer 50, 147-152. Phillips, T.E., Huet, C., Bilbo, P.R., Podolsky, D.K., Louvard, D. and Neutra, M.R. (1988) Human intestinal goblet cells in monolayer culture: characterisation of a mucus-secreting subclone derived from the HT29 colon adenocarcinoma cell line. Gastroenterology 94, 13901403. Pinto, M., Appay, M.D., Simon-Assman, P., Chevalier, G., Dracopoli, N., Fogh, J. and Zweibaum, A. (1982) Enterocytic differentiation of cultured human colon cancer cells by replacement of glucose by galactose in the medium. Biol. Cell 44, 193-196. Pinto, M., Robine-Leon, S., Appay, M.-D., Kedinger, M., Triadou, N., Dussaulx, E., Lacroix, B., Simon-Assman, P., Haffen, K., Fogh, J. and Zweibaum, A. (1983) Enterocyte-like differentiation and polarization of the human colon carcinoma cell line Caco-2 in culture. Biol. Cell 47, 323-330. Polak-Charcon, S., Hekmati, M. and Ben-Shaul, Y. (1989) The effect of modifying the culture medium on cell polarity in a human colon carcinoma cell line. Cell Differ. Dev. 26, 119-129. Robards, A.W. and Wilson, A.J. (1993) Basic biological preparation techniques for TEM. In: Procedures in Electron Microscopy. Wiley, Chichester, UK. Roumagnac, I. and Laboisse, C. (1987) A mucus-secreting human colonic epithelial cell line responsive to cholinergic stimulation. Biol. Cell 61, 65-68. Stevenson, B.R., Anderson, J.M., Braun, I.D. and

A. Wikman-Larhed, P. Artursson / European Journal of Pharmaceutical Sciences 3 (1995) 171-183

Mooseker, M.S. (1989) Phosphorylation of the tight junction protein ZO-1 in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance. Biochem. J. 263, 597-599. Wikman, A., Karlsson, J., Carlstedt, I. and Artursson, P. (1993) A drug absorption model based on the mucus layer producing human intestinal goblet cell line HT29-H. Pharm. Res. 10, 843-852. Woodcock, S., Williamson, I., Hassan, I. and Mackay, M.

183

(1991) Isolation and characterisation of clones from the Caco-2 cell line displaying increased taurocholic acid transport. J. Cell. Sci. 98, 323-332. Zweibaum, A., Pinto, M., Chevalier, G., Dussaulx, E., Triadou, N., Lacroix, B., Haffen, K., Brun, J.-L. and Rousset, M. (1985) Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT29 selected for growth in sugar-flee medium and its inhibition by glucose. J. Cell Physiol. 122, 21-29.