This study was made possible by funding from the Dutch Cancer Society (KWF; EMCR 2012-5473) and the World Cancer Research Funds International (WCRF; project no. 2014-1181)

All procedures were done according to local animal welfare laws and guidelines.
1. Preparation of Instruments, Culture Media, and Dishes
<ol>
	<li>Autoclave 1 set of intestinal scissors, normal scissors, and forceps in a sterile container.</li>
	<li>Place a 96-well (flat bottom) dish in an incubator at 37 &#176;C.</li>
	<li>Prepare 10 mL of complete culture medium with the reagents listed in table of materials.</li>
	<li>Incubate the complete medium at 37 &#176;C in a water bath.</li>
	<li>Thaw reconstituted basement membrane by placing it in an ice bucket. The reconstituted basement membrane will become liquid at 4 &#176;C .</li>
	<li>Fill 4 Petri dishes with cold phosphate-buffered saline or abbreviated PBS (4 &#176;C).</li>
</ol>
2. Isolation of Small Intestinal Crypts
<ol>
	<li>Sacrifice by CO2 inhalation a Lgr5-eGFP-IRES-CreERt2 mouse on a C57BL6/J background. Dissect the peritoneum longitudinally with a pair of scissors.</li>
	<li>Hold the stomach with the forceps and cut it transversally in half.</li>
	<li>Using the intestinal scissors from now on, pull out the intestine and place it in a Petri dish containing PBS. 
	NOTE: Intestinal scissors have a sharp and a blunt tip. The blunt tip of these scissors is meant to not damage the crypt-villus architecture while opening the intestine.</li>
	<li>Start by placing the blunt tip into the stomach and gently push it through the pylorus. Proceed by cutting with the scissors and pulling with the forceps.</li>
	<li>Once the whole small intestine is opened longitudinally, wash it in cold PBS by holding it with the forceps and gently rinse it in the PBS solution with U-shaped movements.</li>
	<li>Once all the stool remnants are cleared, proceed to flatten the intestine on a cutting board, luminal side up. The luminal side is easily recognizable by the absence of blood vessels and by its pale appearance when compared with the outer part.</li>
	<li>With a glass slide, gently remove the villi by scraping the flattened intestine. Perform this step twice along the whole length of the tissue.</li>
	<li>Cut the small intestine with a sterile surgical blade into 2 - 5 mm pieces.</li>
	<li>Place the small intestinal fragments in a 50 mL tube containing 10 mL of ice cold PBS.</li>
	<li>Clean the tissue fragments, removing any remaining impurities by pipetting them up and down in the PBS. Discard supernatant and repeat this step until the PBS is completely clear.</li>
	<li>Add 15 mL cold PBS, to bring the total final volume of PBS to 25 mL. Add 2 mM ethylenediaminetetraacetic acid (EDTA) and incubate for 45 min on a roller at 4 &#176;C.</li>
	<li>Discard the PBS/EDTA.</li>
	<li>Add 10 mL of PBS and detach the crypts by harshly pipetting the tissue fragments up and down (at least three times). Collect the supernatant.</li>
	<li>Repeat step 2.13 four times. Add culture medium to reach a final volume of 50 mL.</li>
	<li>Pellet the cells by centrifugation (300 x g for 5 min).</li>
	<li>Resuspend the pellet in 10 mL of culture medium and pellet the crypts by centrifugation (80 x g for 3 min).</li>
</ol>
3. Single Cell Preparation
<ol>
	<li>Discard the supernatant of the pelleted crypts and resuspend them in 1 mL trypsin like-enzymes together with 50 &#181;L DNAse (50 &#181;g/mL).</li>
	<li>Incubate the cells in a water bath at 32 &#176;C for 2 min.</li>
	<li>Add 10 mL of culture medium. Dissociate the crypts into single cells by harshly pipetting up and down at least 5 times.</li>
	<li>Filter the solution through a 40 &#181;m strainer (to eliminate clumps and other impurities) and pellet the single cells at 300 x g for 5 min.</li>
</ol>
4. Flow Cytometry and Plating
<ol>
	<li>Prepare 50 mL of 1x&#160;Hank&#39;s buffered salt solution or HBSS/2% fetal cow serum (FCS) solution and resuspend the pellet in 1 mL for every 106 cells</li>
	<li>Add to the cell suspension BV421 Lin- (CD31, CD45, TER119) at a concentration of 1:100, and CD24-APC and CD117-PE both at a concentration of 1:250 antibodies for 30 min.</li>
	<li>Sort Lgr5+ stem cells and Paneth cells into separate low bind tubes. For details on sorting protocols for Lgr5+ and Paneth cells, please see Roth et al.7 and Schewe et al.6
	<ol>
		<li>Centrifuge the sorted cells at 300 x g for 5 min. Resuspend the cells in 100 &#181;L medium with the components described as in 1.3 (see the Table of Materials).</li>
	</ol>
	</li>
	<li>Treat (e.g., by chemical or genetic modification) the Paneth cells (n = 2000) or the Lgr5+ stem cells (n = 2000).
	<ol>
		<li>To treat cells, use 5 &#181;L of liposome-mediated transfection reagent in a total volume of 100 &#181;L culture medium and small interfering RNA (siRNA) at a concentration of 100 nM. Incubate for 30 min at 37 &#176;C for siRNA or for the time necessary for the chosen modification.</li>
	</ol>
	</li>
	<li>Wash the cells twice in 500 &#181;L culture medium.</li>
	<li>Centrifuge at 300 x g for 5 min and resuspend the cells in 10 &#181;L culture medium.</li>
	<li>Pool the two cellular components (Paneth or Lgr5+ cells) in a 20 &#181;l total volume and centrifuge at 300 x g for 5 min at RT.</li>
	<li>Co-incubate the Paneth and Lgr5+ cells for 10 min at RT.</li>
	<li>Remove 10 &#181;L of the supernatant and leave a meniscus of liquid to be sure to not aspirate the cell pellet.</li>
	<li>Add 30 &#181;L of reconstituted basement membrane to the cells for a total volume of 40 &#181;L, resuspend the cells and plate in a pre-warmed 96 well plate. After 10 min, add 200 &#181;L of complete medium (see the Table of Materials). Change medium every 48 h.</li>
	<li>Count organoid multiplicity at day 5.</li>
</ol>

