Name: 3d culturing of organoids from the intestinal villi epithelium undergoing dedifferentiation
Uploaded: 2021-04-01T13:00:00
Description: Watch this Scientific Journal Video about 3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation at JoVE.com

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

All the mouse experiments conducted, including the use of Tamoxifen and euthanasia by cervical dislocation, had the approval of the Institutional Animal Care and Use Committee at Stevens Institute of echnology.
1. Mice
NOTE: The generation of Smad4f/f; Catnblox(ex3)/+; Villin-CreERT2 mice have been previously described3. Adult female mice between eight to twelve weeks of agewere used...

<ol>
	<li>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 (7244), 262-265 (2009).</li><li>Schwitalla, 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=Intestinal+tumorigenesis+initiated+by+dedifferentiation+and+acquisition+of+stem-cell-like+properties.">Intestinal tumorigenesis initiated by dedifferentiation and acquisition of stem-cell-like properties.</a> Cell. 152 (1-2), 25-38 (2013).</li><li>Perekatt, A. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SMAD4+Suppresses+WNT-Driven+Dedifferentiation+and+Oncogenesis+in+the+Differentiated+Gut+Epithelium.">SMAD4 Suppresses WNT-Driven Dedifferentiation and Oncogenesis in the Differentiated Gut Epithelium.</a> Cancer Research. 78 (17), 4878-4890 (2018).</li><li>Roche, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+a+purified+epithelial+cell+population+from+human+colon.">Isolation of a purified epithelial cell population from human colon.</a> Methods in Molecular Medicine. 50, 15-20 (2001).</li><li>Aoki, R., 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=Foxl1-expressing+mesenchymal+cells+constitute+the+intestinal+stem+cell+niche.">Foxl1-expressing mesenchymal cells constitute the intestinal stem cell niche.</a> Cellular and Molecular Gastroenterology and Hepatology. 2 (2), 175-188 (2016).</li><li>Seiler, K. 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=Tissue+underlying+the+intestinal+epithelium+elicits+proliferation+of+intestinal+stem+cells+following+cytotoxic+damage.">Tissue underlying the intestinal epithelium elicits proliferation of intestinal stem cells following cytotoxic damage.</a> Cell and Tissue Research. 361 (2), 427-438 (2015).</li><li>Stzepourginski, I., 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=CD34++mesenchymal+cells+are+a+major+component+of+the+intestinal+stem+cells+niche+at+homeostasis+and+after+injury.">CD34+ mesenchymal cells are a major component of the intestinal stem cells niche at homeostasis and after injury.</a> Proceedings of the National Academy of Sciences of the United States of America. 114 (4), 506-513 (2017).</li><li>Roulis, 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=Paracrine+orchestration+of+intestinal+tumorigenesis+by+a+mesenchymal+niche.">Paracrine orchestration of intestinal tumorigenesis by a mesenchymal niche.</a> Nature. 580 (7804), 524-529 (2020).</li><li>Haramis, A. P., 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=De+novo+crypt+formation+and+juvenile+polyposis+on+BMP+inhibition+in+mouse+intestine.">De novo crypt formation and juvenile polyposis on BMP inhibition in mouse intestine.</a> Science. 303 (5664), 1684-1686 (2004).</li><li>Madison, B. B., 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=Epithelial+hedgehog+signals+pattern+the+intestinal+crypt-villus+axis.">Epithelial hedgehog signals pattern the intestinal crypt-villus axis.</a> Development. 132 (2), 279-289 (2005).</li><li>van Es, J. 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=Dll1++secretory+progenitor+cells+revert+to+stem+cells+upon+crypt+damage.">Dll1+ secretory progenitor cells revert to stem cells upon crypt damage.</a> Nature Cell Biology. 14 (10), 1099-1104 (2012).</li><li>Tetteh, P. W., 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=Replacement+of+Lost+Lgr5-Positive+Stem+Cells+through+Plasticity+of+Their+Enterocyte-Lineage+Daughters.">Replacement of Lost Lgr5-Positive Stem Cells through Plasticity of Their Enterocyte-Lineage Daughters.</a> Cell Stem Cell. 18 (2), 203-213 (2016).</li><li>Baulies, A., 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=The+Transcription+Co-Repressors+MTG8+and+MTG16+Regulate+Exit+of+Intestinal+Stem+Cells+From+Their+Niche+and+Differentiation+Into+Enterocyte+vs+Secretory+Lineages.">The Transcription Co-Repressors MTG8 and MTG16 Regulate Exit of Intestinal Stem Cells From Their Niche and Differentiation Into Enterocyte vs Secretory Lineages.</a> Gastroenterology. 159 (4), 1328-1341 (2020).</li><li>Zeilstra, 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=Stem+cell+CD44v+isoforms+promote+intestinal+cancer+formation+in+Apc(min)+mice+downstream+of+Wnt+signaling.">Stem cell CD44v isoforms promote intestinal cancer formation in Apc(min) mice downstream of Wnt signaling.</a> Oncogene. 33 (5), 665-670 (2014).</li><li>Gracz, A. D., 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=Brief+report:+CD24+and+CD44+mark+human+intestinal+epithelial+cell+populations+with+characteristics+of+active+and+facultative+stem+cells.">Brief report: CD24 and CD44 mark human intestinal epithelial cell populations with characteristics of active and facultative stem cells.</a> Stem Cells. 31 (9), 2024-2030 (2013).</li><li>Gao, N., White, P., Kaestner, K. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+intestinal+identity+and+epithelial-mesenchymal+signaling+by+Cdx2.">Establishment of intestinal identity and epithelial-mesenchymal signaling by Cdx2.</a> Developmental Cell. 16 (4), 588-599 (2009).</li><li>Verzi, M. P., Shin, H., Ho, L. L., Liu, X. S., Shivdasani, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Essential+and+redundant+functions+of+caudal+family+proteins+in+activating+adult+intestinal+genes.">Essential and redundant functions of caudal family proteins in activating adult intestinal genes.</a> Molecular and Cellular Biology. 31 (10), 2026-2039 (2011).</li><li>Verzi, M. P., Shin, H., San Roman, A. K., Liu, X. S., Shivdasani, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+master+transcription+factor+CDX2+controls+chromatin+access+for+partner+transcription+factor+binding.">Intestinal master transcription factor CDX2 controls chromatin access for partner transcription factor binding.</a> Molecular and Cellular Biology. 33 (2), 281-292 (2013).</li><li>Grainger, S., Hryniuk, A., Lohnes, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cdx1+and+Cdx2+exhibit+transcriptional+specificity+in+the+intestine.">Cdx1 and Cdx2 exhibit transcriptional specificity in the intestine.</a> PLoS One. 8 (1), 54757 (2013).</li><li>Stringer, E. 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=Cdx2+determines+the+fate+of+postnatal+intestinal+endoderm.">Cdx2 determines the fate of postnatal intestinal endoderm.</a> Development. 139 (3), 465-474 (2012).</li></ol>

