Ian Francis Hassan

Transport and permeability properties of human Caco-2 cells: An in vitro model of the intestinal epithelial cell barrier

Abstract When on nitrocellulose filters, in chambers, Caco- 2 cells form a confluent monolayer with several properties characteristic of differentiated intestinal epithelial cells. The cell layers are morphologically polar and have well-developed brush borders at their apical surface. The monolayers are functional in the carrier-mediated transport of taurocholic acid and in the receptor-mediated endocytic transport of cobalamin-intrinsic factor. These active transport pathways, characteristic of the distal ileum, are specific and unidirectional (apical to basolateral). The Caco- 2 model displays low permeability to the non-specific transepithelial passage of macromolecules. In addition, our results indicate that the model has potential for predicting in vivo absorption of low molecular weight drugs. The reproducibility and long-term viability of this human model, together with the ability to conveniently perform qualitative and quantitative transport studies across an intact epithelial layer, confer significant advantages over other in vitro models used f or absorption studies.

The Transport of Vitamin B12 Through Polarized Monolayers of Caco-2 Cells

Caco-2 cells grown on 0.45-micron filters, in Millicell chambers, form intact monolayers with many of the properties of polarized intestinal epithelial cells. It is reported here that these cells bind and internalize intrinsic factor-cobalamin complexes and that after 14-28 days in culture this specific binding is exclusively located on the apical membrane. Caco-2 cells also synthesize and secrete a protein with properties similar to transcobalamin II. This protein is secreted from the basolateral side of the cells after 20 days in culture. Specific apical-to-basolateral transcellular transport of [57Co]cobalamin also occurs between 20 and 28 days in culture. Thus, Caco-2 cells provide the first polarized human cell system for studying the transepithelial transport of cobalamin.

Proline uptake by monolayers of human intestinal absorptive (Caco-2) cells in vitro

Monolayers of the Caco-2 human intestinal cell line exhibit active and passive uptake systems for the imino acid L-proline. The active transport component is saturable and it is responsible for about two thirds of the observed flux over the nanomolar concentration range, at 37 degrees C and pH 7.4. In contrast to L-phenylalanine, specific L-proline uptake has a high degree of sodium dependency and the efficiency of the carrier system is significantly reduced when protein synthesis (cycloheximide), Na+/K(+)-ATPase (ouabain) or cellular metabolism (sodium azide) are inhibited. The expression of the L-proline carrier by Caco-2 cells was under some degree of nutritional control. Glucose deficiency, over the time scale of the experiment, had no effect. The temperature-dependence of the specific uptake process followed the Arrhenius model with an apparent activation energy of 93.5 kJ nmol-1. This pathway also displayed Michaelis-Menten concentration-dependence with a Ksdm of 5.28 mM and a maximal transport flux (Jsdmax) of 835 pmol min-1 (10(6) cells)-1. Although the passive component was unchanged, the pH of the donor phase exerted a profound effect on the active carrier component. Within the physiological pH range a local maximum efficiency was found at pH 7.4 but dramatic increases were noted as pH 5.0 was approached. In competition studies, with 100-fold excess of a second amino acid, strong inhibition of uptake was found with alpha-aminoisobutyric acid, L-alanine and L-serine whereas moderate inhibition was observed with glycine, D-proline and gamma-aminoisobutyric acid. Aromatic and branched amino acids showed weak (L-valine) or no interaction (L-phenylalanine, L-leucine) with the carrier system. These data indicate that the carrier system for the uptake of L-proline has many features in common with the A system for amino acid transport.

Permeable support type influences the transport of compounds across Caco-2 cells

Abstract The influence of aluminium oxide and nitrocellulose inserts on the uptake and transport of taurocholic acid in Caco-2 cells has been determined. Caco-2 cells grown on nitrocellulose inserts displayed a higher rate of taurocholic acid transport than those grown on aluminium oxide inserts; 59.3 ± 4.1 ng 4 h −1 insert −1 compared to 29.7 ± 4.1 ng 4 h −1 insert −1 at day 14. In addition, Caco-2 cells grown on aluminium oxide and nitrocellulose inserts were not comparable with respect to adsorption potential, cell morphology, cell numbers and transepithelial electrical resistance. The low adsorption potential of aluminium oxide inserts, particularly for high molecular weight or lipophilic ligands such as propranolol and testosterone, allowed basolateral uptake events to be studied with a precision not possible with nitrocellulose inserts. In addition, the translucent nature of aluminium oxide inserts offered significant advantages over the nitrocellulose insert in terms of visualising the cells by light microscopy. Caco-2 cells grown on either type of insert appear fully differentiated when observed by light and electron microscopy. Occasional dome-like structures were present when Caco-2 monolayers were grown on aluminium oxide but not nitrocellulose inserts. Cell numbers were significantly lower on aluminium oxide inserts than on nitrocellulose inserts. Moreover, Caco-2 cells grown on aluminium oxide inserts displayed a higher transepithelial electrical resistance than those grown on nitrocellulose inserts; 871 ± 149 compared to 513 ± 32 at day 14. The reasons for these differences and the respective merits of each type of insert are considered.

Receptor-Mediated Transport of Cobalamin in Caco-2 Cells: Intracellular Localisation of Transcobalamin II

A number of in vitro models have been developed to study the absorption of drugs across the intestinal epithelium (Osiescka et al., 1985). The majority of these models do not have the morphological and functional properties of normal adult human epithelial cells. Recently, a human adenocarcinoma cell line (Caco-2) that reproducibly displays properties of differentiated distal ileal epithelial cells has been characterised (Wilson et al., 1989; Hidalgo et al., 1989). When cultured on permeable supports (nitrocellulose filters), in chambers, Caco-2 cells form a confluent monolayer which is morphologically polar with a well developed brush border at their apical surface. The monolayers are functional in the specific and unidirectional transport of taurocholic acid, (Wilson et al., 1989), and the active transport of phenylalanine (Hidalgo and Borchardt, 1988).