Proteomic and functional profiling of suspended jejunal enteroids reveals pharmacokinetic fidelity to native intestine.

Intestinal enteroids are "mini-guts" that recapitulate essential features of the intestinal epithelium in vitro. We developed and characterized native basal-out (BO) and morphogenically everted apical-out (AO) jejunal enteroids cultured in suspension. Using quantitative proteomics and a range of advanced microscopy techniques, we show that these enteroids progressively adopt protein profiles closely resembling those of freshly isolated villus enterocytes and human jejunal tissue. Optimally differentiated enteroids exhibit mature epithelial morphology, intact barrier function, nutrient absorption, and clinically relevant transporter activity (P-glycoprotein; Pgp/ABCB1, breast cancer resistance protein; BCRP/ABCG2), reflecting in vivo physiology. Metabolic assessment of orally administered cytochrome P450 3A4 (CYP3A4) probe drugs, terfenadine and midazolam, accurately predicted first-pass metabolism and clinical pharmacokinetics via physiologically based pharmacokinetic (PBPK) modeling. These findings establish suspension-grown 3D jejunal enteroids as a translationally relevant, human-derived platform that is advantageous over conventional cell line and animal models for studying drug disposition, nutrient transport, and intestinal barrier function.