Name: real time analysis of metabolic profile in ex vivo mouse intestinal crypt organoid cultures
Uploaded: 2014-11-03T15:00:00.000+00:00
Duration: 8 min 53 s
Description: Watch this Scientific Journal Video about Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures at JoVE.com

This study was supported by grants RO1 CA 135561, R01 CA151494, R01 CA174432 and P3013330 from the National Institutes of Health.
We would like to thank Michele Houston, Elena Dhima and Dr. Anna Velcich for their valuable comments in developing the crypt isolation protocol.
We also thank the Diabetes Training and Research Center of the Albert Einstein College of Medicine supported by NIH P60DK20541, and Dr. Michael Brownlee and Dr. Xue-Liang Du, who direct and operate the Seahorse facility, respectively.

This study was performed in accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics of Animal Experiments of the Albert Einstein College of Medicine.
1. Crypt Isolation and Culture
<ol>
	<li>Isolation of Crypts from the Small Intestine:
	<ol>
		<li>Isolate intestinal crypts from any mice model of interest. Euthanize the mice with CO2 followed by cervical dislocation.</li>
		<li>Open up the abdomen longitudinally and fill the small intestine (SI) with ice cold phosphate buffered saline, PBS (-Ca2+; -Mg2+) with 2x antibiotic&#8211;antimycotic (anti&#8211;anti). Rapidly isolate small intestine.</li>
		<li>Thoroughly dissect free of mesenteric fat using a scalpel. Be careful not to perforate the tissue &#8211; at all times keep the tissue moist with ice cold PBS. Open up intestine longitudinally and wash thoroughly with ice cold PBS.</li>
		<li>Cut small intestine into two sections and flatten out using a wet cotton tip, gently scrape off the villi using a pre-cooled slide. Wash vigorously in ice-cold PBS several times.</li>
		<li>Incubate 3 min in 20 ml 1x PBS per 3 mM ethylenediaminetetraacetic acid (EDTA)/0.5 mM dithiothreitol (DTT) for non-enzymatic dissociation of the tissue.</li>
		<li>Cut tissue into small pieces (2-4 mm) using a razor blade on a pre-cooled slide. Transfer the pieces into a 50 ml tube containing 20 ml ice cold PBS.</li>
		<li>Pipette up and down gently 10x using a 10 ml sterile disposable pipette. Let tissue fragments sediment by gravity. Remove the supernatant with a pipette. Repeat three additional times; make sure supernatant is clear.</li>
		<li>Add 20 ml of PBS per 2 mM EDTA. Swirl and incubate at 4 &#176;C for 30 min with gentle rocking.</li>
		<li>Let tissue sediment and discard supernatant. Add 15 ml ice cold PBS and pipette up and down 5x with a 10 ml pipette. Let the tissue fragments sediment and collect the supernatant as Fraction 1 (F1). Repeat collecting F2-F5, each fraction maintained separately.</li>
		<li>Add 15 ml ice cold PBS and this time shake vigorously by hand for 15 sec. Collect F6 and repeat for F7, F8. If tissue pieces start to float, tap to help them settle.</li>
		<li>Inspect an aliquot of each fraction under the microscope. Pool those fractions containing crypts.</li>
		<li>Pass the pooled fractions through a 70 &#181;m nylon cell strainer, collecting crypts in a 50 ml tube.</li>
		<li>Centrifuge at 100 x g for 5 min at 4 &#176;C, discard the supernatant and resuspend pellet in 10 ml ice cold PBS with 2x anti&#8211;anti and transfer crypts to a 15 ml tube. Repeat the wash one more time.</li>
		<li>Wash the crypts once with 10 ml ice-cold ADF, Advanced DMEM/F-12 (Dulbecco&#34;s Modified Eagle Medium/Ham&#34;s F-12) &#8211; 2x anti&#8211;anti.</li>
		<li>Wash the crypts once with 10 ml ADF &#8211; 1x anti&#8211;anti.</li>
		<li>Count the crypts using a hemocytometer. Adjust the crypt solution volume and transfer to a 1.5 ml tube so that the final crypt concentration when resuspended will be 100-500 crypts per 50 &#181;l of gelatinous protein mixture (Matrigel). Centrifuge at 100 x g for 5 min at 4 &#176;C and resuspend crypts in the gelatinous protein mixture.</li>
		<li>Plate 50 &#181;l of crypt &#8211; gelatinous protein mixture suspension per well in a 24-well plate (growth area: 2 cm2), carefully placing the suspension drop in the center of each well. Maintain the plate in a CO2, 37 &#176;C incubator for ~30 min until the suspension solidifies.</li>
		<li>Add 500 &#181;l ice cold complete culture media (Advanced DMEM/F12 with 1x anti &#8211; anti, 10 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1x GlutaMAX, 1x B27 Supplement, 1x N2 Supplement, 1 mM N-Acetyl-L-Cysteine (NAC), 50 ng/ml Epidermal growth factor (EGF), 100 ng/ml Noggin, 500 ng/ml R-Spondin).
		<ol>
