Name: Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids
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This work was supported by National Institutes of Health Grants R01DK120986 (to K.P.M.).

All the experimentation using murine tissues described herein was approved by the Institutional Review Board at the University of Pittsburgh and conducted in accordance with the guidelines set forth by the Animal Research and Care Committee at the University of Pittsburgh and UPMC.
1. Preparation for culture
NOTE: All the reagents are listed in the Table of Materials section and all solution compositions can be found in the solution c...

Epithelial Effects of Intestinal Inflammation on Established Murine Colonoids

<ol>
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Organoid development has revolutionized the way the scientific community studies organ systems in vitro with the ability to partially recapitulate cellular structure and function from an animal or human in a dish. Further, organoid systems derived from humans with diseases offer a promising tool for personalized medicine that could guide therapeutic decision-making. Here, we&#160;describes a crypt isolation protocol that works well and introduces key steps that allow for cleaning up excess debris in the isolatio...

Inflammatory bowel disease (IBD) is a chronic, remitting, and relapsing disease characterized by episodes of inflammation and clinical quiescence. The etiology of IBD is multifactorial, but key characteristic features of the disease include defective barrier function and increased permeability of the intestinal epithelium, in addition to proinflammatory signaling cascades activated within the epithelial compartment1,2. Several in vitro and in vivo models have been used to recapitulate the epithelial response during IBD, including cell culture and murine models of inflammation3. However, all these systems have important shortcomings that limit their ability to recapitulate the epithelial changes during IBD4. Most cell lines used to study IBD are transformed, have the ability to form a monolayer, and can differentiate3 but intrinsically propagate differently than non-transformed intestinal epithelial cells in the host. Several different murine models of inflammation are used to study IBD, some of which include knockout models, infectious models, chemical inflammatory models, and T-cell transfer models5,6,7,8. While each can study certain etiological aspects of IBD, such as genetic predispositions, barrier dysfunction, immune deregulation, and the microbiome, they are limited in their ability to study the multifactorial nature of the disease.
Intestinal organoids, including enteroids and colonoids, have been established over the last decade as a useful in vitro model for studying not only the dynamics of intestinal stem cells but also their role the barrier integrity and function of the intestinal epithelium play in intestinal homeostasis and disease. These entities have significantly contributed to our understanding of the pathogenesis of IBD9 and have opened new opportunities for personalized medicine. Colonoids, or stem cell-derived, self-organizing tissue cultures from the colon, have been developed from both murine and human tissue in a process that allows stem cells located within intestinal crypts to propagate and be maintained indefinitely10. The stem cell niche in vivo relies on extracellular factors to support its growth, notably the canonical Wnt signaling and bone-morphogenic protein signaling pathways11. The addition of these factors promotes the health and longevity of colonoids but also drives the culture toward a stem cell-like state that is not reflective of the in vivo epithelial cellular architecture, which consists of both self-renewing and terminally differentiated cells12,13. While the functionality of the intestinal epithelium is dependent upon the continual crosstalk between the stem cell compartment and differentiated cells, the ability to have both in a colonoid culture system is fairly limited. Despite these limitations, the organoid culture system remains the gold standard to study the intrinsic properties of the epithelium ex vivo. Nonetheless, alternative culture strategies may need to be considered to answer the scientific question at hand.
It has been shown that mice on a continuous 7 day regimen of dextran sodium sulfate (DSS) develop both epithelial inflammation and barrier dysfunction14. Furthermore, mitochondrial biogenesis failure and metabolic reprogramming within the intestinal epithelium, which have been shown to be evident in human IBD, have also been captured in this DSS model of colitis15. However, our preliminary data demonstrate that the characteristics of mitochondrial biogenesis failure are not retained in colonoids derived from the crypts of DSS-treated animals (Supplementary Figure 1). Thus, alternative culture methods must be used when examining how inflammation drives epithelial changes during murine intestinal inflammation. Here, we outline a protocol we have developed that describes 1) how to isolate crypts from whole colonic tissue for the establishment of murine colonoids, 2) how to terminally differentiate this cell population to reflect the cell population as it stands in vivo, and 3) how to induce inflammation in this in vitro model. To study drug interactions within the inflamed epithelium, we have developed a protocol to establish 2-dimensonal (2D) monolayers from murine colonoids that can be basally treated with inflammatory mediators and apically treated with drug therapies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.4-&#956;M transparent transwell, 24-well</td>
			<td>Greiner Bio-one</td>
			<td>662-641</td>
			<td></td>
		</tr>
		<tr>
			<td>15-mL conical tubes</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-565-269</td>
			<td></td>
		</tr>
		<tr>
			<td>50-mL conical tubes</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-565-271</td>
			<td></td>
		</tr>
		<tr>
			<td>70-&#956;M cell strainer</td>
			<td>VWR</td>
			<td>76327-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634-010</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>B-27 supplement&#160;</td>
			<td>Invitrogen</td>
			<td>12587-010</td>
			<td>Stock Concentration (50x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Chopsticks Electrode Set for EVO</td>
