Human organotypic colon in vitro microtissue: unveiling a new window into colonic drug disposition

Abstract

The purpose of this study was to evaluate EpiColon, a novel human organotypic 3D colon microtissue prototype, developed to assess colonic drug disposition, with a particular focus on permeability ranking, and compare its performance to Caco-2 monolayers. EpiColon was characterized for barrier function using transepithelial electrical resistance (TEER), morphology via histology and immunohistochemistry, and functionality through drug transport studies measuring apparent permeability (P_app). Cutoff thresholds for the permeability of FITC-dextran 4 kDa (FD4), FITC-dextran 10 kDa (FD10S), and [¹⁴C]mannitol were established to monitor microtissue integrity. Permeability of EpiColon for 20 benchmark drugs was compared with Caco-2 data, and the activity of pivotal efflux transporters, including multidrug resistance protein 1/P-glycoprotein (MDR1/P-gp), along with multidrug resistance protein 2 (MRP2) and breast cancer resistance protein (BCRP), was evaluated using selective substrates. EpiColon exhibited a physiological barrier function (272.0 ± 53.05 Ω x cm²) and effectively discriminated between high (e.g., budesonide and [³H]metoprolol) and low permeable compounds (e.g., [³H]atenolol and [¹⁴C]mannitol). The model demonstrated functional activity for key efflux transporters, with efflux ratios of 2.32 for [³H]digoxin (MDR1/P-gp) and 3.34 for sulfasalazine (MRP2 and BCRP). Notably, EpiColon showed an enhanced dynamic range in the low permeability range, differentiating P_app between FD4 and FD10S, in contrast to Caco-2 monolayers. Significant positive correlations were observed between human fraction absorbed (f_abs) and logarithmically transformed P_app [AP-BL] values for both EpiColon (r_s = 0.68) and Caco-2 (r_s = 0.68). Furthermore, EpiColon recapitulates some essential phenotypic and cellular features of the human colon, including the expression of critical marker genes (Pan-Cytokeratin⁺: epithelial/colonocytes, Vimentin⁺: mesenchymal/fibroblast, and Alcian Blue⁺: goblet cell/mucus). In conclusion, EpiColon is a promising platform that offers a valuable complement to conventional Caco-2 monolayers for studying colonic drug disposition. However, the presence of flat and some cuboidal cells, along with low throughput, must be addressed to improve its applicability in both academic research and pharmaceutical industry.

Keywords: Drug absorption; Drug disposition; Intestinal in vitro tools; Intestinal permeability; Organotypic colon microtissues; Pharmacokinetics.