Assessment of different fixation protocols on the presence of membrane-bound vesicles in Caco-2 cells: a multidimensional view by means of correlative light and 3-D transmission electron microscopy.

Herein, we present a comparative analysis of a variety of chemical and physical fixation protocols for the specific visualisation of the membrane-bound vesicles (MBVs) in the Caco-2 colorectal cancer (CRC) cell line. In so doing, we validated the applicability of specific specimen preparation protocols for the preservation and contrasting of membrane-associated vesicles. Next, by employing the best respective chemical (GOT) and physical (SHPF) fixation methods for the application of transmission electron tomography and modelling we were able to characterise MBVs in three-dimensions and at the nanometer scale. In the second part of this study, we employ a correlative light and electron microscopy (CLEM) approach in order to determine which vesicular compartments are implicated in the uptake of FITC-BSA as a model protein drug. In so doing, we provide a solid foundation for future studies investigating chemotherapeutic drug uptake, transport and fate in cancer cell lines.

Ultrastructural aspects of melatonin cytotoxicity on Caco-2 cells in vitro.

Colon adenocarcinoma is a disease expanding worldwide. Cancer of colon and rectum are among the top ten most insidious types in Brazil. In vitro and in vivo studies have demonstrated the efficacy of the hormone melatonin to prevent and reduce tumor growth. However, there are only few studies addressing the action of melatonin on Caco-2 cells. Thus, the cytotoxic effect of melatonin on the ultrastructure of Caco-2 cells was investigated. The MTT colorimetric method was used to assess the cytotoxicity. A total of 2×10(6)cells/mL were seeded in microplates and incubated at 50, 25, 12.5, 6.25, 3.125, 1.56, 0.78 and 0.0 (control) µg/mL of melatonin. For ultrastructural analysis concentrations with low, medium and high cytotoxicity plus the control were used for ultrastructural analysis. The concentrations 50, 1.56 and 0.78 µg/mL of melatonin showed low, medium and high cytotoxicity, respectively. Ultrastructurally, the control tumor cells were shown to be preserved. Caco-2 cells showed morphological changes at 50 µg/mL of melatonin, with numerous vacuoles, mitochondrial degeneration and reduced glycogen. However, Caco-2 cells also showed altered morphology in treatments at 1.56 and 0.78 µg/mL of melatonin with characteristics of cells in degeneration by the presence of numerous vacuoles, absence of microvilli, mitochondrial degeneration and nuclear fragmentation. Thus, one can infer that concentrations of 1.56 and 0.78 µg/mL of melatonin promote cytotoxicity in Caco-2 cells, which can probably be related to the generation of reactive oxygen species (ROS).

Dulbecco's modified eagle's medium and minimum essential medium--which one is more preferred for establishment of Caco-2 cell monolayer model used in evaluation of drug absorption?

The Caco-2 cell monolayer model is widely used in drug absorption studies. Dulbecco's Modified Eagle's Medium (DMEM) and Minimum Essential Medium (MEM) have been used alternatively in the development of this model, although they are different in composition which may affect the differentiation and junction formation of the Caco-2 cell monolayer. Two Caco-2 cell monolayers cultured in both media were compared herein in order to underlay the standardization of this model. These two monolayers were comparatively evaluated regarding reliability and stability by morphology, transepithelial electrical resistance (TEER), alkaline phosphatase (AKPase) activity and transport experiments. Although the results showed that characteristic microvilli were present at the apical side of both monolayers, the dynamic change of TEER of the monolayer cultured in DMEM was more stable than that cultured in MEM, and AKPase activity of the former was stronger than that of the latter. Furthermore, the quantity of atenolol, a key indicator usually used for assessment of this model, across the monolayer cultured in MEM was significantly more than that cultured in DMEM. Therefore, the Caco-2 monolayer cultured in DMEM was more reliable and stable than that cultured in MEM, and thus the former was preferred for drug absorption investigation in vitro.

Entrapment of intracytosolic bacteria by septin cage-like structures.