			<li>Reconstitute growth factors in 0.1% Bovine Serum Albumin (BSA) in PBS.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Crypt Organoid Culture and Passage:
	<ol>
		<li>Passage crypt organoids 14-21 days post seeding (or as needed) as follows:
		<ol>
			<li>Change media every Friday and Monday. On Wednesdays, replace half the media with fresh media.</li>
		</ol>
		</li>
		<li>Remove the culture medium. Wash the sample twice with 500 &#181;l PBS at 5 min intervals.</li>
		<li>Remove PBS and gently break up the gelatinous protein mixture using a sterile P1000 micro pipette tip.</li>
		<li>Add 1 ml complete culture media to a single well and resuspend the organoids in 1 ml media.</li>
		<li>Gently disrupt the organoids using a P1000 by pipetting up and down 20-30x (check under the microscope for proper organoid dissociation with a good crypt yield until a consistent technique is developed).</li>
		<li>Transfer the crypts to a 1.5 ml microcentrifuge tube; centrifuge at 100 x g at 4 &#176;C, 5 min.</li>
		<li>Wash the pellet twice with 1 ml complete culture media.</li>
		<li>Split at a ratio of 1:3 or 1:6 as needed, resuspending crypts in 50 &#181;l Matrigel per well. Proceed as described above (section 1.1.16-18).</li>
	</ol>
	</li>
	<li>Crypt Organoid Freezing:
	<ol>
		<li>Remove the culture medium. Wash the sample in 500 &#181;l PBS.</li>
		<li>Disperse the gelatinous protein mixture using a P1000 pipette tip.</li>
		<li>Add 1 ml media to a single well and resuspend the organoids in 1 ml media.</li>
		<li>Gently break up the organoids using a P1000 by pipetting up and down 20-30x.</li>
		<li>Transfer the organoids to a 1.5 ml tube. Centrifuge at 100 x g at 4 &#176;C, 5 min.</li>
		<li>Wash the pellet twice with 1 ml complete culture media.</li>
		<li>Resuspend crypts in 500 &#181;l freezing media.</li>
		<li>Transfer crypts to a cryotube, place the tube in a freezing container and store in a -80 &#176;C freezer overnight.</li>
		<li>Transfer crypts to liquid N2 tank.
		<ol>
			<li>Recover frozen crypt organoids by rapidly thawing in a 37 &#176;C water bath. Wash with 500 &#181;l complete culture media once, centrifuge at 100 x g, 5 min and resuspend in gelatinous protein mixture, and culture as described above. For better recovery, add ROCK inhibitor (Y-27632) to the freezing media.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Crypt Organoid Metabolism Assay
<ol>
	<li>24-well Plate Preparation:
	<ol>
		<li>Isolate and count crypts according to the protocol (see section 1.1 for crypt isolation and section 1.2 for crypt passage).</li>
		<li>Resuspend crypts in gelatinous protein mixture (100-200 crypts per 20 &#181;l).</li>
		<li>Plate crypt-gelatinous protein mixture suspension in a 24-well assay plate (make sure there are at least triplicates for each sample) and allow the suspension to solidify at 37 &#176;C in a CO2 incubator; then add 500 &#181;l complete culture media.</li>
		<li>Culture crypts (as described in section 1.1) and observe under microscope until crypts grow into fully developed organoids.</li>
	</ol>
	</li>
	<li>Extracellular Flux Assay:
	<ol>
		<li>Hydrate cartridge overnight in a non-CO2 (0% CO2), 37 &#176;C incubator.</li>
		<li>Remove the culture medium and wash organoids twice with 500 &#181;l DMEM (without: glucose, L-glutamine, sodium pyruvate and sodium bicarbonate; and with: phenol red). Wait 5 min.</li>
		<li>Add 675 &#181;l per well assay medium (DMEM with 2 mM L-glutamine and 5 mM D-glucose) to each well.</li>
		<li>Check crypt organoids microscopic morphology to ensure that organoids and the gelatinous protein mixture are intact after the washes. Incubate 1 hr in a non-CO2, 37 &#176;C incubator.</li>
		<li>Prepare 10 &#181;M injectable compounds (oligomycin, carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP), rotenone) in assay medium.</li>
		<li>Set-up the cartridge by loading 75 &#181;l of 10 &#181;M injectable compounds into the ports of the cartridge sequentially: Port A - oligomycin; Port B - FCCP; and Port C - rotenone (The final concentrations during the assay will be 1 &#181;M).</li>
		<li>Incubate cartridge 30 min &#8211; 1 hr at 37 &#176;C in a non-CO2 incubator.</li>
		<li>Simultaneously, turn on the XF Analyzer and create the assay protocol template.</li>
		<li>Place cartridge and utility plate into XF Analyzer and run &#8220;calibrate&#8221; cartridge.</li>
		<li>Check crypt organoids microscopically to make sure they are attached to the gelatinous protein mixture.</li>
		<li>Load cell culture plate into instrument and run assay protocol.</li>
	</ol>
	</li>
</ol>

