Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.
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, we describe two methods that facilitate access to the apical side of the organoid epithelium and support the differentiation of specific intestinal cell types. First, we show how ECM removal induces an inversion of the epithelial cell polarity and allows for the generation of apical-out 3D organoids. Second, we 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.
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 insults1. 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 tissues2. 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 challenging3. 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 applications4,5.
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 epithelium6. 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 generation of monolayer cultures7. 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 interactions8,9. 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 Cryptosporidium10, H. pylori11, and C. difficile12 on the gastrointestinal epithelium in vitro. Using similar techniques, the mutagenic potential of the pks+ strain of E. coli on intestinal epithelium was determined13. 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.
Recent advances with intestinal organoids have also provided methods for the establishment of 2D monolayer organoid cultures, thereby exposing their apical surface14,15,16,17. 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 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 permeability18,19.
Studies have shown that epithelial cell polarity is tightly regulated by ECM proteins in MDCK spheroids20,21 and recently in human intestinal organoids22. 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 medium22. 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 assays. Here, we describe a modified protocol based on the recent work by the Amieva lab22, that facilitates the generation of 3D intestinal organoids that readily expose their apical side. We also outline a protocol that can efficiently and reproducibly generate intestinal 2D monolayers derived from intestinal organoids.
Epithelial organoid models have become powerful platforms that can be used to model tissue organization, disease progression, and identify therapeutics23,28,29. Organoid microinjection has added value to the organoids' capability of modeling infectious diseases as it allows pathogen interaction with the apical side of the host epithelium. Recent advances in microinjection techniques have optimized the injection speed in organoids and have achieved a rate of up to 90 injected organoids per hour. The barrier function in injected organoids was preserved, and the low oxygen concentration inside the lumen allowed for the survival of obligatory-anaerobic injected bacteria30. However, studies have noted the presence of heterogeneity in organoid populations within the same well. These differences were observed in size and shape31, expression levels of key genes32, as well as proliferation rates33. Differential responses within the same organoid population to compounds such as forskolin and PGE2, or to cholera toxin, have also been described28,33. These results highlight the need for high organoid numbers in studies and limit the utilization of luminal injection.
Conventional organoid culture is based upon the encapsulation and propagation of organoids in a hydrogel. However, hydrogels can pose limitations on diffusion and introduce concentration gradients, which can increase heterogeneity34. Moreover, high variability has been documented, not only between cultures and donors but also within individual experimental conditions. Donor source, biochemical properties of the hydrogel, and intrinsic heterogeneity of the organoid as a culture system are important factors that can increase experimental variability and limit the reproducibility of results obtained in downstream applications. Both of the methods described here provide a simple means of exposing the apical side of the epithelium, allowing for the modeling of compounds and pathogens of interest by directly adding them to the culture medium. The reduction in hydrogel utilization can limit experimental variability from technical sources of error.
The apical-out intestinal organoids retain key characteristics of the organoid model system and their scalability make them more amenable to high throughput assays, compared to the 2D monolayer. However, as the organoids retain their 3D structure, the accessibility of the basal side is limited and may hinder studies requiring access to both sides simultaneously.
We have demonstrated that the polarity inversion of intestinal organoids relies on the efficient and absolute removal of ECM, while also preserving the intact structure of the organoids. Both the use of the dissociation solution to remove ECM and the anti-adherent solution to prevent organoids adherence to the plasticware contributed to ameliorating the overall efficiency of the protocol published by Co, J. Y. and colleagues22, particularly in regard to the number of apical-out organoids produced for downstream applications.
Furthermore,we observed that our protocol supports more efficient inversion of organoids smaller than 250 µm and using larger organoids may result in a reduced organoid output, due to the fragmentation caused by pipetting. Wide-bore tips, such as those indicated in the Table of Materials, may allow the utilization of larger organoids. However, wide-bore tips are less effective in ECM dissociation when compared to standard tips due to the lower applied mechanical force. Therefore, repeating steps 1.2.9-1.2.11 may be required for sufficient disruption and complete removal of all ECM remnants when working with larger organoids.
Organoids in suspension can survive for at least 2 weeks. After this period of time, we observed morphology changes and an increased number of cell death. The presence of proliferating cells in the apical-out organoids22 allows for the re-establishment of apical-in intestinal organoid cultures. This can be achieved by dissociating apical-out organoids to single cells and embedding them in ECM with Intestinal Organoid Expansion Medium.
