This publication was supported by Award Number K22 CA218462-03 from the NIH National Cancer Institute. The HEK293-T cells expressing R-Spondin1 was a generous gift from Dr. Michael P. Verzi.

All the mouse experiments conducted, including the use of Tamoxifen and euthanasia by cervical dislocation, had the approval of the Institutional Animal Care and Use Committee at Stevens Institute of echnology.
1. Mice
NOTE: The generation of Smad4f/f; Catnblox(ex3)/+; Villin-CreERT2 mice have been previously described3. Adult female mice between eight to twelve weeks of agewere used.
<ol>
	<li>Induce the Smad4KO:&#946;-cateninGOF mutation in the Smad4f/f; Catnblox(ex3)/+; Villin-CreERT2 with intraperitoneal injection of Tamoxifen in corn oil for four consecutive days.</li>
	<li>Sacrifice the miceby cervical dislocation ten days after the first tamoxifen injection.</li>
	<li>Spray the abdomen with 70% ethanol to prevent mouse fur getting into the peritoneal cavity.</li>
	<li>Open the abdominal cavity with dissection scissors to expose the intestine. Isolate the intestine with the aid of the scissors and forceps. 
	&#8203;NOTE: Concomitant loss of Smad4 along with gain of function mutation of &#946;-catenin (Smad4KO;&#946;-cateninGOF) in intestinal epithelium was attained by injectingSmad4f/f; Catnblox(ex3)/+; Villin-CreERT2 mice every day for four consecutive days with 0.05 g Tamoxifen/kg body weight in corn oil. These mice are harvested ten days after the first tamoxifen injection to ensure the presence of cells expressing stem cell-associated markers in the dedifferentiating villi.</li>
</ol>
2. Duodenum isolation and preparation
<ol>
	<li>Dissect out the proximal half of the duodenum.</li>
	<li>Flush the duodenum with 5 mL of ice-cold PBS in a 10 mL syringe to clear the luminal content.</li>
	<li>Open the duodenum longitudinally with an angled scissor and lay the duodenum flat on a 15 cm Petri plate on ice with the lumen of the duodenum facing the operator.</li>
</ol>
3. Villi isolation by scraping
<ol>
	<li>Prior to beginning the scraping, place a 70 &#181;m mesh strainer in one of the wells of a 6-well tissue culture plate. Fill all the wells with 4 mL of 1x PBS and place the 6-well tissue culture plate on ice (Figure 1).</li>
	<li>Scrape the villi as follows using two microscopic glass slides: one to hold the duodenum down and the other to scrape (Figure 1B1).
	<ol>
		<li>Scrape the luminal side of the duodenum superficially to-and-fro twice to remove the mucus. Apply pressure such that this step removes the mucus layer, but not the villi.</li>
		<li>Scrape the duodenum again, to-and-fro twice applying the same pressure as in 3..1, during which villi can be seen collecting on the slides (Figure 1B2). This is the optimal pressure (which must be determined empirically by the operator) to yield the villi without the crypts tethered.</li>
	</ol>
	</li>
	<li>Use a 1 mL transfer pipet containing PBS to transfer the villi (that are collected on the slide from step 3.2.2.) to a 70 &#181;m mesh strainer placed in a 6-well dish. The villi are collected thus, after every scrape (Figure 1B2).</li>
	<li>Wash the villi collected in the 70 &#181;m strainer (from step 3.3) by transferring the strainer (with the villi) through a series of wells in a 6-well dish containing cold PBS (~ 4 mL/well). This is to remove loose crypts, if any.</li>
	<li>Using a p1000 pipet, transfer the villi suspension in PBS (~ 3 mL) from the 70 &#181;m strainer to a new 15 mL tube on ice.</li>
	<li>Use a 0.1% BSA coated blunt-ended p200 pipet tip to aspirate 50 &#181;L volume of the villi suspension onto a glass slide. Count the number of villi in the 50 &#181;L droplet under 4x magnification to determine the concentration of villi in the PBS suspension. For example, if there are 10 villi in the 50 &#181;L suspension examined, then the villi concentration is 0.2 villi/&#181;L. This is also the time to confirm the absence of tethered crypts.</li>
	<li>Calculate the volume of villi suspension required to plate at a concentration of 0.5 villi/&#181;L of BME-R1 matrix. Add an extra 100 &#181;L to account for pipetting error and the volume required for microscopic examination required to ensure the purity of the villi. 
	&#8203;CAUTION: The pressure used in the first two scrapes, that removes the mucus but not the villi, should be used in the subsequent scrapes during which villi will be released. Limit the number of to-and-fro scrapings to two after the villi release is first observed. This measure avoids release of villi with the crypts tethered. It is essential to microscopically evaluate the villi to ensure the absence of the tethered crypts.</li>
</ol>
4. Plating of villi on BME-R1 matrix
<ol>
	<li>Using a 0.1% BSA coated p200 blunt-ended pipet tip, transfer to a microcentrifuge tube the volume of villi (from step 3.6) required to plate at a density of ~ 6 villi per well in 12.5 &#181;L of BME-R1 matrix. The use of BSA-coated blunt-ended tip at this point ensures that the villi, which are too large for a pointed tip are aspirated without being blocked or lost from being stuck to the sides of the tip.</li>
	<li>Spin down the villi for 2 min at 200 x g in a refrigerated centrifuge (4 &#176;C) and remove the supernatant.</li>
	<li>Repeat step 4.2 to remove any residual PBS and proceed to the next step under a laminar flow-hood.</li>
	<li>Resuspend the villi pellet gently in the required amount of cold BME-R1 thawed on ice.</li>
	<li>Using a p20 pipet, plate 12.5 &#181;L/well of the villi in BME-R1 matrix in 3D in a prewarmed 96-well U-bottom plate.</li>
	<li>Incubate the plate in a tissue culture incubator at 37 &#176;C for 15 minutes to allow solidification of the BME-R1 matrix.</li>
	<li>Add 125 &#181;L/well of pre-warmed ENR (Epidermal growth factor/Noggin/R-spondin1) media: advanced DMEM F-12 media, supplemented with 1x Penicillin: Streptomycin, 10 mM HEPES, Glutamax, 50 ng/mL EGF, 100 ng/mL Noggin, 5% conditioned media from HEK293-T cells expressing R-Spondin1, 1x N2, 1x B27, 1 mM N-acetyl cysteine, and 0.05 mg/mL Primocin.</li>
	<li>Incubate the plated villi in a tissue culture incubator maintained at 37 &#176;C with 5% CO2, and change the media every other day.</li>
	<li>Discard any well in which organoids appear before two days of plating, since the earliest time point at which villi-derived organoids are expected is after two days. 
	NOTE: Any villi that has half or more than half the length of villi are counted as one villus. Unlike crypt-derived organoids, villi-derived organoids are not expected within 24 hours after plating. The organoid-initiating villi appear to be darkening and shrinking prior to formation of organoids. Hence, any wells with organoids developing within a day of plating should be discarded to avoid the plausibility of having derived from plausible crypt contamination. The methodology used to produce the conditioned media is available upon request.</li>
</ol>

