Gut organoids: mini-tissues in culture to study intestinal physiology and disease

Abstract

In vitro, cell cultures are essential tools in the study of intestinal function and disease. For the past few decades, monolayer cellular cultures, such as cancer cell lines or immortalized cell lines, have been widely applied in gastrointestinal research. Recently, the development of three-dimensional cultures known as organoids has permitted the growth of normal crypt-villus units that recapitulate many aspects of intestinal physiology. Organoid culturing has also been applied to study gastrointestinal diseases, intestinal-microbe interactions, and colorectal cancer. These models are amenable to CRISPR gene editing and drug treatments, including high-throughput small-molecule testing. Three-dimensional intestinal cultures have been transplanted into mice to develop versatile in vivo models of intestinal disease, particularly cancer. Limitations of currently available organoid models include cost and challenges in modeling nonepithelial intestinal cells, such as immune cells and the microbiota. Here, we describe the development of organoid models of intestinal biology and the applications of organoids for study of the pathophysiology of intestinal diseases and cancer.

Keywords: colon cancer; genetic editing; gut physiology; intestinal organoids; organoid culture.

Fig. 1.

Origin of gastrointestinal organoids. Organoids can be derived from different segments of the gastrointestinal tract, including esophagus (20), stomach (87), small intestine (72, 74, 116), and colon (18, 97).

Fig. 2.

Anatomy of an intestinal organoid. A three-dimensional section of an intestinal organoid embedded into extracellular matrix in vitro. The organoid consists of spatially arranged cells, with the luminal side facing the inside of the organoid and the apical side facing the extracellular matrix. Intestinal organoids comprise all of the intestinal cellular types, including stem cells, Paneth cells, transit amplifying cells, enterocytes, goblet cells, and enteroendocrine cells. The architecture of the organoid includes a crypt domain where stem cells reside and a villus domain containing differentiated cells.

Fig. 3.

Imaging of mouse small intestinal organoids. A: light microscopy of the small intestine. B: intestinal epithelium organization from crypt to villus with stem cells (S) and Paneth cells (P) at the bottom of the crypt. C: light microscopy image of a mouse small intestinal organoid. D: one-micron section of mouse small intestinal organoids counterstained with Toluidine Blue. E: electron microscopy image of mouse small intestinal organoids. F: immunofluorescence for chromogranin A, mucin 2, and lysozyme in mouse small intestinal organoids (blue = DAPI, red = cell-specific antibody). C and D scale bars = 20 μm; E scale bar = 2 μm, F scale bar = 50 μm. [Reproduced from Beyaz et al. (5), with permission].

Fig. 4.

Evolution of a human intestinal crypt in culture. Human colonic crypts embedded in Matrigel and cultured in media containing Wnt3a, Rspondin-1, Noggin, and other growth factors form organoids within 24 h. Scale bar = 50 μm.

Fig. 5.

Applications of organoid culture. Organoid cultures are established from murine or human intestinal crypts, repurposed embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), or frozen organoids. Potential applications of organoid culture include use in drug discovery by high-throughput screening, characterizing tissue by genetic profiling, exploring host-microbe interaction by coculturing with pathogens, inducing somatic mutations using genetic editing techniques such as CRISPR-Cas9 to target specific mutations, creating models of intestinal diseases, implementing multiorgan cultures in an organoid-on-a-chip model, and modeling intestinal diseases and cancer through in vivo transplantation.

Fig. 6.

Novel organoid culture models. A: isolation of intestinal crypt and culturing onto planer scaffold yields an organoid monolayer that can be further modeled into a scaffold that resembles in vivo architecture of the crypts. EM, expansion medium; SM, stem medium; DM, differentiation medium. B: light microscopy of the organoid monolayers (left) and fluorescent microscopy stained for EdU (green), Muc2 (red), and nuclei (blue). C: fluorescent microscopic side sections stained for EdU pulse (green), ALP stain (red), and Hoechst-DNA labeling (blue) (121, 122). D: intestinal organoids embedded in Matrigel are digested and embedded onto a three-dimensional silk scaffold alongside myofibroblasts, forming a polarized culture along the silk scaffold. E: scanning electron microscopy showing the microvilli brush border on the apical surface of the scaffold. F: fluorescent microscopy of the apical surface staining for zonula occludens-1 (green) and DAPI (blue) (13). B and C scale bars =100 μm. E and F scale bars = 250 μm.