Name: Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling (Video) | JoVE Journal
Uploaded: 2025-03-28T14:57:00.000+00:00
Duration: 5 min 38 s
Description: Learn to generate apical-out intestinal organoids from adult dog stem cells and assess proliferation using EdU-labeling. This method enables accessible apical surface analysis while preserving 3D structure.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This research was supported using resources of the VetImaging Core Facility (VetCore, Vetmeduni, Austria). We want to thank Ursula Reichart for her support with semi-quantitative image analysis. GC is a recipient of a DOC fellowship (grant number 26349) of the Austrian Academy of Sciences (O&#776;AW) at the Division for Small Animal Internal Medicine at Vetmeduni.

Adult stem cell-derived intestinal organoids from dogs, established according to Kramer et al., 202014 were used. Based on the institutional ethics committee guidelines, the use of tissue material collected during therapeutic excision or post-mortem is included in the university&#39;s &#39;owner&#39;s consent for treatment&#39;, which was signed by all patient owners.
1. Organoid culture
<ol>
	<li>Culture adult stem cell-derived intestinal organoids embedded in the basement membrane extract (BME) matrix of choice in 24-well plates and split them mechanically using glass pipettes as described in detail in Pleguezuelos et al., 202015. 
	NOTE: For the protocol described here, Geltrex was used as the BME.</li>
	<li>Carefully remove the refined medium (Table 1) from the BME-embedded organoids.</li>
	<li>Add 500 &#181;L of 0.05 % trypsin-EDTA per well and detach all matrix domes from the well by pipetting up and down repeatedly. Transfer the organoids into a 15 mL tube and resuspend well to fully dissociate the hydrogel matrix.</li>
	<li>Incubate organoids with 0.05 % trypsin-EDTA at 37 &#176;C in a water bath until all organoids are dissociated into single cells or small clusters of cells.</li>
	<li>Dilute the trypsin/cell suspension with basal medium (Table 1). Use at least twice as much basal medium than trypsin.</li>
	<li>Centrifuge cells at 8 &#176;C at 420 &#215; g for 5 min. Remove as much supernatant as possible.</li>
	<li>Seed cells embedded in BME in as many wells as were used for trypsinization in step 1.3. and supply them with refined medium (Table 1).</li>
	<li>Incubate cells for 3 days in a cell culture incubator and proceed with step 2.1.</li>
</ol>
2. Induction of polarity reversal and EdU labeling
<ol>
	<li>Carefully remove the medium from the BME-embedded organoids.</li>
	<li>Add 500 &#181;L of organoid harvesting solution to each well and detach all matrix domes from the well by pipetting up and down repeatedly. Transfer the organoids into a 15 mL tube and resuspend well to fully dissociate the hydrogel matrix.</li>
	<li>Incubate the 15 mL tube on ice for 1.5 h. Shake the tube well every 10 min to prevent clumping of the organoids and ensure even dissociation of remaining hydrogel components.</li>
	<li>During incubation, coat a 96-well plate with anti-adherence solution. For each well of a 24-well plate trypsinized in step 1.3., coat 8 wells of a 96-well plate. Incubate for at least 1 h at room temperature (RT).</li>
	<li>Add at least twice as much PBS as organoid harvesting solution was used to the 15 mL tube and centrifuge at 8 &#176;C at 150 &#215; g for 5 min.</li>
	<li>Remove the supernatant and resuspend the organoids in 1 mL of PBS.</li>
	<li>Transfer 500 &#181;L of the organoid/PBS suspension in a separate 15 mL tube and centrifuge both tubes again at 8 &#176;C at 150 &#215; g for 5 min.</li>
	<li>Remove all anti-adherence solution from the 96-well plate.</li>
	<li>Remove as much supernatant as possible after centrifugation.</li>
	<li>Use one of the tubes for the generation of floating basal-out (BO) organoids and the other tube for apical-out (AO) organoids.</li>
	<li>For BO organoids, add 100 &#181;L of refined medium containing 7.5% BME per well of the 96-well plate to one tube. Mix well and disperse the organoids evenly across the desired number of wells of the pre-coated 96-well plate.</li>
	<li>For AO organoids, add 100 &#181;L plain refined medium (without any BME added) per well of the 96-well plate to the other tube. Mix well and disperse the organoids evenly across the desired number of wells of the pre-coated 96-well plate.</li>
	<li>Incubate the organoids at 37 &#176;C and 5% CO2&#160;for 3 days. 