Actin-based motility is used by various pathogens for dissemination within and between cells. Yet host factors restricting this process have not been identified. Septins are GTP-binding proteins that assemble as filaments and are essential for cell division. However, their role during interphase has remained elusive. Here, we report that septin assemblies are recruited to different bacteria that polymerize actin. We observed that intracytosolic Shigella either become compartmentalized in septin cage-like structures or form actin tails. Inactivation of septin caging increases the number of Shigella with actin tails and enhances cell-to-cell spread. TNF-α, a host cytokine produced upon Shigella infection, stimulates septin caging and restricts actin tail formation and cell-to-cell spread. Finally, we show that septin cages entrap bacteria targeted to autophagy. Together, these results reveal an unsuspected mechanism of host defense that restricts dissemination of invasive pathogens.

Macrophages increase microparticle uptake by enterocyte-like Caco-2 cell monolayers.

Caco-2 cells form an enterocyte-like monolayer that has been used to explore small intestinal microparticle uptake. They are a useful functional model for the investigation of in vivo drug delivery systems and the uptake of particulate environmental pollutants. The aim of this paper was to determine if the previously reported decrease in Caco-2 transepithelial resistance following exposure to macrophages was matched by increased microparticle uptake, especially as macrophage phagocytosis simulates removal of particles from the subepithelial compartment. Caco-2 cells were grown as a monoculture for 21 days on insert membranes. A compartmentalised model involved Caco-2 cells in the upper compartment, with THP-1-derived macrophages adhering to the base of the underlying well, the two cell populations communicating only through the shared culture medium. Caco-2 cells were also cultured in macrophage-conditioned medium and all groups were exposed apically to 2 µm latex particles for 5 or 60 min. Parameters measured were: transepithelial resistance; cytokine levels; cell dimensions and the distribution of nuclei, actin and junctional proteins. Subepithelial particle numbers, defined as those located below the insert membrane, were also counted and were significantly increased in the Caco-2/macrophage model, with over 90% associated with the macrophages. Other changes induced by the presence of macrophages included decreased transepithelial resistance levels, diffuse localisation of some junctional proteins, higher proinflammatory cytokine levels, disorganisation of cell shape and decreased cell height associated with actin reorganisation. Macrophage-conditioned medium produced a smaller transepithelial resistance decrease than the Caco-2/macrophage model and there were few other changes. In conclusion, culture of Caco-2 cells with underlying macrophages produced a lower, less organised epithelium and greater microparticle uptake.

Unlocking the ultrastructure of colorectal cancer cells in vitro using selective staining.

AIM: To characterise differences between three widely used colorectal cancer cell lines using ultrastructural selective staining for glycogen to determine variation in metastatic properties. METHODS: Transmission electron microscopy was used in this investigation to help identify intracellular structures and morphological features which are precursors of tumor invasion. In addition to morphological markers, we used selective staining of glycogen as a marker for neoplastic cellular proliferation and determined whether levels of glycogen change between the three different cell lines. RESULTS: Ultrastructural analysis revealed morphological differences between the cell lines, as well as differentiation into two sub-populations within each cell line. Caco-2 cells contained large glycogen deposits as well as showing the most obvious morphological changes between the two sub-populations. SW480 cells also contained large glycogen stores as well as deep cellular protrusions when grown on porous filter membranes. HT-29 cells had trace amounts of glycogen stores with few cellular projections into the filter pores and no tight junction formation. CONCLUSION: Morphology indicative of metastatic properties coincided with larger glycogen deposits, providing strong evidence for the use of selective staining to determine the neoplastic properties of cells.

Changes in macromolecular transport appear early in Caco-2 cells infected with a human rotavirus.

OBJECTIVE: Rotavirus is a major cause of viral gastroenteritis, but its interaction with intestinal mucosa is poorly understood. The aim of this study was to examine the effect of Wa rotavirus (VP7 serotype 1) on barrier function in confluent Caco-2 cell monolayers. Wa is the most common serotype causing severe diarrhoea in humans. MATERIAL AND METHODS. We examined light and electron microscopic morphology, macromolecular transport, paracellular permeability, electrical parameters, disaccharidases and cytoskeletal structure in Wa- and in control sham-infected cells using a homologous human virus-cell system resembling human infection. RESULTS: During the first 48 h following Wa infection, there was no evidence of loss of integrity or of cytopathic effect in the monolayer. A significant cytopathic effect was noticed after 48 h. Further studies examined the initial 24-h period during which there was no evidence of significant injury. Apical-to-basolateral transcytosis of the macromolecule horseradish peroxidase (HRP) was selectively inhibited at 4 and 24 h post-infection with Wa. There were no significant changes in basolateral-to-apical transcytosis, endocytosis or in apical-to-apical recycling of HRP after Wa infection. G- and F-actin levels were significantly reduced within an area corresponding to the viroplasm in Wa-infected cells but not elsewhere in the monolayer. CONCLUSIONS: The early stages of rotavirus infection, before gross epithelial injury, are associated with a selective reduction in the apical uptake and transcytosis of macromolecules. We speculate that this is an epithelial defence mechanism.