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O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMAD4+Suppresses+WNT-Driven+Dedifferentiation+and+Oncogenesis+in+the+Differentiated+Gut+Epithelium.">SMAD4 Suppresses WNT-Driven Dedifferentiation and Oncogenesis in the Differentiated Gut Epithelium.</a> Cancer Research. 78 (17), 4878-4890 (2018).</li><li>Roche, J. 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The authors declare no conflicts of interest.

This method can confirm the self-renewal capacity of dedifferentiating villi epithelium that acquire stem cell markers in vivo. The normal intestinal epithelium can give rise to organoids from the crypt but not the villi compartment, when cultured in 3D because of the presence of stem cells in the crypts1. Thus, organoid formation from the dedifferentiated villi epithelium&#160;cultured in 3D cultures confirms stem cell formation from cell fate reversal. Reports on cell fate reversal in t...

The intestinal crypts but not villi, form organoids when cultured in Matrigel or BME-R1 matrix. These organoids are self-organizing structures, often referred to as the &#34;mini-gut&#34; owing to the presence of the various differentiated lineages, progenitor, and stem cells present in the intestinal epithelium in vivo. The potential to form organoids from crypts is attributed to the presence of stem cells1. The intestinal villi on the other hand consist only of differentiated cells, and hence cannot form organoids. However, mutations2 or conditions that permit dedifferentiation of the villus epithelium may lead to stem cells in the villi2,3. This fate change resulting in stemness in the villi epithelium can be confirmed by plating the dedifferentiating villus epithelium in 3D matrix to determine their organoid-forming potential as an indicator of de-novo stemness in the villus epithelium. Hence, the critical aspect of this procedure is to ensure absence of crypt contamination.
The Smad4KO:&#946;-cateninGOF conditional mutation causes dedifferentiation in the intestinal epithelium marked by the expression of proliferation and stem cell markers in the villi, and eventually the formation of crypt-like structures in the villi referred to as ectopic crypts The presence of stem cells these dedifferentiated villi was determined by the expression of stem cell markers in the ectopic crypts (in vivo) and the ability of the mutant villi to form organoids when plated Matrigel3. The below mentioned procedure elaborates the methodology used to confirm the stemness of the dedifferentiating intestinal epithelium in the Smad4KO:&#946;-cateninGOF mutant mice. A key feature of this methodology for isolating villi was the use of scraping of the intestinal lumen, as opposed to the EDTA chelation method4. Unlike in the EDTA chelation method, villi isolation by scraping retains majority of the underlying mesenchyme and allows adjust the pressure of scraping to yield villi without tethered crypts. The pressure of scraping is subjective to the operator, hence, the optimal pressure to yield villi without crypts tethered must be determined empirically by the operator. The critical aspect of this procedure is to ensure the absence of crypt contamination by microscopic examination of the villi both before and after plating in the BME-R1 matrix.
