The gastrointestinal epithelium is not only a central hub for nutrient uptake and an important barrier shielding the organism from external hazards, but also the site of many pathophysiological processes putting the epithelial functions at risk. Hence, a thorough understanding of the defensive, protective, and adaptive mechanisms the epithelium employs under different circumstances is decisive for improving the prophylactic and therapeutic options to treat gastrointestinal disease. With organoids, or, more specifically, enteroids, i.e., intestinal epithelial organoids, modern in vitro techniques have recently been developed that allow investigating epithelial pathophysiology without the need for animal experiments and with a high cellular resolution. However, there is only little evidence that the enteroids cultivated from intestinal stem cells under the influence of manifold growth factors actually reflect the intestinal epithelium in situ. Therefore, our study aimed to define growth conditions for equine jejunum and colon enteroids (eqJE and eqCE) mimicking the cellular composition and differentiation grade of the respective original tissue.

To cultivate eqJE and eqCE, intestinal epithelial crypts were isolated from intestinally healthy horses euthanized for unrelated reasons. After propagation, eqJE and eqCE (N ≥ 4) were cultivated either with proliferation medium (PM) or one of four differentiation media (DM1-4) with varying compositions. The enteroids were characterized histomorphologically, on gene expression and protein level, and functionally using the Ussing chamber technique in comparison to the native epithelium of the donor horses.

Morphologically, enteroids cultivated in PM and DM1 and 2 showed extensive budding, whereas DM3 and 4 appeared to induce rather spherical growth. Together with an increased number of goblet cells this might indicate a wider variety and higher grade of differentiation of epithelial cell types. In line with this, eqJE and eqCE cultivated in DM3 and 4 showed the highest similarity to the tissue in situ regarding the mRNA expression of specific markers for stem cells (olfactomedin), goblet cells (mucin 2) and enterocytes (villin, Na⁺/K⁺-ATPase, epithelial cell adhesion molecule, Na⁺ coupled glucose transporter). Ussing chamber analyses revealed that the electrophysiological parameters (short-circuit current [I_sc], transepithelial conductance [G_t]) were within similar ranges for eqCE-derived monolayers (G_t: 33.47 ± 1.30 mS cm^-2, I_sc 0.30 ± 0.07 μEq cm^-2 h^-1) and isolated equine colon epithelia (G_t 26.82 ± 6.22 mS cm^-2, I_sc 0.89 ± 0.15 μEq cm^-2 h^-1). Incubation with Na-butyrate (10 mM), forskolin (10 µM), and ouabain (100 µM) provoked similar electrophysiological responses of the organoid-derived monolayers and isolated epithelium, indicating the presence of transepithelial Na⁺-dependent transport, cAMP-dependent Cl^– secretion and Na⁺/K⁺-ATPase activity to a similar extent in eqCE and the ex vivo epithelium.

In conclusion, we succeeded in isolation, propagation and differentiation of eqJE and eqCE consisting of multiple cell types that resembled the equine jejunum and colon epithelium in situ. Thus, enteroids can serve as a suitable model to investigate the pathomechanisms underlying (equine) gastrointestinal disorders. However, the influence of growth conditions on the cellular composition and differentiation must not be neglected when enteroids are used for in vitro experiments meant to be translated to the in vivo situation.