Kefiran antagonizes cytopathic effects of Bacillus cereus extracellular factors.

Kefiran, the polysaccharide produced by microorganisms present in kefir grains, is a water-soluble branched glucogalactan containing equal amounts of D-glucose and D-galactose. In this study, the effect of kefiran on the biological activity of Bacillus cereus strain B10502 extracellular factors was assessed by using cultured human enterocytes (Caco-2 cells) and human erythrocytes. In the presence of kefiran concentrations ranging from 300 to 1000 mg/L, the ability of B. cereus B10502 spent culture supernatants to detach and damage cultured human enterocytes was significantly abrogated. In addition, mitochondrial dehydrogenase activity was higher when kefiran was present during the cell toxicity assays. Protection was also demonstrated in hemolysis and apoptosis/necrosis assays. Scanning electron microscopy showed the protective effect of kefiran against structural cell damages produced by factors synthesized by B. cereus strain B10502. Protective effect of kefiran depended on strain of B. cereus. Our findings demonstrate the ability of kefiran to antagonize key events of B. cereus B10502 virulence. This property, although strain-specific, gives new perspectives for the role of bacterial exopolysaccharides in functional foods.

New methodological approach to induce a differentiation phenotype in Caco-2 cells prior to post-confluence stage.

BACKGROUND: Various differentiation-inducing agents or harvesting of spontaneously late post-confluence cultures have been used to differentiate the human colon carcinoma Caco-2 cell line. We report a new procedure to generate pre-confluent subcultures of Caco-2 population at various stages of differentiation without altering culture conditions. MATERIALS AND METHODS: Ultrastructural analysis, cell proliferation activity and biochemical markers of differentiation were evaluated at different passages. RESULTS: Subcultures of Caco-2 cells at pre-confluence, exhibiting progressive acquisition of a more benign differentiation phenotype, were generated. Early passages of Caco-2 cells showed a well-developed brush border and incomplete junctional apparatus; subsequent subcultures yielded cell populations with well-developed junctions similar to those of small intestinal cells. CONCLUSION: These culture conditions represent a new versatile model not only to progressively induce the differentiation program in Caco-2 cells at pre-confluence without changes of culture media, but also to explore mechanistic modes of drug transport and tumor development.

PATJ connects and stabilizes apical and lateral components of tight junctions in human intestinal cells.

The Crumbs complex that also contains the cortical proteins Stardust and DPATJ (a homologue of PATJ), is crucial for the building of epithelial monolayers in Drosophila. Although loss of function of the Crumbs or Stardust genes prevents the stabilization of a belt of adherens junctions at the apico-lateral border of the cells, no phenotype has been described for the Dpatj gene and its role in epithelial morphogenesis and polarity remains unknown. We have produced downregulated PATJ stable lines of Caco2 to clarify its role in epithelial morphogenesis. In PATJ knockdown cells, Pals1 (a Stardust homologue) is no longer associated with tight junctions whereas Crumbs3 (Crb3) is accumulated into a compartment spatially close to the apical membrane and related to early endosomes. Furthermore, occludin and ZO-3, two proteins of tight junctions are mislocalized on the lateral membrane indicating that PATJ plays a novel role in the building of tight junctions by providing a link between their lateral and apical components. Thus, PATJ stabilizes the Crb3 complex and regulates the spatial concentration of several components at the border between the apical and lateral domains.

Influence of polarisation and differentiation on interaction of 43-kDa outer-membrane protein of Aeromonas caviae with human enterocyte-like Caco-2 cell line.

