Organoid culture to study epithelial cell differentiation and barrier formation in the colon: bridging the gap between monolayer cell culture and human subject research

Abstract

Organoid culture provides a powerful technology that can bridge the gap between monolayer cell culture on the one hand and whole animal or human subject research on the other. Tissues from many different organs from multiple species, including human, have already been successfully adapted to organoid growth. While optimal culture conditions have not yet been established for all tissue types, it seems that most tissues will, ultimately, be amenable to this type of culture. The colon is one of the tissues in which organoid culture was first established as a technology and which has been most successfully employed. The ready availability of histologically normal tissue as well as both premalignant and malignant tissue (often from the same individual) makes this possible. While individual tumors are highly variable relative to one another in organoid culture, a high degree of genotypic consistency exists between the tumor tissue and the histologically normal counterpart from a given source. Further, source material and tumor tissue in organoid culture demonstrate a high degree of genotypic consistency. Even after 6-9 mo in continuous culture, drift in the mutational profile has been shown to be minimal. Colon tissue maintained in organoid culture, thus, provides a good surrogate for the tissue of origin-a surrogate, however, that is as amenable to intervention with molecular, pharmacological, and immunological approaches as are more-traditionally studied cell lines.

Keywords: Adenoma; Colonic mucosa; Organoid; Proteomics.

Conflict of interest statement

Compliance with Ethical Standards

Conflict of Interest All the authors (JV, SDM, MNA) have declared “no conflict of interest” except grants from National Institutes of Health to JV.

Figures

Figure 1.. Appearance of histologically normal human colon tissue in organoid culture.

(a) Scanning electron micrograph of highly pure human colonic crypts upon isolation. (b) Scanning electron micrograph of a preparation of colon organoids. Most of the organoids are spherical or oval in shape with a smooth surface. (c) Highpower scanning electron microscopic view of a colon organoid that has been “cut” open to reveal the central lumen surrounded by a single layer of columnar epithelial cells. (d) Light microscopic image through a preparation of colon organoids. A single layer of mostly columnar epithelial cells surrounds the central lumens. Magnification bars for b = 100 μm, c = 20 μm, and d = 100 μm.

Figure 2.

Appearance of organoids derived from human colon adenoma tissue, (a) Phase-contrast microscopy reveals a dense central core structure with multiple tiny buds emanating from the core surface. (b) At the light microscopic level, the surface buds are shown to be tiny crypts. (c) Adenoma organoids stained with an antibody to Ki67 (red color). Most of the staining is in the outer surface of the tiny crypts (away from the central core). Magnification bars for a = 200 μm, b = 70 μm, and c = 100 μm.

Figure 3.

Mutational variants in a human colon adenoma. (a) Venn plot comparing source material with organoid culture after 2 and 6 mo in culture. (b) Allele frequency in source tumor and in organoid cultures. Insert: Important alleles detected in source material but not organoids.

Figure 4.

Effects of calcium supplementation on histological/ immunohistological features in normal human colon organoids. Upper panels: Lumen diameter and wall thickness. Middle panels: Ki67 expression. Lower panels: CK20 expression. Lumen diameter increased modestly with calcium supplementation but the other three measurements demonstrated minimal or no change. See Attili et al. 2019 for details. Magnification bar = 70 μm.

Figure 5.

Effects of calcium supplementation on occludin and desmoglein-2 expression in histologically normal human colon organoids. Upper left panels: Immunohistology. Middle left panels: Confocal fluorescence microscopy. Lower left panels: Transmission electron microscopy. Upper right panels: Quantitative Immunohistology. Middle right panels: Western blotting. Lower right panels: Quantitative TEM results. With all approaches, calcium supplementation had minimal effect on occludin (OCLN) but resulted in large increase in desmoglein-2 (DSG-2) and increased desmosome count. See Attili et al. 2019 and McClintock et al. 2020 for details. Magnification bars for IHC panels = 70 μm, confocal fluorescence panels = 50 μm, and TEM panels = 200 nm.

Figure 6.

Functional consequences of calcium-supplementation in human colon organoids. Upper panel: Transepithelial electrical resistance (TEER). This measurement reflects resistance to intercellular small molecule permeability and is thought to depend primarily on tight junctions. Lower panel: Cohesion index. This measurement assesses tissue strength (a function of desmosomes) and is based on resistance to mechanical fragmentation. TEER increased modestly in response to calcium-supplementation while the cohesive index increased substantially with treatment. See McClintock et al. 2020 for details. Magnification bar for phase contrast panels = 200 μm.

Figure 7.

Effects of calcium-supplementation on human colon adenoma organoid structure. (a, b) Phase-contrast images of individual adenoma organoids in control medium and in calcium-supplemented culture medium, respectively. (c, d) Light microscopic appearance of hematoxylin and eosin-stained adenoma organoids in control medium and in calcium-supplemented culture medium, respectively. (e, f) Scanning electron microscopic appearance of adenoma organoids in control medium and in calcium-supplemented culture medium, respectively. (g, h) Transmission electron microscopic appearance of adenoma organoids in control medium and in calcium-supplemented culture medium, respectively. Magnification bars for a and b = 200 μm, c and d = 50 μm, e and f = 100 μm, and g and h = 2 μm.

Figure 8.

Effects of calcium supplementation on immunohistological features in human colon adenoma organoids. Upper panels: Ki67 expression. Lower panels: CK20 expression. Calcium supplementation reduced Ki67 expression but increased CK20 expression. See McClintock et al. 2018 for details. Magnification bar for IHC panels = 70 μm.