			<td>World Precision Instruments</td>
			<td>STX2</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning Matrigel GFR Membrane Mix</td>
			<td>Corning</td>
			<td>354-230</td>
			<td>Stock Concentration (100%); Final Concentration (100%)</td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>Sigma-Aldrich</td>
			<td>D0632-5G</td>
			<td>Stock Concentration (1 M); Final Concentration (1.5 mM); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>DMEM high glucose</td>
			<td>Thermo Fisher</td>
			<td>11960-069</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline without Calcium and Magnesium</td>
			<td>Gibco&#160;</td>
			<td>14190-144</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (ETDA)</td>
			<td>Sigma-Aldrich</td>
			<td>E7889</td>
			<td>Stock Concentration (0.5 M); Final Concentration (30 mM)</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Bio-Techne</td>
			<td>S11150H</td>
			<td>Stock Concentration (100%); Final Concentration (1%)</td>
		</tr>
		<tr>
			<td>Fisherbrand Superfrost Plus Microscope Slides, White, 25 x 75 mm</td>
			<td>Thermo Fisher&#160;</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>G418</td>
			<td>InvivoGen</td>
			<td>ant-ga-1</td>
			<td>Final Concentration (400 &#181;g/&#181;L)</td>
		</tr>
		<tr>
			<td>Gentamicin Reagent</td>
			<td>Gibco/Fisher</td>
			<td>15750-060</td>
			<td>Stock Concentration (50 mg/mL); Final Concentration (250 &#956;g/mL)</td>
		</tr>
		<tr>
			<td>GlutaMAX-1</td>
			<td>Fisher Scientific</td>
			<td>35050-061</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>HEPES 1 M</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td>Stock Concentration (1 M); Final Concentration (10 mM)</td>
		</tr>
		<tr>
			<td>hIFN&#947;</td>
			<td>R&#38;D Systems</td>
			<td>285-IF</td>
			<td>Stock Concentration (1000 ng/&#181;L); Final Concentration (10 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hIL-1&#946;</td>
			<td>R&#38;D Systems</td>
			<td>201-LB</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (20 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>hTNF&#945;</td>
			<td>R&#38;D Systems</td>
			<td>210-TA</td>
			<td>Stock Concentration (10 ng/&#181;L); Final Concentration (40 ng/mL); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>Hydrogen Peroxide&#160;</td>
			<td>Sigma</td>
			<td>H1009</td>
			<td>Stock Concentration (30%); Final Concentration (0.003%); Solvent (Mouse wash media)</td>
		</tr>
		<tr>
			<td>Hygromycin B Gold</td>
			<td>InvivoGen</td>
			<td>ant-hg-1</td>
			<td>Final Concentration (400 &#181;g/&#181;L)</td>
		</tr>
		<tr>
			<td>L-WRN Cell Line</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>mEGF</td>
			<td>Novus</td>
			<td>NBP2-35176</td>
			<td>Stock Concentration (0.5 &#181;g/&#181;L); Final Concentration (50 ng/mL); Solvent (D-PBS + 1% BSA)</td>
		</tr>
		<tr>
			<td>N-2 supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>N-Acetyl-L-cysteine</td>
			<td>Sigma&#160;</td>
			<td>A9165-5G</td>
			<td>Stock Concentration (500 mM); Final Concentration (1 mM); Solvent (ultrapure water)</td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td>Stock Concentration (0.1 ng/&#181;L); Final Concentration (100 ng/mL); Solvent (UltraPure water + 0.1% BSA)</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Thermo Fisher</td>
			<td>15140-122</td>
			<td>Stock Concentration (100x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Petri dishes (sterilized; 100 mm x 15 mm) Polystrene disposable</td>
			<td>VWR</td>
			<td>25384-342</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene Microplates, 24 well tissue culture treated, sterile</td>
			<td>Greiner Bio-one</td>
			<td>5666-2160</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Spondin</td>
			<td>R&#38;D Systems</td>
			<td>3474-RS-050</td>
			<td>Stock Concentration (0.25 &#181;g/&#181;L); Final Concentration (500 ng/mL); Solvent (D-PBS + 1% BSA)</td>
		</tr>
		<tr>
			<td>Tryp LE Express</td>
			<td>Thermo Fisher</td>
			<td>12604-013</td>
			<td>Stock Concentration (10x); Final Concentration (1x); Solvent (1 mM EDTA)</td>
		</tr>
		<tr>
			<td>UltraPure Water&#160;</td>
			<td>Invitrogen</td>
			<td>10977-023</td>
			<td>Stock Concentration (1x); Final Concentration (1x)</td>
		</tr>
		<tr>
			<td>Y-27632 dihyddrochloride&#160;</td>
			<td>Abcam</td>
			<td>ab120129</td>
			<td>Stock Concentration (10 mM); Final Concentration (10 &#181;M); Solvent (UltraPure Water)</td>
		</tr>
	</tbody>
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studying the epithelial effects of intestinal inflammation in vitro on established murine colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

Author Spotlight: Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids  

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The 3D intestinal colonoid culture system is an invaluable tool to study the intrinsic contribution of the epithelium to intestinal mucosal homeostasis. The described protocol provides detailed instructions on how to isolate crypts from C57BL/6J (WT) mice at 8 weeks of age and establish a long-term colonoid culture system that can be manipulated for multiple downstream applications. Upon the isolation and plating of crypts in the basement membrane matrix, the crypts appear dense and multicellular in structure when visual...

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We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...

Through our research, we aim to explore how murine colonoids could be modified from a conventional colonoid culture system, to study certain aspects of intestinal physiology and their alteration in the presence of an inflamed disease state. Studying barrier function and intestinal physiology in disease states like inflammatory bowel disease is limited when using a traditional murine colonoid culture system that reflects only stem cell physiology. Our protocol is advantageous in that it provides the reader with multiple techniques to study how inflammatory mediators affect the intestinal epithelium, by either terminally differentiating them, or creating a 2D monolayer system.In the future, our laboratory will concentrate on comprehending how the human colonoid culture system can be altered to investigate different aspects of intestinal physiology and diseases, particularly inflammatory bowel disease.