It has been recognised that adherence and invasion to host cells are important steps in the pathogenesis of entero-pathogenic bacteria, including Aeromonas caviae. However, the virulence factors of A. caviae remain, for the most part, poorly known. This study examined the interaction of A. caviae isolates to Caco-2 cells in different polarisation and differentiation conditions. The adherence of A. caviae may be related to accessibility of host cell basolateral receptors. Aggregative A. caviae isolates, grown at 22 degrees C, were more adherent in both non-polarised and undifferentiated Caco-2 cells and EGTA-treated polarised and differentiated Caco-2 cells. Furthermore, monolayers pre-incubated with 43-kDa outer-membrane protein (OMP) or A. caviae strains pre-incubated with rabbit IgG anti-43-kDa OMP decreased adherence of some A. caviae strains to EGTA-treated polarised and differentiated Caco-2 cells, suggesting an interaction of 43-kDa OMP with basolateral cell receptors. Bacterial cells were observed adhering to microvilli and to plasma membrane on both the apical and basal surfaces of the monolayer. Pedestal-like formation with cytoskeletal rearrangement was also observed. The bacteria entered the Caco-2 cells and were observed enclosed in single and multiple membrane-bound vacuoles within the host cell cytoplasm. Furthermore, A. caviae were observed free in the cytosol of Caco-2 cells, suggesting escape form cytoplasmatic vacuoles.

Glutamine deprivation facilitates tumour necrosis factor induced bacterial translocation in Caco-2 cells by depletion of enterocyte fuel substrate.

BACKGROUND AND AIMS: Factors that induce luminal bacteria to cross the intestinal epithelium following injury remain poorly defined. The aim of this study was to investigate the interaction between glutamine metabolism, energy supply, and inflammatory mediators in determining the translocation of non-pathogenic bacteria across cultured enterocytes. METHODS: The effect of tumour necrosis factor alpha (TNF-alpha) on translocation of Escherichia coli C25 across Caco-2 epithelial monolayers was studied in the presence of products and inhibitors of glutamine metabolism. Simultaneous measurements of transepithelial electrical resistance (TEER) and flux of lucifer yellow were used to assess effects on the paracellular pathway. Lactate dehydrogenase release was used to monitor enterocyte integrity. Imaging of monolayers in these experimental conditions was undertaken with transmission electron microscopy. RESULTS: Exposure to basolateral TNF-alpha (20 ng/ml) for six hours induced translocation of E coli across Caco-2 but only if accompanied by simultaneous glutamine depletion (p<0.01). Translocation was inhibited by addition of glutamine for two hours (p<0.01) but not by an isonitrogenous mixture of non-glutamine containing amino acids. Inhibition of glutamine conversion to alpha-ketoglutarate, but not blockade of glutathione or polyamine synthesis, also induced translocation in the presence of TNF-alpha. Manipulations that induced bacterial translocation were associated with a marked reduction in enterocyte ATP levels. No effect of these treatments on paracellular permeability or lactate dehydrogenase release was observed. Conditions in which translocation occurred were associated with the presence of bacteria within enterocyte vacuoles but not the paracellular space. CONCLUSIONS: In inflammatory conditions, the availability of glutamine as an enterocyte fuel substrate is essential for the preservation of a functional barrier to microorganisms. In conditions of acute glutamine depletion, cytokine mediated bacterial translocation appears to be primarily a transcellular process.

Adherence of yeast and filamentous forms of Candida albicans to cultured enterocytes.

OBJECTIVE: Systemic candidiasis is a major cause of complicating infections in intensive care units. Morbidity and mortality are high, even in those who receive appropriate antifungal therapy. Because the intestinal tract is considered a major portal of entry for systemic candidiasis, experiments were designed to clarify the ability of yeast and filamentous forms, as well as the INT1 gene product, to influence adherence of Candida albicans to the intestinal epithelium. DESIGN: Controlled. SETTING: University teaching hospital research laboratory. SUBJECTS: Mature Caco-2 and HT-29 cultured enterocytes. INTERVENTIONS: C. albicans INT1 mutant strains, defective in filament production, were used to observe the ultrastructural surface interactions of C. albicans with cultured intestinal epithelial cells, namely Caco-2 and HT-29 cells. These mutant strains also were used to quantify the effect of the INT1 gene product on C. albicans adherence (yeast and filamentous forms) to cultured enterocytes. Ultrastructural surface interactions of C. albicans with cultured enterocytes were observed with high resolution scanning electron microscopy. C. albicans adherence to cultured enterocytes was quantified by using a colorimetric enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Both yeast and filamentous forms of C. albicans appeared tightly adherent to the apical surface of cultured enterocytes, and INT1 appeared to have little, if any, effect on these ultrastructural surface interactions. The distal ends of C. albicans filaments appeared to mediate adherence to enterocyte apical microvilli, and thigmotropism (contact guidance) appeared to play a role in C. albicans adherence. The absence of functional INT1 was associated with decreased adherence of C. albicans yeast forms to cultured enterocytes. CONCLUSIONS: Although functional INT1 appeared to facilitate adherence of C. albicans yeast forms to cultured enterocytes, the role of INT1 in adherence of filamentous forms was unclear, and both yeast and filamentous forms could adhere to, and perhaps invade, the apical surface of cultured enterocytes.