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

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

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Advanced DMEM F-12 media</td>
			<td>Gibco</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>3,3-diaminobenzidine</td>
			<td>Vector Labs</td>
			<td>SK-4105</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well U-bottom plate</td>
			<td>Fisher Scientific</td>
			<td>FB012932</td>
			<td></td>
		</tr>
		<tr>
			<td>ABC kit</td>
			<td>Vector Labs</td>
			<td>&#160;PK4001</td>
			<td></td>
		</tr>
		<tr>
			<td>Angled scissor</td>
			<td>Fisher Scientific</td>
			<td>11-999</td>
			<td></td>
		</tr>
		<tr>
			<td>Animal-Free Recombinant Human EGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27 Supplement (50X), minus vitamin A</td>
			<td>Gibco</td>
			<td>12587010</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA) Protease-free Powder</td>
			<td>Fisher Scientific</td>
			<td>BP9703100</td>
			<td></td>
		</tr>
		<tr>
			<td>CD44 antibody</td>
			<td>BioLegend</td>
			<td>1030001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cdx2 antibody</td>
			<td>Cell Signaling</td>
			<td>12306</td>
			<td></td>
		</tr>
		<tr>
			<td>Corn oil</td>
			<td>Sigma-Aldrich</td>
			<td>C8267-500ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning 70-micron cell strainer</td>
			<td>Life Sciences</td>
			<td>431751</td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Reduced Growth Factor Basement Membrane Extract, Type R1</td>
			<td>R&#38;D</td>
			<td>3433-005-R1</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection scissors</td>
			<td>Fisher Scientific</td>
			<td>22-079-747</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fisher Scientific</td>
			<td>17-456-209</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax (100X)</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK 293-T cells expressing RSPO-1</td>
			<td>Gift from Dr. Michael Verzi</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1M)</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>Histogel</td>
			<td>Thermoscientific</td>
			<td>HG-4000-012</td>
			<td></td>
		</tr>
		<tr>
			<td>Mesh filter</td>
			<td>Fisher Scientific</td>
			<td>07-201-431</td>
			<td></td>
		</tr>
		<tr>
			<td>Micrscope glass slide</td>
			<td>VWR</td>
			<td>89218-844</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2 Supplement (100X)</td>
			<td>Gibco</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetyl cysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>p200 Blunt tips</td>
			<td>VWR</td>
			<td>46620-642</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin (10,000 U/mL)</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>Primocin (50mg/mL)</td>
			<td>Invivogen</td>
			<td>ant-pm-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Quality Biological Inc PBS (10X)</td>
			<td>Fisher Scientific</td>
			<td>50-146-770</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Noggin</td>
			<td>Peprotech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Signal diluent</td>
			<td>Cell Signaling</td>
			<td>8112L</td>
			<td></td>
		</tr>
		<tr>
			<td>Tamoxifen</td>
			<td>Sigma-Aldrich</td>
			<td>T5648-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>6-well tissue culture plate</td>
			<td>Fisher Scientific</td>
			<td>50-146-770</td>
			<td></td>
		</tr>
	</tbody>
</table>