<ol>
	<li>Barker, N., 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=Identification+of+stem+cells+in+small+intestine+and+colon+by+marker+gene+Lgr5.">Identification of stem cells in small intestine and colon by marker gene Lgr5.</a> Nature. 449, 1003-1007 (2007).</li><li>Sato, T., 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=Paneth+cells+constitute+the+niche+for+Lgr5+stem+cells+in+intestinal+crypts.">Paneth cells constitute the niche for Lgr5 stem cells in intestinal crypts.</a> Nature. 469, 415-418 (2011).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459, 262-265 (2009).</li><li>Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+Development+and+Disease+with+Organoids.">Modeling Development and Disease with Organoids.</a> Cell. 165, 1586-1597 (2016).</li><li>Cantrell, M. A., Kuo, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoid+modeling+for+cancer+precision+medicine.">Organoid modeling for cancer precision medicine.</a> Genome Med. 7, 32 (2015).</li><li>Schewe, M., 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=Secreted+Phospholipases+A2+Are+Intestinal+Stem+Cell+Niche+Factors+with+Distinct+Roles+in+Homeostasis,+Inflammation,+and+Cancer.">Secreted Phospholipases A2 Are Intestinal Stem Cell Niche Factors with Distinct Roles in Homeostasis, Inflammation, and Cancer.</a> Cell Stem Cell. 19, 38-51 (2016).</li><li>Roth, S., 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=Paneth+cells+in+intestinal+homeostasis+and+tissue+injury.">Paneth cells in intestinal homeostasis and tissue injury.</a> PLoS One. 7, e38965 (2012).</li><li>Rodriguez-Colman, M. J., 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=Interplay+between+metabolic+identities+in+the+intestinal+crypt+supports+stem+cell+function.">Interplay between metabolic identities in the intestinal crypt supports stem cell function.</a> Nature. 543, 424-427 (2017).</li><li>Dow, L. E., 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=Apc+Restoration+Promotes+Cellular+Differentiation+and+Reestablishes+Crypt+Homeostasis+in+Colorectal+Cancer.">Apc Restoration Promotes Cellular Differentiation and Reestablishes Crypt Homeostasis in Colorectal Cancer.</a> Cell. 161, 1539-1552 (2015).</li><li>Yilmaz, O. H., 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=mTORC1+in+the+Paneth+cell+niche+couples+intestinal+stem-cell+function+to+calorie+intake.">mTORC1 in the Paneth cell niche couples intestinal stem-cell function to calorie intake.</a> Nature. 486, 490-495 (2012).</li><li>Igarashi, M., Guarente, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mTORC1+and+SIRT1+Cooperate+to+Foster+Expansion+of+Gut+Adult+Stem+Cells+during+Calorie+Restriction.">mTORC1 and SIRT1 Cooperate to Foster Expansion of Gut Adult Stem Cells during Calorie Restriction.</a> Cell. , 436-450 (2016).</li></ol>