	NOTE: Morphological differences will begin to be visible after day 1. However, previously published results show that it takes around 3 days for the vast majority of organoids to present apical-out polarity13.</li>
	<li>At defined time points, e.g., 24 h/48 h/72 h after induction of polarity reversal, add 50 &#181;L of 3 &#181;M EdU diluted in refined medium to all wells of organoids to be labeled, to receive a final concentration of 1 &#181;M EdU in each well.</li>
	<li>Incubate at 37 &#176;C and 5% CO2 for 1.5 h. </li>
	<li>Collect EdU-labelled organoids using wide-bore tips and transfer them into a 15 mL tube.</li>
	<li>Add an equal amount of 4% PFA to each tube (BO and AO organoids; final concentration of PFA = 2%) and mix carefully.</li>
	<li>Incubate at RT for 15 min.</li>
	<li>Add at least twice the amount of PBS as there is volume in the 15 mL tube and centrifuge at 8 &#176;C at 80 &#215; g for 5 min.</li>
	<li>Remove the supernatant and resuspend the organoids in 1 mL of PBS.</li>
	<li>Centrifuge again at 8 &#176;C at 80 &#215; g for 5 min.</li>
	<li>Remove the supernatant, resuspend the organoids in 1 mL of PBS, and transfer them into a 1.5 mL tube. Store the organoids at 4 &#176;C until performing the EdU staining reaction (see section 3).</li>
	<li>Repeat steps 2.14-2.22 for every time point to be analyzed.</li>
</ol>
3. Click-it EdU staining reaction and Hoechst 33342 staining
<ol>
	<li>Centrifuge the fixed organoids (from step 2.22.) in a 1.5 mL tube at 4 &#176;C at 50 &#215; g for 5 min.</li>
	<li>Remove the supernatant and resuspend organoids in 1 mL of 3% bovine serum albumin (BSA) diluted in PBS using wide bore tips.</li>
	<li>Repeat steps 3.1. and 3.2 (washing steps).</li>
	<li>Remove the supernatant and resuspend organoids in 1 mL of 0.5% Triton X-100 diluted in PBS.</li>
	<li>Incubate at RT for 20 min.</li>
	<li>Centrifuge organoids at RT at 50 &#215; g for 5 min.</li>
	<li>In a fresh tube, prepare 1x Buffer Additive by mixing 45 &#181;L of H2O with 5 &#181;L of 10x Buffer Additive.</li>
	<li>In another tube, prepare the Reaction Cocktail by mixing the following components: 385.8 &#181;L of H2O, 43 &#181;L of 10x Reaction Buffer, 20 &#181;L of CuSO4,1.2 &#181;L of Alexa Fluor 647 azide, and 50 &#181;L o 1x Buffer Additive (from step 3.7.). 
	NOTE: This mix is sufficient for five reactions and can be scaled up and down according to the required number of staining reactions.</li>
	<li>Repeat steps 3.1. and 3.2. twice (washing steps).</li>
	<li>Remove the supernatant and add 100 &#181;L of Reaction Cocktail (step 3.8.) into each tube with organoids.</li>
	<li>Incubate at RT for 30 min in the dark.</li>
	<li>Repeat steps 3.1. and 3.2 (washing steps).</li>
	<li>Centrifuge organoids at 50 &#215; g for 5 min at RT.</li>
	<li>Resuspend organoids in 1 mL of 10 &#181;g/mL Hoechst33342 diluted in PBS.</li>
	<li>Incubate at RT for 45 min in the dark.</li>
	<li>Repeat steps 3.1. and 3.2 (washing steps).</li>
	<li>Centrifuge organoids at 50 &#215; g for 5 min at 4 &#176;C.</li>
	<li>Seed organoids in the BME matrix of choice into a slide compatible with confocal microscopy (e.g., &#181;-Slide 18 well)</li>
	<li>Image organoids under a confocal microscope.</li>
</ol>
4. Semiautomatic image analysis
NOTE: For this analysis, arivis Pro version 4.2.2 (image analysis software) was used.