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

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

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

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

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

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

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

This method can confirm the organoid-forming capacity of de-differentiating villi epithelium that acquire stem cell markers in vivo. The collection of villi is performed via scraping rather than the EDTA chelation method preventing the complete loss of the underlying mesenchyme that may provide the niche signals if required for organoid initiation. This method is applicable in epithelial tissues where proliferative and differentiated compartments are physically delineated and the de-differentiating cells express stem cell markers in vivo.Some factors in this protocol need to be determined empirically, including the optimal stage for harvesting the villi and the optimal pressure for scraping the villi, which will take time and practice. Induce the Smad4 knockout and beta-catenin gain of function mutation in mutant mice with intraperitoneal injection of tamoxifen and corn oil for four consecutive days. After euthanizing the mouse, spray the abdomen with 70%ethanol to prevent mouse fur from getting into the peritoneal cavity.Open the abdominal cavity with dissection scissors to expose the intestine and isolate the intestine using scissors and forceps. Dissect out the proximal half of the duodenum, then flush the duodenum with five milliliters of ice cold PBS in a 10 milliliter syringe to clear the luminal content. Open the duodenum longitudinally with an angled scissor and lay the duodenum flat on a 15 centimeter Petri dish on ice with the lumen of the duodenum facing the operator.Prior to beginning the scraping, place a 70 micrometer mesh strainer in one of the wells of a six-well tissue culture plate. Fill all the wells with four milliliters of 1X PBS and place the plate on ice. Scrape the villi using two microscopic glass slides, one to hold the duodenum down and the other to scrape.Scrape the luminal side of the duodenum superficially twice to remove the mucus without removing the villi, then scrape the duodenum two more times to collect the villi on the slides without tethering the crypts. Use a one milliliter transfer pipette containing PBS to transfer the villi to a 70 micrometer mesh strainer in the six-well dish. Collect the villi after every scrape.To remove loose crypts, wash the villi collected with the 70 micrometer strainer by transferring the strainer through a series of wells in the six-well dish containing four milliliters of ice cold PBS per well. Using a P1000 pipette, transfer the villi suspension in about three milliliters of PBS from the 70 micrometer strainer to a new 15 milliliter tube on ice. Use a 0.1%BSA-coated blunt-ended P200 pipette tip to transfer a 50 microliter volume of the villi suspension onto a glass slide.Count the number of villi in the 50 microliter droplet under 4X magnification to determine the concentration of villi in the PBS suspension and to confirm the absence of tethered crypts. To plate the villi, use a 0.1%BSA-coated P200 blunt-ended pipette tip to transfer the villi to a microcentrifuge tube for a plating density of six villi per well in 12.5 microliters of BME-R1 matrix. Spin down the villi for two minutes at 200 times G at four degrees Celsius.Remove the supernatant and repeat the centrifugation to remove any residual PBS. In a laminar flow hood, resuspend the villi pellet gently in the required amount of cold BME-R1 thawed on ice. Using a P20 pipette, plate 12.5 microliters of the villi in BME-R1 matrix per well of a 96-well U-bottomed plate pre-warmed to 37 degrees Celsius.Incubate the plate in a tissue culture incubator at 37 degrees Celsius for 15 minutes to allow solidification of the BME-R1 matrix. To each well, add 125 microliters of pre-warmed ENR media supplemented as described in the text manuscript. Incubate the plated villi in a tissue culture incubator at 37 degrees Celsius with 5%carbon dioxide and change the media every other day.Discard any well in which organoids appear before two days. There's a difference in appearance of the organoids emerging from the crypts versus the de-differentiated villi epithelium from the same mouse intestine. The crypt-derived organoids appear overnight as spherical structures with well-defined borders.The kinetics and morphological appearance of the organoids initiated from villi was examined, which appear irregularly shaped at first and take two to five days before they can be seen under a microscope. Organoid initiation from two different villi is shown. The villi with organoid-forming potential appears dense, possibly due to the retention of the underlying mesenchyme.Organoid initiation from the villas is apparent at day two from one of the villi. While in the other villas, the organoid appears at day four. It's crucial to take the proper steps to avoid crypt contamination when harvesting the villi and growing the resulting organoids.Phenotypic differences between the organoids emerging from the crypts and villi of the same mutant were observed following this procedure, so further experimentation could be carried out to look for molecular differences between the two. This protocol could be used to study the differences between organoids arising from endogenous crypts versus ectopic crypts arising from de-differentiation, thus implications of de-differentiation could be addressed.