Intestinal villi are scraped off the intestinal lumen with glass slides and placed in a 70 &#181;m filter and washed with PBS to get rid of loose cells or crypts, if any, prior to plating in BME-R1 matrix. The method stresses on the following criteria to avoid crypt contamination: a) confining the villi harvest the proximal half of the duodenum where the villi are the longest, b) minimizing the number of villi-yielding scrapes, c) washing the filter containing the villi through a series of PBS in a six-well dish, and d) confirming the absence of crypt contamination by microscopic examination prior to and after plating in BME-R1 matrix. Villi isolation by scraping, rather than by EDTA chelation, prevents the complete loss of the underlying mesenchyme that may provide the niche signals5, 6, 7, 8, if required, for organoid initiation from the villus epithelium.

<table><tbody><tr><td>Advanced DMEM F-12 media</td><td>Gibco</td><td>12634010</td><td></td></tr><tr><td>3,3-diaminobenzidine</td><td>Vector Labs</td><td>SK-4105</td><td></td></tr><tr><td>96 well U-bottom plate</td><td>Fisher Scientific</td><td>FB012932</td><td></td></tr><tr><td>ABC kit</td><td>Vector Labs</td><td>&amp;nbsp;PK4001</td><td></td></tr><tr><td>Angled scissor</td><td>Fisher Scientific</td><td>11-999</td><td></td></tr><tr><td>Animal-Free Recombinant Human EGF</td><td>Peprotech</td><td>AF-100-15</td><td></td></tr><tr><td>B-27 Supplement (50X), minus vitamin A</td><td>Gibco</td><td>12587010</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA) Protease-free Powder</td><td>Fisher Scientific</td><td>BP9703100</td><td></td></tr><tr><td>CD44 antibody</td><td>BioLegend</td><td>1030001</td><td></td></tr><tr><td>Cdx2 antibody</td><td>Cell Signaling</td><td>12306</td><td></td></tr><tr><td>Corn oil</td><td>Sigma-Aldrich</td><td>C8267-500ML</td><td></td></tr><tr><td>Corning 70-micron cell strainer</td><td>Life Sciences</td><td>431751</td><td></td></tr><tr><td>Cultrex Reduced Growth Factor Basement Membrane Extract, Type R1</td><td>R&amp;amp;D</td><td>3433-005-R1</td><td></td></tr><tr><td>Dissection scissors</td><td>Fisher Scientific</td><td>22-079-747</td><td></td></tr><tr><td>Forceps</td><td>Fisher Scientific</td><td>17-456-209</td><td></td></tr><tr><td>Glutamax (100X)</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>HEK 293-T cells expressing RSPO-1</td><td>Gift from Dr. Michael Verzi</td><td></td><td></td></tr><tr><td>HEPES (1M)</td><td>Gibco</td><td>15630-080</td><td></td></tr><tr><td>Histogel</td><td>Thermoscientific</td><td>HG-4000-012</td><td></td></tr><tr><td>Mesh filter</td><td>Fisher Scientific</td><td>07-201-431</td><td></td></tr><tr><td>Micrscope glass slide</td><td>VWR</td><td>89218-844</td><td></td></tr><tr><td>N-2 Supplement (100X)</td><td>Gibco</td><td>17502048</td><td></td></tr><tr><td>N-acetyl cysteine</td><td>Sigma-Aldrich</td><td>A9165</td><td></td></tr><tr><td>p200 Blunt tips</td><td>VWR</td><td>46620-642</td><td></td></tr><tr><td>Penicillin-Streptomycin (10,000 U/mL)</td><td>Gibco</td><td>15140-122</td><td></td></tr><tr><td>Primocin (50mg/mL)</td><td>Invivogen</td><td>ant-pm-1</td><td></td></tr><tr><td>Quality Biological Inc PBS (10X)</td><td>Fisher Scientific</td><td>50-146-770</td><td></td></tr><tr><td>Recombinant Murine Noggin</td><td>Peprotech</td><td>250-38</td><td></td></tr><tr><td>Signal diluent</td><td>Cell Signaling</td><td>8112L</td><td></td></tr><tr><td>Tamoxifen</td><td>Sigma-Aldrich</td><td>T5648-1G</td><td></td></tr><tr><td>6-well tissue culture plate</td><td>Fisher Scientific</td><td>50-146-770</td><td></td></tr></tbody></table>