<ol>
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We tested the oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of crypts isolated from 8-month old mice and grown into organoids ex vivo. After measurement of the basal rate, crypt metabolism was evaluated by adding oligomycin, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP) and rotenone, sequentially.
Basal OCR and basal ECAR were recorded from 0 - 29 min (Figure 2A and 2B). At the 29th min, oligomyci...

Colorectal cancer (CRC) is the third leading cause of cancer related deaths in the United States. Sporadic colon cancer &#8211; i.e. that arising later in life (&#62;50 years of age) and with no clear predisposing genetic factors &#8211; accounts for ~80% of all cases, with incidence strongly influenced by long term dietary patterns1,2. These tumors exhibit a metabolic shift towards dependence on oxidative glycolysis, known as the Warburg effect, which may in part make higher concentrations of cellular building blocks and energy available (through glutaminolysis) to permit and perhaps drive high rates of tumor cell proliferation3-5. Studies of colon cancer as well as other gastrointestinal cancers including small intestine cancers provide important insight into the cause of tumor formation. Investigating the metabolic differences between normal, pro-tumorigenic and tumorigenic states of gastrointestinal organ systems may assist determination of relative risk for tumor development as well as early detection of neoplasia. Moreover, understanding bioenergetic metabolism involving mitochondrial respiration and glycolysis will provide fundamental insight into how cell physiology, aging and disease state perturbs intestinal homeostasis. Utilization of the bioenergetics assay technology for extracellular flux analysis can assess the rates of mitochondrial respiration and glycolysis simultaneously in cells growing in culture in real time6,7.
Until recently, in vitro studies of small intestine were limited to cell lines derived from either benign or malignant tumors8,9 and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. In 2009, Sato et al.10 introduced an ex vivo culture system to grow three-dimensional (3D) mouse intestinal epithelial organoids, or epithelial &#8220;mini-guts&#8221;, suitable for experimental, diagnostic and therapeutic investigations10,11. Moreover, crypts isolated from calorically restricted mice maintain their altered growth properties as organoids in such cultures12. Compared to transformed cell lines, crypt organoid cultures can be used to generate physiologically relevant data presenting a far better model to understand the in vivo state.
We adapted bioenergetics analysis technology to assay energy metabolism of intestinal crypt organoids. Mouse intestinal crypt organoids were cultured ex vivo to develop the crypt organoid energy metabolism studies presented. The oxygen consumption rate (OCR) and the extracellular acidification rate (ECAR) of crypt organoids were measured in the absence and presence of two different metabolic inhibitors (oligomycin, rotenone) and an ion carrier (carbonyl cyanide-p-trifluoromethoxyphenylhydrazone). The crypt organoid metabolic response to these chemical compounds were successfully reflected through changing ECAR and OCR values.
Cellular bioenergetic studies will elucidate the reciprocal interactions between metabolic state and disease risk and phenotype in cancer, obesity, diabetes, metabolic disorders and mitochondrial diseases and help advance screening methods with direct implications for translational medicine. Here, we describe a detailed protocol to isolate small intestinal crypts and to culture crypt organoids. Moreover, we introduce a novel method to use crypt organoid cultures for metabolic assays.

