Self-organized intestinal epithelial monolayers in crypt and villus-like domains show effective barrier function

Abstract

Intestinal organoids have emerged as a powerful in vitro tool for studying intestinal biology due to their resemblance to in vivo tissue at the structural and functional levels. However, their sphere-like geometry prevents access to the apical side of the epithelium, making them unsuitable for standard functional assays designed for flat cell monolayers. Here, we describe a simple method for the formation of epithelial monolayers that recapitulates the in vivo -like cell type composition and organization and that is suitable for functional tissue barrier assays. In our approach, epithelial monolayer spreading is driven by the substrate stiffness, while tissue barrier function is achieved by the basolateral delivery of medium enriched with stem cell niche and myofibroblast-derived factors. These monolayers contain major intestinal epithelial cell types organized into proliferating crypt-like domains and differentiated villus-like regions, closely resembling the in vivo cell distribution. As a unique characteristic, these epithelial monolayers form functional epithelial barriers with an accessible apical surface and physiologically relevant transepithelial electrical resistance values. Our technology offers an up-to-date and novel culture method for intestinal epithelium, providing an in vivo -like cell composition and distribution in a tissue culture format compatible with high-throughput drug absorption or microbe-epithelium interaction studies.

Intestinal enteroids recapitulate the effects of short-chain fatty acids on the intestinal epithelium

“…3D enteroids were cultured on chamber slides (Nunc Lab-Tek II™; ThermoFisher Scientific, Waltham MA) while 2D monolayers were grown on Transwell™ inserts prior to staining. Enteroids in both conditions were fixed in 10% NBF at room temperature for 20 min, followed by permeabilization in Triton x 100 for 20 min [21]. 3D murine enteroids were then stained with F-actin marker, ActinRed™ 555 ReadyProbes Reagent (Invitrogen, ThermoFisher Scientific, Waltham, MA), while 2D enteroids were stained with tight junction proteins claudin-3 (CLDN3) and zonula occludens-1 (ZO1; Abcam, Cambridge, MA).…”

Architecture of the intestinal crypts of the payers’ patches of the albino rats’ small intestine.

“…Следовательно, согласно существующим представлениям, длина кишечных крипт является соразмерной с толщиной собственной пластинки слизистой оболочки. Действительно, гистологические срезы, полученные в поперечном сечении тонкой кишки, выявляют данные трубчатые образования именно в таком ракурсе [15][16][17][18][19][20]. Но в литературе имеются данные, согласно которым в пейеровых бляшках кишечные крипты подвержены топографической дифференцировке относительно к их лимфоидным узелкам, в связи с чем среди них выделяются две разновидности, одна из которых ассоциирована с лимфоидной тканью соответствующих узелков, тогда как вторая -находится за их пределами.…”

Spheres of Influence: Insights into Salmonella Pathogenesis from Intestinal Organoids

“…These monolayers can be cultured for up to three weeks (Stefania Senger, unpublished observations). Recent studies [60][61][62] have developed techniques to generate monolayers that exhibited compartmentalization of proliferative crypt-like domains and differentiated villus-like regions closely resembling in vivo distribution. Liu et al [60] generated self-renewing 2D monolayers by plating intestinal stem cells on a thin layer (10 µM) of Matrigel coated onto glass sheets, while Wang et al [63] relied on the presence of collagen hydrogels in tissue culture plates.…”

“…These models do not provide access to the basolateral side of the monolayers. To overcome this limitation, Altay and colleagues [62] cultured mouse-derived intestinal enteroids on Transwells coated with Matrigel that provided proper mechanical stiffness. The authors also boosted the proliferation of the stem cells by supplementing the culture medium for the basolateral compartment with conditioned medium from intestinal sub-epithelial myofibroblasts.…”