A limitation frequently encountered in protocols describing the establishment of intestinal organoids in suspension cultures is the generation of large aggregates. This impacts several variables such as efficiency, reproducibility of the morphological features, permeability to compounds, and paracrine signaling. Similar to the protocol published by Co, J. Y. and colleagues, here we confirm to have obtained polarity inversion of at least 97% of all the suspended organoids without shearing after 3 days in suspension. However, in contrast to the publication, we have introduced a mechanical dissociation step in order to reduce the formation of large aggregates and increase yield. Since this procedure may damage the epithelium of the organoids, we extended the organoids incubation period for an additional 2 days to allow complete epithelium recovery and ensure high quality cultures for downstream applications. The introduction of constant agitation with the use of an incubator shaker or a spinner flask could potentially reduce fusion events, minimize fragmentation, and increase oxygenation. These alternative approaches may maintain the cultures for longer durations, reduce cell death, and allow for further differentiation of the apical-out intestinal organoids.
The establishment of an organoid-derived 2D monolayer provides several advantages and disadvantages compared to the inverted polarity organoids. The protocol described here allows for the rapid establishment of a confluent monolayer culture, typically, in less than 7 days and the option of long-term maintenance of the cultures for an extended period of time (up to 10-weeks). The protocol and the media used here also allow for the efficient differentiation of significant numbers of cells, not always found in other organoid-derived monolayer cultures16. Establishing a monolayer on a cell culture insert membrane allows for simultaneous access to both the apical and basolateral sides of the epithelium making them ideal for studies in barrier integrity and epithelial transport. This simplified access also renders them more amenable to infection and drug treatment studies. Furthermore, these cultures maintain many of the characteristics unique to the donor, maintaining their relevance for patient-specific studies. The ALI culture method also facilitates the differentiation of a more functional epithelium composed of both secretory and absorptive cell types, making it more representative of the human intestinal epithelium. The relative stability of these cultures also allows for them to be maintained for a prolonged period of time, providing the possibility for long-term studies. However, limitations of this approach are the high number of cells required to establish a confluent monolayer and the need to maintain complete confluency to have a functional separation between the apical and basolateral chambers. The characteristic crypt architecture, which can be modeled in the 3D organoid cultures is also lost upon establishing a monolayer culture. Nevertheless, the experimentally friendly format of the culture and the ease at which the apical and basolateral sides of the epithelium can be accessed, make it a powerful tool for the study of intestinal physiology.
The authors have nothing to disclose.
This research was funded by the Horizon 2020 grant OrganoVIR 812673 on the project Organoids for Virus Research – An innovative training-ITN programme.
Anti-Adherence Rinsing Solution | STEMCELL Technologies Inc. | 7010 | For coating cultureware. Referred as anti-adherent solution into the main text. |
Conical tubes, 15 mL | STEMCELL Technologies Inc. | 38009 | |
Corning Matrigel Matrix, Growth Factor Reduced (GFR), Phenol Red-Free | Corning | 356231 | Extracellular matrix (ECM) for maintenance and establishment of organoid lines. |
Costar 6.5 mm or 12 mm Transwell inserts | STEMCELL Technologies Inc. | 38023/38024 | For 2D Monolayer culture. |
Costar 24 Well Flat-Bottom, Tissue culture-treated plate | STEMCELL Technologies Inc. | 38017 | For maintenance and establishment of organoid lines. |
D-PBS (Without Ca++ and Mg++) | STEMCELL Technologies Inc. | 37350 | For washing |
Dimethyl sulfoxide (DMSO) | Millipore Sigma | D2650 | Reconstitution of small molecules |
DMEM/F-12 with 15 mM HEPES | STEMCELL Technologies Inc. | 36254 | For washing |
Gentle Cell Dissociation Reagent (GCDR) | STEMCELL Technologies Inc. | 7174 | For Matrigel removal. Referred as dissociation reagent into the main text. |
IntestiCult Organoid Growth Medium (Human) | STEMCELL Technologies Inc. | 6010 | For expansion of organoid lines prior to differentiation. Referred as Intestinal Organoid Expansion Medium into the main text. |
IntestiCult Organoid Differentiation Medium (Human) | STEMCELL Technologies Inc. | 100-0214 | For establishment of monolayers and 3D differentiation. Referred as Intestinal Organoid Differentiation Medium into the main text. |
Trypsin-EDTA (0.05%) | STEMCELL Technologies Inc. | 7910 | For 2D Monolayer establishment. |
Y-27632 | STEMCELL Technologies Inc. | 72302 | RHO/ROCK pathway inhibitor, Inhibits ROCK1 and ROCK2. Used for 2D monolayer establishment. |
Wide bore tips | Corning | #TF-1005-WB-R-S | Organoids handling |