Effect of Bacillus cereus exocellular factors on human intestinal epithelial cells.

To gain insight on the biological effects of the exocellular factors produced by Bacillus cereus, culture filtrate supernatants of different strains were coincubated with differentiated Caco-2 cells. Exocellular factors were able to detach enterocyte-like cells from the substratum after 1 h of incubation. In addition, microvilli effacing and dramatic changes on the cellular surface of enterocytes were found after incubation periods as short as 20 min. Since cell detachment was not inhibited by fetal calf serum, thiol activated cholesterol-binding cytolysin, cereolysin O, does not seem to be involved. Also, translocation of phosphatidylserine from the inner to the outer leaflets of the plasma membrane was demonstrated by using fluorescein isothiocyanate (FITC)-Annexin V. In contrast to the high capability of detaching Caco-2 cells shown by all the strains under study, the mitochondrial dehydrogenase activity was lowered by culture filtrate supernatants in a strain-dependent manner. For strain M2, the decrease in dehydrogenase activity was already evident after 30 min of incubation. Production of biologically active factors depends on the growth phase, and maximal activity was found in late exponential-early stationary phases. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of concentrated exocellular factors showed a very complex scenery supporting the multifactorial character of the biological activity of B. cereus.

Characteristics of L-glutamine transport during Caco-2 cell differentiation.

Glutamine is the main fuel of intestinal epithelial cells, as well as a precursor for the intense nucleotide biosynthesis which arises with the rapid turnover of enterocytes. In order to determine whether glutamine uptake may vary as a function of metabolic demand, glutamine transport across the brush-border membrane of differentiating Caco-2 cells has been investigated. The uptake of L-[(3)H]glutamine was measured between day 7 and day 21 post-seeding. Kinetic analysis with glutamine concentrations ranging from 6.25 microM to 12.8 mM revealed the involvement of high affinity Na(+)-dependent (K(t)=110 microM) and low affinity Na(+)-independent (K(t)=900 microM) transport components at day 7. Both components were partially inhibited by L-lysine in a competitive fashion, suggesting that four different systems were responsible for glutamine uptake: B(0), B(0,+), b(0,+) and L. All four systems were present during the differentiation process, with systems L and B(0) being responsible for up to 80% of glutamine uptake. Caco-2 cell differentiation was associated with a marked decrease in L-glutamine uptake, which affected both the Na(+)-dependent and the Na(+)-independent components. In contrast to glucose uptake, the development of L-glutamine uptake across the brush-border membrane of Caco-2 cells may reflect an adjustment to cell metabolic demand rather than the progressive appearance of a vectorial transport process.

Infection of human enterocyte-like cells with rotavirus enhances invasiveness of Yersinia enterocolitica and Y. pseudotuberculosis.

Mixed infection with rotavirus and either Yersinia enterocolitica or Y. pseudotuberculosis was analysed in Caco-2 cells, an enterocyte-like cell line highly susceptible to these pathogens. Results showed an increase of bacterial adhesion and internalisation in rotavirus-infected cells. Increased internalisation was also seen with Escherichia coli strain HB101 (pRI203), harbouring the inv gene from Y. pseudotuberculosis, which is involved in the invasion process of host cells. In contrast, the superinfection with bacteria of Caco-2 cells pre-infected with rotavirus resulted in decreased viral antigen synthesis. Transmission electron microscopy confirmed the dual infection of enterocytes. These data suggest that rotavirus infection enhances the early interaction between host cell surfaces and enteroinvasive Yersinia spp.