<ol>
	<li>Import the confocal microscope images into the image analysis software and define a folder into which the analysis file will be saved. Use the option Images as Time Points to import multiple images (replicate pictures, different time points, different treatments etc.). Each image will then be treated as a single time point, enabling easy shifting between the individual images.</li>
	<li>Open the analysis panel and import the analysis pipeline provided as Supplementary File 1&#160;(arivis_EdU_pipeline).</li>
	<li>Use the Place New Objects tool and Sphere with the tag Organoid to easily encircle all well-separated organoids.</li>
	<li>Switch to the next image, i.e., the next time point, and proceed to mark organoids for all images.</li>
	<li>For organoids that are very close to each other and can therefore not be separated using the Sphere mode, open the object list and activate the tag Organoid.</li>
	<li>Continue using the Draw Objects tool in Polygon mode to mark organoid outlines by hand. 
	NOTE: In the object list, all organoids are now marked and assigned to a specific time point. This time point corresponds to the order of images imported in step 4.1. These time points can now be renamed to match the original image name if preferred.</li>
	<li>In the object list, mark all objects (=organoids) and right click, then click Remove Tags to remove the Manual tag from all objects.</li>
	<li>Inactivate the Organoid tag in the object list and mark all areas within previously defined objects that should be excluded from the analysis using the Draw Objects tool in Polygon mode. These areas may include dead cells within the organoid lumen or blurry areas, which might skew the analysis.</li>
	<li>When opening the object list, ensure all organoids are listed with the Organoid tag and all areas that should be excluded have the tag Manual.</li>
	<li>Start the analysis pipeline by clicking the arrow in the top left panel. 
	NOTE: The software now segments all nuclei in the Hoechst33342 and EdU channels. This analysis pipeline only considers segmented Hoechst33342+ and EdU+ nuclei larger than 15 &#181;m2 in size (i.e., area in the nucleus segment) to ensure small nuclei, most likely originating from dead cells, are excluded from the analysis.</li>
	<li>Find the results of the analysis in the object list by clicking the Feature Columns tag.</li>
	<li>Switch to Master Detail View and select the Organoids tag in the upper panel and the feature First Timepoint.</li>
	<li>In the lower panel, select the features to display per organoid. Use Projection (x/y/z) Area (voxel), Mean intensity # 1&#160;(EdU channel), and SD Intensities #1.</li>
	<li>Export the results (Master Detail Report) using the Excel Export function.</li>
	<li>Save the analysis file and close the software.</li>
</ol>
5. Determination of organoid proliferation rates
<ol>
	<li>Open the Master Detail Report file exported in step 4.14.</li>
	<li>There is one separate tab for each analyzed image. Calculate the area/voxel sum for nuclei and EdU stain for each image. Then, copy all resulting data into a new collective tab and group all data referring to the same time point.</li>
	<li>Then, calculate the total area of cell nuclei (i.e., the sum of Hoechst33342+ area and EdU+ area) for each image.</li>
	<li>Subsequently, divide the total area by itself (= 100%) and the mean EdU+ area by the total area (=percentage of EdU+ DNA).</li>
	<li>Plot the percentage of proliferative DNA of BO and AO organoids on the y-axis and the different time points on the x-axis.</li>
</ol>