<table><tbody><tr><td>BD Matrigel Basement Membrane Matrix, GFR, Phenol Red-free, LDEV-free</td><td>BD Biosciences</td><td>356231</td><td></td></tr><tr><td>PBS (phosphate buffered saline), no magnesium, no calcium, pH 7.2</td><td>Life Technologies</td><td>20012-027</td><td></td></tr><tr><td>Advanced DMEM/F-12 (1x)</td><td>Life Technologies</td><td>12634-028</td><td></td></tr><tr><td>Dulbecco&amp;prime;s Modified Eagle&amp;prime;s Medium w/o glucose, L-glutamine, phenol red, sodium pyruvate, and sodium bicarbonate</td><td>Sigma-Aldrich</td><td>D5030</td><td></td></tr><tr><td>Phenol red sodium salt</td><td>Sigma-Aldrich</td><td>P4758</td><td>Final Concentration 15 mg/l in DMEM (D5030) - &lt;strong&gt;step 2.2.2&lt;/strong&gt;</td></tr><tr><td>Antibiotic-Antimycotic, 100x, 100 ml</td><td>Life Technologies</td><td>15240-062</td><td>Final concentration 1x or 2x</td></tr><tr><td>Penicilin-Streptomycin, liquid</td><td>Life Technologies</td><td>15140-122</td><td>Final concentration 1x</td></tr><tr><td>Gibco&amp;reg; GlutaMAX&amp;trade; supplement</td><td>Life Technologies</td><td>35050061</td><td>Final concentration 1x</td></tr><tr><td>Gibco&amp;reg; HEPES (N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid), 1 M</td><td>Life Technologies</td><td>15630-080</td><td>Final concentration 10 mM</td></tr><tr><td>N-acetyl-L-cysteine, 25 g</td><td>Sigma-Aldrich</td><td>A9165-25G</td><td>Final concentration 1 mM</td></tr><tr><td>100x N-2 supplement, liquid</td><td>Invitrogen</td><td>17502-048</td><td>Final concentration 1x</td></tr><tr><td>50x B-27&amp;reg; supplement minus Vitamin A, liquid</td><td>Invitrogen</td><td>12587-010</td><td>Final concentration 1x</td></tr><tr><td>Recombinant Mouse R-Spondin 1, CF, 50 &amp;mu;g</td><td>R&amp;amp;D Systems</td><td>3474-RS-050</td><td>Final concentration 500 ng/ml</td></tr><tr><td>Recombinant Murine EGF, 100 &amp;mu;g</td><td>Peprotech</td><td>315-09</td><td>Final concentration 50 ng/ml</td></tr><tr><td>Recombinant Murine Noggin, 20 &amp;mu;g</td><td>Peprotech</td><td>250-38</td><td>Final concentration 100 ng/ml</td></tr><tr><td>Gibco&amp;reg; L-glutamine, 200 mM</td><td>Life Technologies</td><td>25030-081</td><td>Final concentration 2 mM</td></tr><tr><td>Gibco&amp;reg; glucose powder</td><td>Life Technologies</td><td>15023-021</td><td>Final concentration 5 mM</td></tr><tr><td>Ambion&amp;reg; 0.5 M EDTA (ethylenediaminetetraacetic acid), pH 8.0</td><td>Life Technologies</td><td>AM9260G</td><td>Final concentration 3 mM for &lt;strong&gt;step 1.1.5&lt;/strong&gt;; 2 mM for &lt;strong&gt;step 1.1.8&lt;/strong&gt;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>DTT (dithiothreitol), 1M</td><td>Life Technologies</td><td>P2325</td><td>Final concentration 3 mM</td></tr><tr><td>Albumin from bovine serum (BSA)</td><td>Sigma-Aldrich</td><td>A2058</td><td>0.1% in PBS</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Life Technologies</td><td>16000-044</td><td>1% in PBS</td></tr><tr><td>Recovery&amp;trade; Cell Culture Freezing Medium</td><td>Life Technologies</td><td>12648-010</td><td></td></tr><tr><td>ROCK inhibitor (Y-27632)</td><td>Sigma-Aldrich</td><td>Y0503</td><td>Final concentration 10 &amp;mu;M</td></tr><tr><td>Oligomycin</td><td>Sigma-Aldrich</td><td>O4876</td><td>Final concentration 1 &amp;mu;M</td></tr><tr><td>Carbonyl cyanide-p-trifluoro-methoxy-phenyl-hydrazone (FCCP)</td><td>Sigma-Aldrich</td><td>C2920</td><td>Final concentration 1 &amp;mu;M</td></tr><tr><td>Rotenone</td><td>Sigma-Aldrich</td><td>R8875</td><td>Final concentration 1 &amp;mu;M</td></tr><tr><td>Sodium hydroxide</td><td>Sigma-Aldrich</td><td>221465</td><td>Final concentration 0.1 N in PBS</td></tr><tr><td>XF24 Extracellular Flux Analyzer (XF Analyzer)</td><td>Seahorse Bioscience</td><td></td><td></td></tr></tbody></table>