The authors have no competing financial interest or other conflicts of interest

The ORA allows the refined functional analysis of the two essential components of the intestinal stem cell niche, namely Lgr5+ and Paneth cells. This approach has been previously employed by us and others with slight modifications6,10,11. Here, we present the ORA procedure as a reproducible and standardized laboratory protocol. Also, we report on the effects of Apc downregulation in Lgr5<sup...

The intestinal epithelium is the most rapidly self-renewing tissue in the mammalian body and, as such, has been the object of a plethora of studies aimed at the identification and functional characterization of the adult stem cells residing at the bottom of the crypt of Lieberk&#252;hn, earmarked by expression of the Lgr5 gene and dependent on canonical Wnt signals1. Notably, Lgr5+ stem cells are flanked and supported by specialized niche cells, i.e. Paneth cells, which also depend on Wnt signaling for their maturation2. Together, these two cell types underlie self-renewal of the intestinal epithelial lining and preserve the daily homeostatic equilibrium: Lgr5+ stem cells rapidly divide and give rise to progenitor and more specialized intestinal epithelial cells; Paneth cells provide essential niche factors (e.g., Dll1, Wnt3, EGF) to Lgr5+ stem cells2. The capacity of intestinal crypts when plated ex vivo to form organized and self-renewing structures called organoids, or &#34;mini-guts&#34;, has been exploited as an experimental tool to provide insight into processes such as self-renewal and differentiation in normal and pathological conditions including cancer4. Organoid cultures have been established from several tissues, including the intestine, pancreas, liver, and kidney, from both mouse and human samples4. The extraction method and the growth factors employed to develop these organoid cultures are tissue-specific and designed to drive multi-lineage differentiation and mimic as closely as possible the original stem cell niche in vivo. Organoids may have potential applications including the treatment of genetic diseases, the assessment of therapeutic efficacy in cancer, the analysis of drug toxicity, or the study of organogenesis in vitro5.
Overall, the main limitation of organoid cultures when established from tissue samples is a lack of cell-specificity. For example, intestinal organoids established from whole intestinal crypts do not allow the analysis of individual cellular components encompassed within the tissue source (e.g., whole crypts contain Lgr5+, Paneth, and progenitor cells).
Here, we describe a novel method, referred to as ORA, that combines the advantages of intestinal organoid cultures with the functional analysis of its most fundamental components, namely stem (Lgr5+) and niche (Paneth) cells. This is achieved through the unique ability of Paneth and Lgr5+ cells to physically associate with each other when co-incubated and give rise to organoids2,6,10. We took advantage of this feature and pre-treated the two cell types individually before allowing them to reconstitute organoids. When doing so, each cell component can be exposed to any given drug, growth factor, biochemical inhibitor, genetic modification, or chemical treatment prior to reconstitution and organoid formation. Therefore, using the ORA assay will allow the determination of whether a specific drug treatment or genetic modification has a specific effect on the stem cells or their niche counterpart.