Comparative study of the invasiveness of Salmonella serotypes Typhimurium, Choleraesuis and Dublin for Caco-2 cells, HEp-2 cells and rabbit ileal epithelia.

Patterns of invasiveness of Salmonella serotypes Typhimurium, Choleraesuis and Dublin in Caco-2 cells (without centrifugation) were compared with previously published studies of the rabbit ileal invasion assay (RIIA) and (where relevant) a HEp-2 cell invasion assay. Optimal conditions for the use of Caco-2 cell monolayers in bacterial invasion assays were defined. Centrifuge-assisted attachment of bacteria to cells was not used routinely as this increased the invasiveness of known hypo-invasive strains and detachment of Caco-2 cells. Inocula with too high bacterial numbers resulted in rapid acidification of media and detachment of the monolayers. The invasiveness of Typhimurium strains TML, WAKE, WII8, LT7, SL1027 and M206 in Caco-2 cells reflected that seen in the RIIA. The invasiveness of Choleraesuis strain A50 was similar to that in the RIIA except that bacteria grown at 37 degrees C and used without storage at 4 degrees C were slightly more invasive than those grown at 37 degrees C and stored at 4 degrees C before use. Dublin strain 3246 showed no apparent temperature-regulated invasiveness in Caco-2 cells, in contrast to the results observed in the RIIA. Dublin strain 3246 did not cleave tight junctions in the Caco-2 cell monolayer as it did in rabbit ileal epithelia both in vitro and in vivo. Three TnphoA insertion LPS mutants of Typhimurium TML were uniformly hypo-invasive in both Caco-2 cells and the RIIA; in contrast, they were differentially invasive in HEp-2 cells. Three smooth TnphoA insertion mutants of Typhimurium TML (invH, invG and pagC) were hypo-invasive in both the Caco-2 and HEp-2 cell invasion assays but not in the RIIA.

Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.

PURPOSE: The objective of this study was a systematic characterization and evaluation of cell culture models based on mixtures of Caco-2/HT29-MTX co-cultures for their use in screening for drug absorption and intestinal permeability in comparison to the properties of the respective mono-cultures. METHODS: Co-cultures of Caco-2 cells (absorptive-type) and HT29-MTX cells (goblet-type) were set up. Three different co-cultures (initial seeding ratios Caco-2/HT29-MTX: 90/10, 70/30, and 50/50) were grown on permeable filter supports, and monolayers were used for permeability studies with model compounds for paracellular absorption (atenolol, furosemide, H334/75, mannitol, terbutaline), transcellular absorption (antipyrine, ketoprofen, metoprolol, piroxicam), carrier-mediated absorption (D-glucose, Gly-Pro, and L-phenylalanine) as well as substrates for carrier-mediated secretion via P-glycoprotein (cimetidine and talinolol). Electrophysiological and microscopic controls were performed to characterize the cell cultures. RESULTS: For compounds undergoing passive intestinal absorption permeabilities were generally higher in co-cultures than in Caco-2 monolayers, yielding highest values in pure HT29-MTX monolayers. This difference was most obvious for compounds transported via the paracellular pathway, where HT29-MTX cells may be up to 30 times more permeable than Caco-2 cells, whereas for lipophilic and highly permeable compounds, the difference in permeability values was less obvious. For drugs undergoing intestinal secretion mediated by P-glycoprotein, co-cultivation of Caco-2 cells with HT29-MTX cells led to increased apical to basolateral permeability which was decreased in the opposite direction, consistent with the fact that HT29-MTX cells do not express P-glycoprotein. When a carrier-mediated absorption mechanism is involved, the permeabilities observed were lower than the values reported for human small intestine and co-cultivation of HT29-MTX cells with Caco-2 cells resulted in even lower values as compared to the plain Caco-2 cultures. CONCLUSIONS: Co-cultures of HT29-MTX and Caco-2 cells offer the opportunity of modifying the permeability barrier of the cell monolayers both with respect to paracellular resistance and secretory transport via P-gp. Thus, in special cases, they allow more flexibility in adapting the in vitro system to the in vivo situation as compared to the monocultures. Another advantage is the obvious robustness of the method with respect to the reproducibility of the results. A problem remaining, however, is the quantitative expression of carriers involved in intestinal uptake of many nutrients and drugs.