<ol><li>Sato, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459, 262-265 (2009).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((BMP+suppresses+Wnt+signaling+via+the+Bcl11b-regulated+NuRD+complex+to+maintain+intestinal+stem+cells%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">BMP suppresses Wnt signaling via the Bcl11b-regulated NuRD complex to maintain intestinal stem cells.</a> EMBO J. 43 (23), 6032-6051 (2024).</li>
<li>Gaowa, A., Leangpanich, S., Park, E. J., Kawamoto, E., Shimaoka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Irisin+promotes+intestinal+epithelial+cell+proliferation+via+Wnt%2F%CE%B2-catenin+and+focal+adhesion+kinase+signaling+pathways%5BTitle%5D)+AND+%22Sci+Rep%22%5BJournal%5D)">Irisin promotes intestinal epithelial cell proliferation via Wnt/β-catenin and focal adhesion kinase signaling pathways.</a> Sci Rep. 14, 25702(2024).</li>
<li>Basak, O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Induced+quiescence+of+Lgr5%2B+stem+cells+in+intestinal+organoids+enables+differentiation+of+hormone-producing+enteroendocrine+cells%5BTitle%5D)+AND+%22Cell+Stem+Cell%22%5BJournal%5D)">Induced quiescence of Lgr5+ stem cells in intestinal organoids enables differentiation of hormone-producing enteroendocrine cells.</a> Cell Stem Cell. 20 (2), 177-190 (2017).</li>
<li>Ludikhuize, M. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mitochondria+define+intestinal+stem+cell+differentiation+downstream+of+a+FOXO%2FNotch+axis%5BTitle%5D)+AND+%22Cell+Metab%22%5BJournal%5D)">Mitochondria define intestinal stem cell differentiation downstream of a FOXO/Notch axis.</a> Cell Metab. 32 (5), 889-900.e7 (2020).</li>
<li>Ettayebi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Replication+of+human+noroviruses+in+stem+cell-derived+human+enteroids%5BTitle%5D)+AND+%22Science+%281979%29%22%5BJournal%5D)">Replication of human noroviruses in stem cell-derived human enteroids.</a> Science (1979). 353 (6306), 1387-1393 (2016).</li>
<li>Zhou, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infection+of+bat+and+human+intestinal+organoids+by+SARS-CoV-2%5BTitle%5D)+AND+%22Nat+Med%22%5BJournal%5D)">Infection of bat and human intestinal organoids by SARS-CoV-2.</a> Nat Med. 26 (7), 1077-1083 (2020).</li>
<li>Joo, S. -S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Porcine+intestinal+apical-out+organoid+model+for+gut+function+study%5BTitle%5D)+AND+%22Animals%22%5BJournal%5D)">Porcine intestinal apical-out organoid model for gut function study.</a> Animals. 12 (3), 372(2022).</li>
<li>Co, J. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Controlling+epithelial+polarity%3A+A+human+enteroid+model+for+host-pathogen+interactions%5BTitle%5D)+AND+%22Cell+Rep%22%5BJournal%5D)">Controlling epithelial polarity: A human enteroid model for host-pathogen interactions.</a> Cell Rep. 26, 2509-2520.e4 (2019).</li>
<li>Sasaki, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Development+of+a+scalable+coculture+system+for+gut+anaerobes+and+human+colon+epithelium%5BTitle%5D)+AND+%22Gastroenterology%22%5BJournal%5D)">Development of a scalable coculture system for gut anaerobes and human colon epithelium.</a> Gastroenterology. 159 (1), 388-390.e5 (2020).</li>
<li>Mayorgas, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+strategy+to+study+the+invasive+capability+of+adherent-invasive+escherichia+coli+by+using+human+primary+organoid-derived+epithelial+monolayers%5BTitle%5D)+AND+%22Front+Immunol%22%5BJournal%5D)">A novel strategy to study the invasive capability of adherent-invasive escherichia coli by using human primary organoid-derived epithelial monolayers.</a> Front Immunol. 12, 646906(2021).</li>