<table><tbody><tr><td>Advanced DMEM F12 *</td><td>Thermo Fisher Scientific</td><td>12634-010</td><td></td></tr><tr><td>Glutamax *</td><td>Thermo Fisher Scientific</td><td>35050061</td><td>Final concentration: 10 mM</td></tr><tr><td>Penicillin/streptomycin *</td><td>Thermo Fisher Scientific</td><td>15140122</td><td>Final concentration: 1%</td></tr><tr><td>Hepes *</td><td>Thermo Fisher Scientific</td><td>15630080</td><td>Final concentration: 10 mM</td></tr><tr><td>Recombinant murine EGF *</td><td>Thermo Fisher Scientific</td><td>PMG8041</td><td>Final concentration: 50 ng/ml</td></tr><tr><td>Recombinant murine Noggin *</td><td>Peprotech</td><td>250-38</td><td>Final concentration: 100 ng/ml</td></tr><tr><td>y27632 *</td><td>Sigma</td><td>Y0503</td><td>Final concentration: 10 &amp;micro;M</td></tr><tr><td>Jagged-1 *</td><td>Anaspec Bio</td><td>AS-61298</td><td>Final concentration: 1 &amp;micro;M</td></tr><tr><td>Recombinant murine R-spondin *</td><td>R&amp;amp;D systems</td><td>3474-RS-050</td><td>Final concentration: 1 mg/ml</td></tr><tr><td>Flexitube Gene solution siRNA APC</td><td>Qiagen</td><td>GS11789</td><td>Final concentration: 100 nM</td></tr><tr><td>Lipofectamine 2000</td><td>Thermo Fisher Scientific</td><td>11668019</td><td></td></tr><tr><td>CD24 APC</td><td>Biolegend</td><td>101814</td><td></td></tr><tr><td>c-kit PE</td><td>Biolegend</td><td>105808</td><td></td></tr><tr><td>BV 421 CD31</td><td>Biolegend</td><td>102424</td><td></td></tr><tr><td>BV 421 CD45</td><td>Biolegend</td><td>103134</td><td></td></tr><tr><td>BV421 TER119</td><td>Biolegend</td><td>116234</td><td></td></tr><tr><td>HBSS</td><td>Thermo Fisher Scientific</td><td>14180046</td><td></td></tr><tr><td>B6.129P2-Lgr5tm1(cre/ERT2)Cle/J</td><td>Jackson Laboratories</td><td>008875</td><td></td></tr><tr><td>Low binding tubes</td><td>Eppendorf</td><td>Z666548-250EA</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>356230</td><td></td></tr><tr><td>* The combination of these reagents constitutes the complete culture medium</td><td></td><td></td><td></td></tr></tbody></table>

the organoid reconstitution assay (ora) for the functional analysis of intestinal stem and niche cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The organoid reconstitution assay allows the separate functional analysis of the essential niche and stem cell components of the intestinal epithelium, here demonstrated by small interfering RNA (siRNA) of the Apc gene.
To achieve this aim, we first utilized fluorescent activated cell sorting (FACS) to separate 8000 Lgr5+ cells and 6000 Paneth cells from inbred C57BL6/J mice. In ...

Watch this Scientific Journal Video about The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells at JoVE.com

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...

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Unit 1

Stem Cell Biology And Renewal in Epithelial Tissue

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10.4K Views.  Erasmus University Medical Center. Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

Video: The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

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

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the complexities of the organoid growth characteristics carry significant caveats for the investigator. Specifically, the growth patterns of each individual organoid are highly variable and create a heterogeneous population of epithelial cells in culture. With such caveats, common tissue culture practices cannot be simply applied to the organoid system due to the complexity of the cellular structure. Counting and plating based solely on cell number, which is common for individually separated cells, such as cell lines, is not a reliable method for organoids unless some normalization technique is applied. Normalizing to total protein content is made complex due to the resident protein matrix. These characteristics in terms of cell number, shape and cell type should be taken into consideration when evaluating secreted contents from the organoid mass. This protocol has been generated to outline a simple procedure to culture and treat small intestinal organoids with microbial pathogens and pathogen associated molecular patterns (PAMPs). It also emphasizes the normalization techniques that should be applied when protein analysis are conducted after such a challenge.