3d culturing of organoids from the intestinal villi epithelium undergoing dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The determining factor for the success of the procedure is preventing crypt contamination. Organoid development from the villi (and not from any contaminating crypts) is ensured by confirming four major criteria: 1) ensuring the purity of the harvested villi by microscopic examination before and after plating the villi in BME-R1, 2) plating limited number of villi per well to allow visualization of all the plated villi individually, 3) onitoring the development of organoid daily; images of the time course show developmen...

Watch this Scientific Journal Video about 3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation at JoVE.com

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...

3d-culturing-organoids-from-intestinal-villi-epithelium-undergoing

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4.8K Views.  Stevens Institute of Technology. The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Video: 3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of the crypt region. IESCs include active stem cells, which self-renew and differentiate into various epithelial cell types, and quiescent stem cells, which serve as the reserve stem cells in the case of injury. Regeneration of the intestinal epithelium is controlled by the self-renewing and differentiating capabilities of these active IESCs. In addition, the balance of the crypt stem cell population and maintenance of the stem cell niche are essential for intestinal regeneration. Organoid culture is an important and attractive approach to studying proteins, signaling molecules, and environmental cues that regulate stem cell survival and functions. This model is less expensive, less time-consuming, and more manipulatable than animal models. Organoids also mimic the tissue microenvironment, providing in vivo relevance. The present protocol describes the isolation of colonic crypts, embedding these isolated crypt cells into a three-dimensional gel matrix system and culturing crypt cells to form colonic organoids capable of self-organization, proliferation, self-renewal, and differentiation. This model allows one to manipulate the environment-knocking out specific proteins such as claudin-7, activating/deactivating signaling pathways, etc.-to study how these effects influence the functioning of colonic stem cells. Specifically, the role of tight junction protein claudin-7 in colonic stem cell function was examined. Claudin-7 is vital for maintaining intestinal homeostasis and barrier function and integrity. Knockout of claudin-7 in mice induces an inflammatory bowel disease-like phenotype exhibiting intestinal inflammation, epithelial hyperplasia, weight loss, mucosal ulcerations, epithelial cell sloughing, and adenomas. Previously, it was reported that claudin-7 is required for intestinal epithelial stem cell functions in the small intestine. In this protocol, a colonic organoid culture system is established to study the role of claudin-7 in the large intestine.

Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

The present protocol describes establishing a murine colonic organoid system to study the activity and functioning of colonic stem cells in a claudin-7 knockout model.

The intestinal epithelium regenerates every 5-7 days, and is controlled by the intestinal epithelial stem cell (IESC) population located at the bottom of ...

Cancer Research

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Medicine

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal epithelium. Due to their closed structures and significant supporting extracellular matrix, 3D cultures are not ideal for host-pathogen studies. Enteroids or colonoids can be grown as epithelial monolayers on permeable tissue culture membranes to allow manipulation of both luminal and basolateral cell surfaces and accompanying fluids. This enhanced luminal surface accessibility facilitates modeling bacterial-host epithelial interactions such as the mucus-degrading ability of enterohemorrhagic E. coli (EHEC) on colonic epithelium. A method for 3D culture fragmentation, monolayer seeding, and transepithelial electrical resistance (TER) measurements to monitor the progress towards confluency and differentiation are described. Colonoid monolayer differentiation yields secreted mucus that can be studied by the immunofluorescence or immunoblotting techniques. More generally, enteroid or colonoid monolayers enable a physiologically-relevant platform to evaluate specific cell populations that may be targeted by pathogenic or commensal microbiota.

