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Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models

The lining of the gut epithelium is made up of a simple layer of specialized epithelial cells that expose their apical side to the lumen and respond to external cues. Recent optimization of in vitro culture conditions allows for the re-creation of the intestinal stem cell niche and the development of advanced 3-dimensional (3D) culture systems that recapitulate the cell composition and the organization of the epithelium. Intestinal organoids embedded in an extracellular matrix (ECM) can be maintained for long-term and self-organize to generate a well-defined, polarized epithelium that encompasses an internal lumen and an external exposed basal side. This restrictive nature of the intestinal organoids presents challenges in accessing the apical surface of the epithelium in vitro and limits the investigation of biological mechanisms such as nutrient uptake and host-microbiota/host-pathogen interactions. Here, they describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, they show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, they describe how to generate 2-dimensional (2D) monolayers from single-cell suspensions derived from intestinal organoids, comprised of mature and differentiated cell types. These techniques provide novel tools to study apical-specific interactions of the epithelium with external cues in vitro and promote the use of organoids as a platform to facilitate precision medicine.


Stages of the apical-out intestinal organoid generation

(A) Representative images of a dome with organoids of the desired size at day 4 (Left Panel, Scale bar = 500 µm). Organoids are thin-walled, with an open luminal compartment (Right Panel, Scale bar = 100 µm). (B) Representative image of a well with extensive aggregation after 3 days in suspension (Left Panel, Scale bar = 200 µm). Image of clump fragments directly after shearing (Right Panel, Scale bar = 200 µm). (C) Representative image of intestinal organoids in dome at day 7. Organoids display an expanded lumen with the formation of small buds on the basolateral side of the epithelium (Left 20x magnification, Right 100x magnification of the marked region, Scale bar = 200 µm). (D) Representative image of intestinal organoids after ECM removal and subsequent suspension culture for 5 days. The organoids obtain a dense morphology with a thickened epithelium and expose their apical side to the medium. (Left 20x magnification, Right 100x magnification of the marked region, Scale bar = 200 µm).


The intestinal epithelium is the second-largest epithelium in the human body and consists of a polarized cell layer that facilitates nutrient uptake and acts as a barrier against environmental insults. This distinction between the apical and basolateral sides allows cells of the epithelium to carry out their diverse functions. The apical compartment is exposed to the lumen and mediates the epithelial interactions with environmental stimuli and microorganisms, while also facilitating nutrient uptake. The basolateral surface houses intercellular junctions and cell-matrix adhesions, while interfacing with cells of the immune system and other tissues. These junctions generate an impermeable monolayer attached to the basement membrane, which acts as a barrier and delivers the absorbed nutrients to the surrounding body tissue.

The establishment of culture systems that are able to recapitulate these intestinal functions in vitro has been challenging. Conventional in vitro models utilize transformed human colorectal cancer cell lines, such as Caco-2, to generate 2D monolayer cultures. Despite being capable of modeling multiple functions of the absorptive compartment, these models cannot fully recapitulate the intestinal epithelium composition and function, which limit key functional characteristics and applications.


Immunofluorescent staining for cell polarity markers in intestinal organoids.

Apical-out (A,B), and apical-in (C,D) oriented intestinal organoids were stained with apical markers ZO-1 and VILLIN, and with epithelial marker E-CADHERIN (red). DAPI (blue) was used to visualize nuclei. Left panels display images taken at 25x magnification and right panels display images of different organoids at 63x magnification (only panel C displays 25x and 63x magnification of the same organoid). (A) Apical-out intestinal organoids stained with VILLIN (green) and E-CADHERIN (red) indicate the exposure of the apical side to the medium. (B) Apical-out intestinal organoids stained with ZO-1 (green) and E-CADHERIN (red) show the presence of tight junctions and reversion of the apicobasal polarity. (C) Matrigel-embedded intestinal organoid stained with VILLIN (green) and E-CADHERIN (red) showing the apical side facing the organoid lumen. (D) Matrigel-embedded intestinal organoids stained with ZO-1 (green) and E-CADHERIN (red) indicating the presence of apical tight junctions facing the lumen of the organoid. (Scale bar = 100 µm).


