Functional Comparison of Human Colonic Carcinoma Cell Lines and Primary Small Intestinal Epithelial Cells for Investigations of Intestinal Drug Permeability and First-Pass Metabolism

Abstract

To further the development of a model for simultaneously assessing intestinal absorption and first-pass metabolism in vitro, Caco-2, LS180, T84, and fetal human small intestinal epithelial cells (fSIECs) were cultured on permeable inserts, and the integrity of cell monolayers, CYP3A4 activity, and the inducibility of enzymes and transporters involved in intestinal drug disposition were measured. Caco-2, T84, and fSIECs all formed tight junctions, as assessed by immunofluorescence microscopy for zonula occludens-1, which was well organized into circumscribing strands in T84, Caco-2, and fSIECs but was diffuse in LS180 cells. The transepithelial electrical resistance value for LS180 monolayers was lower than that for Caco-2, T84, and fSIECs. In addition, the apical-to-basolateral permeability of the paracellular marker Lucifer yellow across LS180 monolayers was greater than in fSIECs, T84, and Caco-2 monolayers. The transcellular marker propranolol exhibited similar permeability across all cells. With regard to metabolic capacity, T84 and LS180 cells showed comparable basal midazolam hydroxylation activity and was inducible by rifampin and 1a,25(OH) 2 D 3 in LS180 cells, but only marginally so in T84 cells. The basal CYP3A4 activity of fSIECs and Caco-2 cells was much lower and not inducible. Interestingly, some of the drug transporters expressed in LS180 and Caco-2 cells were induced by either 1a,25(OH) 2 D 3 or rifampin or both, but effects were limited in the other two cell lines. These results suggest that none of the cell lines tested fully replicated the drug disposition properties of the small intestine and that the search for an ideal screening tool must continue.

Bioprinted 3D Primary Human Intestinal Tissues Model Aspects of Native Physiology and ADME/Tox Functions

“…The 3D intestinal tissue demonstrated the expression and function of key enzymes involved in xenobiotic metabolism including CYP3A4, an enzyme known to be absent in Caco-2 models ( Yamaura et al., 2016 ), consistent with our findings. Enzyme activity was confirmed by modulation of midazolam hydroxylation through both inhibition and induction—CYP3A4 activity and PXR-regulated genes were induced in response to rifampicin.…”

“…Physiological conditions lacking in 2D culture may be improved by microfluidic chip models; however, maintenance of cell-cell and cell-matrix interactions and scale-up for high-throughput applications remain challenging ( Bhatia and Ingber, 2014 , Chi et al., 2016 ). Standard 2D monolayers and more recent gut-on-a-chip systems commonly include cell lines originating from colorectal and duodenal tumor tissue ( Alqahtani et al., 2013 , Kim et al., 2012 ) that may exhibit altered metabolic profiles and expression patterns of xenobiotic-metabolizing enzymes and transporters potentially leading to misrepresentation of the native response ( Prueksaritanont et al., 1996 , Yamaura et al., 2016 ). The colorectal tumor-derived Caco-2 cell line is the most established model to study permeability and predict intestinal absorption.…”

“…The colorectal tumor-derived Caco-2 cell line is the most established model to study permeability and predict intestinal absorption. Known limitations of the Caco-2 model include reduced CYP450 activity ( Prueksaritanont et al., 1996 , Yamaura et al., 2016 ), variability with passage number, and inconsistencies between subclones of the cell line ( Bentz et al., 2013 ).…”

Caco-2 Cells for Measuring Intestinal Cholesterol Transport - Possibilities and Limitations

“…ns not significant, *P < 0.05, **P < 0.01 versus solvent vehicle (determined by one-way ANOVA with Dunnett post-test) protein detection via CLSM, applying Protocol #6 for staining. Several protocols for staining and CLSM with Caco-2 cells are already available [17,[41][42][43]. However, fixation of cells, the blocking solution used as wells as the permeabilization differ between these publications.…”

Physiologically Relevant, Humanized Intestinal Systems to Study Metabolism and Transport of Small Molecule Therapeutics

“…Early attempts to study disposition of small molecules, in vitro, leveraged Ussing chambers (Wallon et al, 2005;Wuyts et al, 2015) with isolated human (endoscopic biopsied) tissue, followed by migration to gut tissue-derived subcellular fractions, and finally, the deployment of human gut "surrogate" reagents such as LS180 and Caco-2 cells (Rogers et al, 1987;Yamaura et al, 2016). Only recently has it been possible to consider using physiologically relevant human intestinal models by analogy with human primary hepatocytes [see Khetani et al (2018) and Bale et al (2018) in this special issue on novel models of drug metabolism, disposition, and toxicity].…”