Here, we present a protocol to culture human enteroid or colonoid monolayers that have intact barrier function to study host epithelial-microbiota interactions at the cellular and biochemical level.

Human 3-dimensional (3D) enteroid or colonoid cultures derived from crypt base stem cells are currently the most advanced ex vivo model of the intestinal ...

Immunology and Infection

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

Here, we describe the preparation of human organoid-derived intestinal epithelial monolayers for studying intestinal barrier function, permeability, and transport. As organoids represent original epithelial tissue response to external stimuli, these models combine the advantages of expandability of cell lines and the relevance and complexity of primary tissue.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

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, 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&#160;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.

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 ...

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, nutrients, metabolites, pathogens, and the microbiota. Methods to culture intestinal organoids are now available for multiple species, including pigs, which is a species of major interest both as a farm animal and as a translational model for humans, for example, to study zoonotic diseases. Here, we give an in-depth description of a procedure used to culture pig intestinal 3D organoids from frozen epithelial crypts. The protocol describes how to cryopreserve epithelial crypts from the pig intestine and the subsequent procedures to culture 3D intestinal organoids. The main advantages of this method are (i) the temporal dissociation of the isolation of crypts from the culture of 3D organoids, (ii) the preparation of large stocks of cryopreserved crypts derived from multiple intestinal segments and from several animals at once, and thus (iii) the reduction in the need to sample fresh tissues from living animals. We also detail a protocol to establish cell monolayers derived from 3D organoids to allow access to the apical side of epithelial cells, which is the site of interactions with nutrients, microbes, or drugs. Overall, the protocols described here is a useful resource for studying the pig intestinal epithelium in veterinary and biomedical research.

Here, we describe a protocol to culture pig intestinal 3D organoids from cryopreserved epithelial crypts. We also describe a method to establish cell monolayers derived from 3D organoids, allowing access to the apical side of epithelial cells.

Intestinal organoids are increasingly being used to study the gut epithelium for digestive disease modeling, or to investigate interactions with drugs, ...

3D Culture of Organoids from Murine Intestinal Crypts and Single Stem Cell

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine small intestinal crypts can form organoids that mimic the intestinal epithelium when cultured in a 3D extracellular matrix. The organoids are composed of all cell types that fulfill various intestinal homeostatic functions. These include Paneth cells, enteroendocrine cells, enterocytes, goblet cells, and tuft cells. Well-characterized molecules are added into the culture medium to enrich the intestinal stem cells (ISCs) labeled with leucine-rich repeats containing G protein-coupled receptor 5 and are used to drive differentiation down specific lineages; these molecules include epidermal growth factor, Noggin (a bone morphogenetic protein), and R-spondin 1. Additionally, a protocol to generate organoids from a single erythropoietin-producing hepatocellular receptor B2 (EphB2)-positive ISC is also detailed. In this methods article, techniques to isolate small intestinal crypts and a single ISC from tissues and ensure the efficient establishment of organoids are described.

Author Spotlight: The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research  

We describe a protocol to isolate murine small intestinal crypts and culture intestinal 3D organoids from the crypts. Additionally, we describe a method to generate organoids from a single intestinal stem cell in the absence of a sub-epithelial cellular niche.

At present, organoid culture represents an important tool for in vitro studies of different biological aspects and diseases in different organs. Murine ...

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. Accordingly, patient-derived organoids are used for disease modeling, drug discovery, and personalized treatment screening. Mouse intestinal organoids are commonly utilized to understand aspects of both intestinal function/physiology and stem cell dynamics/fate decisions. However, in many disease contexts, rats are often preferred over mice as a model due to their greater physiological similarity to humans in terms of disease pathophysiology. The rat model has been limited by a lack of genetic tools available in vivo, and rat intestinal organoids have proven fragile and difficult to culture long-term. Here, we build upon previously published protocols to robustly generate rat intestinal organoids from the duodenum and jejunum. We provide an overview of several downstream applications utilizing rat intestinal organoids, including functional swelling assays, whole mount staining, the generation of 2D enteroid monolayers, and lentiviral transduction. The rat organoid model provides a practical solution to the need of the field for an in vitro model which retains physiological relevance to humans, can be quickly genetically manipulated, and is easily obtained without the barriers involved in procuring human intestinal organoids.

