3D organoids derived from the small intestine: An emerging tool for drug transport research

Abstract

Small intestine in vitro models play a crucial role in drug transport research. Although conventional 2D cell culture models, such as Caco-2 monolayer, possess many advantages, they should be interpreted with caution because they have relatively poor physiologically reproducible phenotypes and functions. With the development of 3D culture technology, pluripotent stem cells (PSCs) and adult somatic stem cells (ASCs) show remarkable self-organization characteristics, which leads to the development of intestinal organoids. Based on previous studies, this paper reviews the application of intestinal 3D organoids in drug transport mediated by P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2). The advantages and limitations of this model are also discussed. Although there are still many challenges, intestinal 3D organoid model has the potential to be an excellent tool for drug transport research.

Keywords: 3D organoid; ASCs, adult somatic stem cells; BCRP, breast cancer resistance protein; BMP, bone morphogenetic protein; CDF, 5(6)-carboxy-2′,7′-dichlorofluorescein; Caco-2 cell monolayer; DDI, drug–drug interactions; Drug transporter; EGF, epidermal growth factor; ER, efflux ratio; ESCs, embryonic stem cells; FGF, fibroblast growth factor; Lgr5+, leucine-rich-repeat-containing G-protein-coupled receptor 5 positive; MCT, monocarboxylate transporter protein; MRP2, multidrug resistance protein 2; NBD, nucleotide-binding domain; OATP, organic anion transporting polypeptide; OCT, organic cation transporter; OCTN, carnitine/organic cation transporter; P-glycoprotein; P-gp, P-glycoprotein; PEPT, peptide transporter protein; PMAT, plasma membrane monoamine transporter; PSCs, pluripotent stem cells; Papp, apparent permeability coefficient; Rh123, rhodamine 123; SLC, solute carrier; Small intestine; TEER, transepithelial electrical resistance; TMDs, transmembrane domains; cMOAT, canalicular multispecific organic anion transporter; iPSCs, induced pluripotent stem cells.

Graphical abstract

Figure 1

Schematic representation of intestinal epithelium (A), intestinal 3D organoid (B) and Caco-2 cell monolayer (C). Small intestinal epithelial cells are mainly divided into two segments: villus and crypt. The villus part is mainly involved in drug transport, and the crypt part is responsible for the renewal of small intestinal epithelium. The 3D organoids are embedded in laminin-rich Matrigel and cultured in serum-free medium with a defined set of niche factors including R-spondin-1, EGF and the BMP inhibitor Noggin, which contain Paneth cells, enterocytes, goblet cells, Lgr5⁺ stem cells and enteroendocrine cells. Different kinds of absorption and efflux transporters are expressed on the cytomembrane of enterocytes. While the Caco-2 cells are cultured on the transwell membrane with DMEM medium, and they will form a well-polarized cell monolayer joined by tight junctions after 21 days cultivation.

Figure 2

Flow chart of the intestinal 3D organoids cultivation and its application in drug transport evaluation. The intestinal 3D organoids are derived from isolated crypts and synthetic induced pluripotent stem cells. After cultivation in Matrigel with a series of essential niche factors, the stem cells will arrange themselves into cystic epithelial structures with a central lumen and can reflect the physiological architecture and function of the intestine. The fluorogenic probe substrates, Rh123, Hoechst 33342 and CDF, are selected as the substrates of P-gp, BCRP and MRP2 for drug transport study, respectively.