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

Research

JoVE Journal

Biology

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 ...

Primary Cell Cultures from the Mouse Retinal Pigment Epithelium

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the eye. It is a single sheet of pigmented cells that are hexagonally packed and connected by tight junctions. The main functions of the RPE include absorption of light, phagocytosis of the shed photoreceptor outer segments, spatial buffering of ions, transport of nutrients, ions and water as well as active involvement in the visual cycle. With such important and diverse functions, it is critically important to study the biology of RPE cells. A number of RPE cell lines have been established; however, passaged and immortalized cells are known to quickly lose some of the morphological and physiological characteristics of natural RPE cells. Thus, primary cells are more suitable for studying different aspects of RPE cell biology and function. Mouse primary RPE cell culture is very useful to researchers since mouse models are widely used in biological studies, however collecting RPE cells from mouse is also very challenging due to their small size. Here, we present a protocol for establishing primary mouse RPE cell cultures which includes enucleation and dissection of the eyes and isolation of the RPE sheets to yield the cells for culturing. This method enables efficient cell recovery. The RPE cells obtained from two mice&#160;can reach confluency on one 12 mm polyester membrane insert pre-loaded in culture plate after one week of culture and display some of the original properties of bona fide RPE cells such as hexagonal shape and pigmentation after two weeks of culture.

The retinal pigment epithelium (RPE) is a multi-functional epithelium of the eye. Here we present a protocol to establish primary cell cultures derived from the murine RPE.

The retinal pigment epithelium (RPE) is a highly polarized multi-functional epithelium that is located between the neural retina and the choroid of the ...

Establishing 3D Endometrial Organoids from the Mouse Uterus

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of luminal and glandular epithelium, a stromal compartment, a vascular network, and a complex immune cell population. Mouse models have been a powerful tool to study the endometrium, revealing critical mechanisms that control implantation, placentation, and cancer. The recent development of 3D endometrial organoid cultures presents a state-of-the-art model to dissect the signaling pathways that underlie endometrial biology. Establishing endometrial organoids from genetically engineered mouse models, analyzing their transcriptomes, and visualizing their morphology at a single-cell resolution are crucial tools for the study of endometrial diseases. This paper outlines methods to establish 3D cultures of endometrial epithelium from mice and describes techniques to quantify gene expression and analyze the histology of the organoids. The goal is to provide a resource that can be used to establish, culture, and study the gene expression and morphological characteristics of endometrial epithelial organoids.

This protocol describes methodologies to establish mouse endometrial epithelial organoids for gene expression and histological analyses.

Endometrial tissue lines the inner cavity of the uterus and is under the cyclical control of estrogen and progesterone. It is a tissue that is composed of ...