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

Here, we present a protocol to generate rat intestinal organoids and use them in several downstream applications. Rats are often a preferred preclinical model, and the robust intestinal organoid system fills the need for an in vitro system to accompany in vivo studies.

When using organoids to assess physiology and cell fate decisions, it is important to use a model that closely recapitulates in vivo contexts. ...

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of the human intestinal epithelium, in which they exhibit crypt/villus-like structures. Here, we describe the generation of hiPSC-derived intestinal organoids by the stepwise differentiation of hiPSCs into definitive endoderm, which is then posteriorized to form hindgut epithelium before being transferred into 3D culture conditions. The 3D culture environment consists of extracellular matrix (ECM) (e.g., Matrigel or other compatible ECM) supplemented with SB202190, A83-01, Gastrin, Noggin, EGF, R-spondin-1 and CHIR99021. Organoids undergo passaging every 7 days, where they are mechanically disrupted before transfer to fresh extracellular matrix and allowed to expand. QPCR and immunocytochemistry confirm that hiPSC-derived intestinal organoids contain mature intestinal epithelial cell types including goblet cells, Paneth cells and enterocytes. Additionally, organoids show evidence of polarization by expression of villin localized on the apical surface of epithelial cells.
The resulting organoids can be used to model human intestinal development as well as numerous human intestinal diseases including inflammatory bowel disease. To model intestinal inflammation, organoids can be exposed to inflammatory mediators such as TNF-&#945;, TGF-&#946;, and bacterial LPS. Organoids exposed to proinflammatory cytokines display an inflammatory and fibrotic phenotype in response. Pairing of healthy versus hiPSCs derived from patients with IBD may be useful in understanding mechanisms driving IBD. This may reveal novel therapeutic targets and novel biomarkers to assist in early disease diagnosis.

This protocol allows for the differentiation of human pluripotent cells into intestinal organoids. The protocol mimics normal human development by differentiating cells into a population of definitive endoderm, hindgut endoderm and then intestinal epithelium. This makes the protocol suitable for studying both intestinal development as well as disease modelling applications.

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of ...

Integrating Single-Cell Transcriptomics with Human Intestinal Organoids

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid cultures provide ex vivo model systems that bridge the gap between animal models and clinical studies. The application of single cell transcriptomics to human intestinal organoid (HIO) models is revealing previously unrecognized cell biology, biochemistry, and physiology of the GI tract. The advanced single cell transcriptomics platforms use microfluidic partitioning and barcoding to generate cDNA libraries. These barcoded cDNAs can be easily sequenced by next generation sequencing platforms and used by various visualization tools to generate maps. Here, we describe methods to culture and differentiate human small intestinal HIOs in different formats and procedures for isolating viable cells from these formats that are suitable for use in single-cell transcriptional profiling platforms. These protocols and procedures facilitate the use of small intestinal HIOs to obtain an increased understanding of the cellular response of human intestinal epithelium at the transcriptional level in the context of a variety of different environments.

Author Spotlight: Integrating Single-Cell Transcriptomics with Organoid Cultures for Advanced Research and Therapeutic Insights

The protocol combines human intestinal organoid technology with single cell transcriptomic analysis to provide significant insight into previously unexplored intestinal biology.

Single cell transcriptomics has revolutionized our understanding of the cell biology of the human body. State-of-the-art human small intestinal organoid ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

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Three-Dimensional Culture of Murine Colonic Crypts to Study Intestinal Stem Cell Function Ex Vivo

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

Human Colonoid Monolayers to Study Interactions Between Pathogens, Commensals, and Host Intestinal Epithelium

Organoid-Derived Epithelial Monolayer: A Clinically Relevant In Vitro Model for Intestinal Barrier Function

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

Culture of Piglet Intestinal 3D Organoids from Cryopreserved Epithelial Crypts and Establishment of Cell Monolayers

The 3D Culturing of Organoids from Murine Intestinal Crypts and a Single Stem Cell for Organoid Research

Generation and Manipulation of Rat Intestinal Organoids

Generation of hiPSC-Derived Intestinal Organoids for Developmental and Disease Modelling Applications

Using Human Intestinal Organoids to Understand the Small Intestine Epithelium at the Single Cell Transcriptional Level