Production and characterization of avian crypt-villus enteroids and the effect of chemicals

Abstract

Background: Three-dimensional models of cell culture such as organoids and mini organs accord better advantage over regular cell culture because of their ability to simulate organ functions hence, used for disease modeling, metabolic research, and the development of therapeutics strategies. However, most advances in this area are limited to mammalian species with little progress in others such as poultry where it can be deployed to study problems of agricultural importance. In the course of enterocyte culture in chicken, we observed that intestinal mucosal villus-crypts self-repair and form spheroid-like structures which appear to be useful as ex vivo models to study enteric physiology and diseases.

Results: The villus-crypts harvested from chicken intestinal mucosa were cultured to generate enteroids, purified by filtration then re cultured with different chemicals and growth factors to assess their response based on their morphological dispositions. Histochemical analyses using marker antibodies and probes showed the enteroids consisting different cell types such as epithelial, goblet, and enteroendocrine cells typical to villi and retain functional characteristics of intestinal mucosa.

Conclusions: We present a simple procedure to generate avian crypt-villous enteroids containing different cell types. Because the absorptive cells are functionally positioned outwards, similar to the luminal enterocytes, the cells have better advantages to interact with the factors present in the culture medium. Thus, the enteroids have the potential to study the physiology, metabolism, and pathology of the intestinal villi and can be useful for preliminary screenings of the factors that may affect gut health in a cost-effective manner and reduce the use of live animals.

Keywords: Avian intestinal mucosa; Effects of chemicals; Enteroids; Immunochemical characterization.

Fig. 1

a Intestinal mucosa with villi following harvest, b purified enteroids after 24 h of culture, and (c) purified enteroids cultured additionally for 24 h showing extrusion and accumulation of cells around the enteroids. Magnification 200X. (Bar =100 μm)

Fig. 2

Immunolocalization of antigens in the villus enteroids: a and b keratin types I and II, c Na-K-ATPase α-2-subunit, d Pan cadherin, e actin binding alexa 535 labelled phalloidin, f goblet cells binding Sambucus nigra lectin SNII-TRITC, g goblet cells binding Anti-mucin antibody and (h) and Fast red positive alkaline phosphatase. The nuclei are stained blue with DAPI in all pictures. The magnification of the images in a, c, and h are 200 X, bar = 80 μm and the rest 400X, bar = 40 μm

Fig. 3

Immunofluorescence localization of antigen specific cells and proliferating cells. a faint green serotonin positive cells without counter stain, b tryptophan hydroxylase positive faint green cells, c chromogranin A positive cells identified in saffron color, d lysozyme positive cells, faint green, and (e) Andy fluor green fluorescent EdU labeled proliferating cells show as fluorescent green nuclei. The nuclei were stained blue with DAPI except in (e) where they were stained with propidium iodide showing orange-red color. Images are magnified to 200X, bar = 40 μm

Fig. 4

Shows the effect of different chemicals on the enteroids following 24 h of incubation with 1 μg/ml of each factor. Magnification 200X

Fig. 5

An illustration of the procedure for generating enteroids from intestinal villi