The Ex Vivo Culture and Pattern Recognition Receptor Stimulation of Mouse Intestinal Organoids

Here, a protocol to harvest, maintain, and treat mouse small intestinal organoids with pathogen associated molecular patterns (PAMPs) and Listeria monocytogenes is described, as well as emphasis on gene expression and proper normalization techniques for protein.

Primary intestinal organoids are a valuable model system that has the potential to significantly impact the field of mucosal immunology. However, the ...

Immunology and Infection

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

Generation, Maintenance, and Characterization of Human Pluripotent Stem Cell-derived Intestinal and Colonic Organoids

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing gastrointestinal (GI) tract. Regional specification in the intestine is driven by multiple developmental pathways, including the bone morphogenetic protein (BMP) pathway. Based on normal regional specification, a method to generate human colonic organoids (HCOs) from human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hES) and induced pluripotent stem cells (iPSCs), was developed. A three-day induction of BMP signaling sufficiently patterns mid/hindgut tube cultures into special AT-rich sequence-binding protein 2 (SATB2)-expressing HCOs containing all of the main epithelial cell types present in human colon as well as co-developing mesenchymal cells. Omission of BMP (or addition of the BMP inhibitor NOGGIN) during this critical patterning period resulted in the formation of human intestinal organoids (HIOs). HIOs and HCOs morphologically and molecularly resemble human developing small intestine and colon, respectively. Despite the utility of HIOs and HCOs for studying human intestinal development, the generation of HIOs and HCOs is challenging. This paper presents methods for generating, maintaining, and characterizing HIOs and HCOs. In addition, the critical steps in the protocol and troubleshooting recommendations are provided.

Here, detailed methods for generating, maintaining, and characterizing human pluripotent stem cell-derived small intestinal and colonic organoids are described. These methods are designed to improve reproducibility, expand scalability, and decrease the working time required for plating and passaging of organoids.

Intestinal regional specification describes a process through which unique morphology and function are imparted to defined areas of the developing ...

Developmental Biology

Combining Human Organoids and Organ-on-a-Chip Technology to Model Intestinal Region-Specific Functionality

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, and metabolism of nutrients and xenobiotics while facilitating a symbiotic relationship with microbiota and restricting the invasion of microorganisms. Functional interaction between various cell types and their physical and biochemical environment is vital to establish and maintain intestinal tissue homeostasis. Modeling these complex interactions and integrated intestinal physiology in vitro is a formidable goal with the potential to transform the way new therapeutic targets and drug candidates are discovered and developed.
Organoids and Organ-on-a-Chip technologies have recently been combined to generate human-relevant intestine chips suitable for studying the functional aspects of intestinal physiology and pathophysiology in vitro. Organoids derived from the biopsies of the small (duodenum) and large intestine are seeded into the top compartment of an organ chip and then successfully expand&#160;as monolayers while preserving the distinct cellular, molecular, and functional features of each intestinal region. Human intestine tissue-specific microvascular endothelial cells are incorporated in the bottom compartment of the organ chip to recreate the epithelial-endothelial interface. This novel platform facilitates luminal exposure to nutrients, drugs, and microorganisms, enabling studies of intestinal transport, permeability, and host-microbe interactions.
Here, a detailed protocol is provided for the establishment of intestine chips representing the human duodenum (duodenum chip) and colon (colon chip), and their subsequent culture under continuous flow and peristalsis-like deformations. We demonstrate methods for assessing drug metabolism and CYP3A4 induction in duodenum chip using prototypical inducers and substrates. Lastly, we provide a step-by-step procedure&#160;for the in vitro modeling of interferon gamma (IFN&#947;)-mediated barrier disruption (leaky gut syndrome) in a colon chip, including methods for evaluating the alteration of paracellular permeability, changes in cytokine secretion, and transcriptomic profiling of the cells within the chip.

Biopsy-derived intestinal organoids and organ-on-a-chips technologies are combined into a microphysiological platform to recapitulate region-specific intestinal functionality.

The intestinal mucosa is a complex physical and biochemical barrier that fulfills a myriad of important functions. It enables the transport, absorption, ...

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 ...

Biology

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 ...

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 ...

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

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

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