real time analysis of metabolic profile in ex vivo mouse intestinal crypt organoid cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Crypt organoids were established from 8 month old C57BL/6 mice fed purified rodent diet American Institute of Nutrition 76A (AIN76A). Intestinal crypt organoids can be grown in culture for extended periods from a single crypt (Figure 1A, single red arrow). Organoids grow out crypt-like structures in 18-20 days in culture (Figure 1B, red arrows). Crypts were passaged every 3 weeks and organoids efficiently recovered following each passage.
Seahorse bioenergetic...

Watch this Scientific Journal Video about Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures at JoVE.com

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

real-time-analysis-metabolic-profile-ex-vivo-mouse-intestinal-crypt

Research

JoVE Journal

Biology

16.2K Views.  Albert Einstein College of Medicine. Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

Video: Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to manipulate gene expression in organoids have made limited progress in this arena. While CRISPR/Cas9 technology allows for precise genome editing of cells for organoid generation, this strategy requires extensive selection and screening by sequence analysis, which is both time-consuming and costly. Here, we provide a detailed protocol for efficient viral transduction of intestinal organoids. This approach is rapid and highly efficient, thus decreasing the time and expense inherent in CRISPR/Cas9 technology. We also present a protocol to generate frozen sections from intact organoid cultures for further analysis with immunohistochemical or immunofluorescent staining, which can be used to confirm gene expression or silencing. After successful transduction of viral vectors for gene expression or silencing is achieved, intestinal stem cell and crypt function can be rapidly assessed. Although most organoid studies employ in vitro assays, organoids can also be delivered to mice for in vivo functional analyses. Moreover, our approaches are advantageous for predicting therapeutic responses to drugs because currently available therapies generally function by modulating gene expression or protein function rather than altering the genome.

We describe step-by-step instructions to: 1) efficiently engineer intestinal organoids using magnetic nanoparticles for lenti- or retroviral transduction, and, 2) generate frozen sections from engineered organoids. This approach provides a powerful tool to efficiently alter gene expression in organoids for investigation of downstream effects.

