Porcine Intestinal Apical-Out Organoid Model for Gut Function Study

Abstract

Pig models provide valuable research information on farm animals, veterinary, and biomedical sciences. Experimental pig gut models are used in studies on physiology, nutrition, and diseases. Intestinal organoids are powerful tools for investigating intestinal functions such as nutrient uptake and gut barrier function. However, organoids have a basal-out structure and need to grow in the extracellular matrix, which causes difficulties in research on the intestinal apical membrane. We established porcine intestinal organoids from jejunum tissues and developed basal-out and apical-out organoids using different sub-culture methods. Staining and quantitative real-time PCR showed the difference in axis change of the membrane and gene expression of epithelial cell marker genes. To consider the possibility of using apical-out organoids for intestinal function, studies involving fatty acid uptake and disruption of the epithelial barrier were undertaken. Fluorescence fatty acid was more readily absorbed in apical-out organoids than in basal-out organoids within the same time. To determine whether apical-out organoids form a functional barrier, a fluorescent dextran diffusion assay was performed. Hence, we successfully developed porcine intestinal organoid culture systems and showed that the porcine apical-out organoid model is ideal for the investigation of the intestinal environment. It can be used in future studies related to the intestine across various research fields.

Keywords: apical-out; barrier integrity; intestinal organoid; nutrient uptake; pig model.

Figure 1

Development of small intestinal organoids in porcine jejunum tissue. (A) Brief scheme for experimental procedure of this study. (B) Representative image of isolated crypts from pig jejunum tissue (magnification: ×100). Scale bar (100 μm). (C) The organoids images show development of organoids from day 0 to day 5 (magnification: ×40). The arrows indicate the organoid budding points. Scale bar (200 μm).

Figure 1

Development of small intestinal organoids in porcine jejunum tissue. (A) Brief scheme for experimental procedure of this study. (B) Representative image of isolated crypts from pig jejunum tissue (magnification: ×100). Scale bar (100 μm). (C) The organoids images show development of organoids from day 0 to day 5 (magnification: ×40). The arrows indicate the organoid budding points. Scale bar (200 μm).

Figure 2

Comparison of morphology for basal-out and apical-out porcine small intestinal organoids. (A) Representative images of two types of organoids culture from day 1 to day 3 (magnification: ×100). The arrows indicate the organoid budding points. Scale bar (200 μm). (B) Images of stained organoids (magnification: ×400). Nuclear and F-actin were stained by DAPI and phalloidin. The images represent switched axis between basal-out organoid and apical-out organoids. Scale bar (100 μm).

Figure 3

The expression of genes for intestinal epithelial cells in established organoids. (A) The image of gel electrophoresis shows expression of intestinal epithelial cell markers (Lgr5, Muc2, Lyz, ChgA, ALPI). The pig jejunum tissue was used for positive control. (B) The mRNA expression of intestinal epithelial cells on both basal-out and apical-out organoids. Data are represented as mean ± SD (n = 3). The significance between basal-out and apical-out organoids was analyzed by two-tailed unpaired Student’s t-test. ** p < 0.01; *** p < 0.001.

Figure 4

The BODIPY uptake for porcine small intestinal organoid. (A) Representative organoid images for basal-out and apical-out organoids treated with fluorescent fatty acid analog (BODIPY) (magnification: ×400). Each organoid was treated with BODIPY for 30 min, then staining was performed. Nuclear was stained by DAPI. Scale bar (100 μm). (B) Quantification of fatty acid analog uptake in porcine organoids. Data are represented as mean ± SD (n = 10, 7–10 fluorescence area/organoids). The significance between basal-out and apical-out organoid was analyzed by two-tailed unpaired Student’s t-test. *** p < 0.001.

Figure 5

Disrupted gut barrier on porcine small intestinal apical-out organoid. The porcine apical-out organoids were treated in FITC–dextran-containing media (2 mg/mL) (magnification: ×400). Five mM EDTA was used for demonstrating disruption of epithelial barrier integrity. Scale bar (200 μm). BF, bright field.