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

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

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

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

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

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

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

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

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

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

Rat Intestinal Crypts Isolation and Intestinal Organoids Generation

Generation and Manipulation of Rat Intestinal Organoids

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

Author Spotlight: Generation and Manipulation of Rat Intestinal Organoids  

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

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

Generation of Rat Intestinal 2D Monolayers from 3D Organoids

To coat the plate with EME, dilute EME 1 to 20 in cold Advanced DMEM+For coating with collagen, dilute five milligrams per milliliter of collagen I in Advanced DMEM+to 100 micrograms per milliliter. Coat with the plate with 200 microliters of diluted EME or collagen to cover the well surface entirely and incubate for one to two hours at 37 degrees Celsius. To generate monolayers, aspirate the media from a 35 millimeter plate containing organoids.Add one milliliter of PBS and disrupt the EME in the wells with a P1000 tip, pipetting up and down approximately 20 times to loosen all the EME. Transfer the contents to a 15 milliliter conical tube. Add one milliliter of PBS to the plate to collect additional organoids and transfer them to the same conical tube.Centrifuge the sample at 350 G for two minutes and remove the supernatant, including any EME residue. Then, add one milliliter of trypsin solution to the organoid pellet and incubate at 37 degrees Celsius for two minutes. Pipette up and down 10 times using a P1000 tip and add two milliliters of Advanced DMEM+to neutralize trypsin.Centrifuge at 350 G for five minutes and aspirate the supernant before resuspending the pellet in 4.8 milliliters of rIOM2D. Remove excess EME or collagen in Advanced DMEM+from the wells. Then, add 200 microliters of organoids in rIOM2D and 10 micromolar Y27632 to each pre-coated well.After 4 to 16 hours, collect the media and centrifuge at 1, 000 G for one minute. Transfer the supernatant into a new 15 milliliter conical tube and discard the pellet. Wash each well with 300 microliters of PBS before adding 200 microliters of the centrifuged rIOM2D to each well.Change the rIOM2D every two to three days, suspending the use of Y27632.2D monolayers plated on the EME readily reformed into small spheroids when EME was added back to the top of the cells. In contrast, a collagen I substrate was insufficient to reform 3D structures.

Generating Human Gastric Organoids from Upper Endoscopy Biopsies

Establishment and Characterization of Patient-derived Gastric Organoids from Biopsies of Benign Gastric Body and Antral Epithelium

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a wide array of research applications. However, a shortage of published approaches exists for producing gastric PDOs from single-cell digests while maintaining a standardized initial cell seeding density. In this protocol, the emphasis is on the initiation of gastric organoids from isolated single cells and the provision of a method for passaging organoids through fragmentation. Importantly, the protocol demonstrates that a standardized approach to the initial cell seeding density consistently yields gastric organoids from benign biopsy tissue and allows for standardized quantification of organoid growth. Finally, evidence supports the novel observation that gastric PDOs display varying rates of formation and growth based on whether the organoids originate from biopsies of the body or antral regions of the stomach. Specifically, it is revealed that the use of antral biopsy tissue for organoid initiation results in a greater number of organoids formed and more rapid organoid growth over a 20-day period when compared to organoids generated from biopsies of the gastric body. The protocol described herein offers investigators a timely and reproducible method for successfully generating and working with gastric PDOs.

Author Spotlight: Generation of and Comparison Between Patient-Derived Gastric Organoids from Different Regions of the Stomach 

Gastric patient-derived organoids find increasing use in research, yet formal protocols for generating human gastric organoids from single-cell digests with standardized seeding density are lacking. This protocol presents a detailed method for reliably creating gastric organoids from biopsy tissue obtained during upper endoscopy.

Gastric patient-derived organoids (PDOs) offer a unique tool for studying gastric biology and pathology. Consequently, these PDOs find increasing use in a ...

Integrating Single-Cell Transcriptomics with Human Intestinal Organoids

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

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

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

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

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

Preparation of Single Cell Suspensions from Differentiated 3D Human Intestinal Organoids for Single Cell Transcriptomics

To begin, thaw the enzymatic cell digestion reagent at 37 degrees Celsius for 30 minutes. Under a brightfield microscope at 10x magnification, observe the differentiated three-dimensional human intestinal organoids to ensure cell health. Replace the media from the wells of a 24-well plate containing differentiated organoids with 500 microliters of cold cell recovery solution.Pipette up and down to release the cells from the basement membrane. Transfer the cell suspension to a 1.5-milliliter microcentrifuge tube. Incubate the tube on ice for 10 minutes, pipetting up and and down every five minutes.Centrifuge the cell suspension at 400 g for three minutes at four degrees Celsius. Remove the supernatant from the tube without disturbing the pellet. Resuspend the pellet in 500 microliters of prewarmed enzymatic cell digestion reagent, pipetting up and down 10 times.Place the organoids for 30 minutes at 37 degrees Celsius in a carbon dioxide incubator. Every 10 minutes, pipette the entire volume 10 times using a P1000 pipette to help dissociate the cells. Centrifuge the cell suspension at 400 g for three minutes at four degrees Celsius, and resuspend the pellet in one milliliter of differentiation media.Rapidly pipette the cells up and down 50 times on ice to dissociate organoids into single cells. Filter the entire volume through a 70 micron tip strainer and collect the eluate in a fresh 1.5-milliliter tube. Then filter the eluate obtained through a 70 micron strainer on a 40 micron tip strainer.Add five microliters of 0.4%Trypan Blue to five microliters of cell suspension, and count viable single cells using Trypan Blue Exclusion. Dilute the sample with cell culture media to obtain 1000 cells per microliter. A total of 4, 402 single cells were isolated from differentiated ileal three-dimensional human intestinal organoids, representing expected cell types, including absorptive enterocytes and secretory cells.