Intestinal organoid cultures provide a unique opportunity to investigate intestinal stem cell and crypt biology in vitro, although efficient approaches to ...

Developmental Biology

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

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for maintaining local and systemic homeostasis; changes to gut microbiota composition (dysbiosis) associate with a wide array of human diseases. Methods for dissecting host-microbiota interactions encompass an inherent tradeoff among preservation of physiological tissue structure (when using in vivo animal models) and the level of control over the experiment factors (as in simple in vitro cell culture systems). To address this tradeoff, Yissachar et al. recently developed an intestinal organ culture system. The system preserves a naive colon tissue construction and cellular mechanisms and it also permits tight experimental control, facilitating experimentations that cannot be readily performed in vivo. It is optimal for dissecting short-term responses of various gut components (such as epithelial, immunological and neuronal elements) to luminal perturbations (including anaerobic or aerobic microbes, whole microbiota samples from mice or humans, drugs and metabolites). Here, we present a detailed description of an optimized protocol for organ culture of multiple gut fragments using a custom-made gut culture device. Host responses to luminal perturbations can be visualized by immunofluorescence staining of tissue sections or whole-mount tissue fragments, fluorescence in-situ hybridization (FISH), or time-lapse imaging. This system supports a wide array of readouts, including next-generation sequencing, flow cytometry, and various cellular and biochemical assays. Overall, this three-dimensional organ culture system supports the culture of large, intact intestinal tissues and has broad applications for high-resolution analysis and visualization of host-microbiota interactions in the local gut environment.

This article presents a unique method for analyzing host-microbiome interactions using a novel gut organ culture system for ex vivo experiments.

The structure of the gut tissue facilitates close and mutualistic interactions between the host and the gut microbiota. These cross-talks are crucial for ...

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in response to infection or injury at the mucosal level as they are involved in antimicrobial responses and wound healing. They are also critical secondary messengers, regulating several pathways, including cell growth and differentiation. On the other hand, excessive ROS levels lead to oxidative stress, which can be deleterious for cells and favor intestinal diseases like chronic inflammation or cancer. This work provides a straightforward method to detect ROS in the intestinal murine organoids by live imaging and flow cytometry, using a commercially available fluorogenic probe. Here the protocol describes assaying the effect of compounds that modulate the redox balance in intestinal organoids and detect ROS levels in specific intestinal cell types, exemplified here by the analysis of the intestinal stem cells genetically labeled with GFP. This protocol may be used with other fluorescent probes.

The present protocol describes a method to detect reactive oxygen species (ROS) in the intestinal murine organoids using qualitative imaging and quantitative cytometry assays. This work can be potentially extended to other fluorescent probes to test the effect of selected compounds on ROS.

Reactive oxygen species (ROS) play essential roles in intestinal homeostasis. ROS are natural by-products of cell metabolism. They are produced in ...

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. ...

Intestinal Crypts Isolation: A Method to Isolate Whole Crypts from Small Intestine of Murine Model

Source: Schewe, M. et al. <a href="https://www.jove.com/video/56329" target="_blank">The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells.</a> J. Vis. Exp. (2017)
In this video, we demonstrate a technique to isolate whole crypts from a mouse&#8217;s small intestine. The isolated whole crypts can be used to propagate long-lived and self-renewing 3D organoids in vitro.

Izolacja krypt jelitowych: metoda izolowania całych krypt z jelita cienkiego modelu myszy

Source: Schewe, M. et al. The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells. J. Vis. Exp. (2017)
In ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

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Genetic Engineering of Primary Mouse Intestinal Organoids Using Magnetic Nanoparticle Transduction Viral Vectors for Frozen Sectioning

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

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

An Intestinal Gut Organ Culture System for Analyzing Host-Microbiota Interactions

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Analyzing Oxidative Stress in Murine Intestinal Organoids using Reactive Oxygen Species-Sensitive Fluorogenic Probe

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

Generation and Manipulation of Rat Intestinal Organoids

Izolacja krypt jelitowych: metoda izolowania całych krypt z jelita cienkiego modelu myszy