The emergence of organoids as an advanced 3D culture system generated from stem cells that can self-organize and differentiate to organ-specific cell types was a breakthrough in the in vitro study of the intestinal epithelium. Intestinal organoids are embedded in an extracellular matrix (ECM) that resembles the basal lamina and form cell-matrix junctions that allow these cultures to retain the apicobasal polarity of the epithelium. Organoids exhibit an enclosed architecture in which the apical side is exposed to the luminal compartment, thus mimicking the structure of the intestine. Although this closed organization offers the opportunity to study orientation-specific functions, it limits investigations that require access to the apical side of the epithelium. Different approaches have been taken to overcome these limitations in both 2D and 3D, including organoid fragmentation, organoid microinjection, and the generation of monolayer cultures. Organoid fragmentation causes the loss of the structural organization and the destruction of cell junctions, which allows the exposure of the apical surface of the epithelium to the medium. This technique takes advantage of the regenerative capacity of the fragments to reform organoids when seeded into an extracellular matrix and has been used to model infectious disease and host-pathogen interactions. However, the simultaneous access to both the apical and basal surface may also elicit non-specific responses to infection.


An alternative approach that allows access to the apical surface and preserves both the structural architecture and cell junctions is represented by the microinjection of factors into the lumen of organoids. This method has been extensively utilized to study host-pathogen interactions and model the effects of Cryptosporidium, H. pylori, and C. difficile on the gastrointestinal epithelium in vitro. Using similar techniques, the mutagenic potential of the pks+ strain of E. coli on intestinal epithelium was determined. Although effective, organoid microinjection is a laborious and inefficient task considering the high number of organoids that are needed to be injected to obtain measurable effects and therefore limits its application for high-throughput assays.



Immunofluorescent staining for differentiated cell markers in monolayer cultures.

(A) Z-stack image of immunofluorescent staining of a submerged monolayer culture for the mucin protein MUC2, indicating the presence of goblet cells within the monolayer culture (green = MUC2, blue = DAPI). (B) Z-stack image of immunofluorescent staining of a submerged monolayer. VILLIN staining (green) along the apical end of the epithelium indicates the presence of a brush border and ZO-1 staining (red) indicates the presence of tight junctions between cells (blue = DAPI). (C) Z-stack image of immunofluorescent staining of an ALI-differentiated monolayer culture for the mucin protein MUC2, indicating the presence of a significantly larger number of goblet cells within the ALI monolayer culture (green = MUC2, blue = DAPI). (D) Cryosection of the ALI-differentiated monolayer culture, stained for the presence of MUC2 (green) and E-CADHERIN (red) indicating the presence of goblet cells in the epithelium and the secretion of mucus along the apical side of the monolayer culture. (Scale bar = 200 µm).


Recent advances with intestinal organoids have also provided methods for the establishment of 2D monolayer organoid cultures, thereby exposing their apical surface. These organoid-derived monolayers recapitulate key in vivo properties of the intestinal epithelium. They exhibit a physiologically relevant cell composition, containing both differentiated and stem cell populations, and model the diversity across the crypt-villus axis. As apicobasal polarity is retained, the inherent monolayer properties allow for easy access of both the apical and basolateral sides and media exchanges can mimic the intestinal flow and waste removal allowing for long-term culture. These features render organoid-derived monolayers amenable for studies focusing on luminal interactions and provide a superior model for epithelial barrier integrity and permeability.


Studies have shown that epithelial cell polarity is tightly regulated by ECM proteins in MDCK spheroids and recently in the human intestinal organoids. Removal of ECM components or inhibition of the integrin receptor that mediates the cell-matrix junctions results in a polarity reversal of intestinal organoids and the exposure of the apical side of the epithelium to the medium. This approach has attracted the interest of researchers working on infectious diseases as it allows easy access to the apical side in 3D and makes it amenable to high throughput assays1.


  1. SStroulios, G., Stahl, M., Elstone, F., Chang, W., Louis, S., Eaves, A., Simmini, S., Conder, R. K. Culture Methods to Study Apical-Specific Interactions using Intestinal Organoid Models. J. Vis. Exp. (169), e62330, doi:10.3791/62330 (2021).