<li>Csukovich, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neutralising+effects+of+different+antibodies+on+clostridioides+difficile+toxins+TcdA+and+TcdB+in+a+translational+approach%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Neutralising effects of different antibodies on clostridioides difficile toxins TcdA and TcdB in a translational approach.</a> Int J Mol Sci. 24 (4), 3867(2023).</li>
<li>Csukovich, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Polarity+reversal+of+canine+intestinal+organoids+reduces+proliferation+and+increases+cell+death%5BTitle%5D)+AND+%22Cell+Prolif%22%5BJournal%5D)">Polarity reversal of canine intestinal organoids reduces proliferation and increases cell death.</a> Cell Prolif. 57 (2), e13544(2023).</li>
<li>Kramer, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Generation+of+differentiating+and+long-living+intestinal+organoids+reflecting+the+cellular+diversity+of+canine+intestine%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">Generation of differentiating and long-living intestinal organoids reflecting the cellular diversity of canine intestine.</a> Cells. 9 (4), 822(2020).</li>
<li>Pleguezuelos-Manzano, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Establishment+and+culture+of+human+intestinal+organoids+derived+from+adult+stem+cells%5BTitle%5D)+AND+%22Curr+Protoc+Immunol%22%5BJournal%5D)">Establishment and culture of human intestinal organoids derived from adult stem cells.</a> Curr Protoc Immunol. 130 (1), e106(2020).</li>
<li>Dutta, D., Heo, I., O'Connor, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Studying+infection+in+3D+tissue-derived+human+organoid+culture+systems+by+microinjection%5BTitle%5D)+AND+%22J+Vis+Exp%22%5BJournal%5D)">Studying infection in 3D tissue-derived human organoid culture systems by microinjection.</a> J Vis Exp. (151), e59610(2019).</li>
<li>Kumar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Decreased+SLC26A3+expression+and+function+in+intestinal+epithelial+cells+in+response+to+Cryptosporidium+parvum+infection%5BTitle%5D)+AND+%22Am+J+Physiol+Cell+Physiol%22%5BJournal%5D)">Decreased SLC26A3 expression and function in intestinal epithelial cells in response to Cryptosporidium parvum infection.</a> Am J Physiol Cell Physiol. 317 (6), C1205-C1212 (2019).</li>
<li>Dutta, D., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Organoid+culture+systems+to+study+host-pathogen+interactions%5BTitle%5D)+AND+%22Curr+Opin+Immunol%22%5BJournal%5D)">Organoid culture systems to study host-pathogen interactions.</a> Curr Opin Immunol. 48, 15-22 (2017).</li>
<li>Csukovich, G., Pratscher, B., Burgener, I. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+world+of+organoids%3A+Gastrointestinal+disease+modelling+in+the+age+of+3R+and+one+health+with+specific+relevance+to+dogs+and+cats%5BTitle%5D)+AND+%22Animals%22%5BJournal%5D)">The world of organoids: Gastrointestinal disease modelling in the age of 3R and one health with specific relevance to dogs and cats.</a> Animals. 12 (18), 2461(2022).</li>
<li>Han, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Creating+a+more+perfect+union%3A+Modeling+intestinal+bacteria-epithelial+interactions+using+organoids%5BTitle%5D)+AND+%22Cell+Mol+Gastroenterol+Hepatol%22%5BJournal%5D)">Creating a more perfect union: Modeling intestinal bacteria-epithelial interactions using organoids.</a> Cell Mol Gastroenterol Hepatol. 12 (2), 769-782 (2021).</li>
<li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Next-generation+porcine+intestinal+organoids%3A+an+apical-out+organoid+model+for+swine+enteric+virus+infection+and+immune+response+investigations%5BTitle%5D)+AND+%22J+Virol%22%5BJournal%5D)">Next-generation porcine intestinal organoids: an apical-out organoid model for swine enteric virus infection and immune response investigations.</a> J Virol. 94 (21), e01006-e01020 (2020).</li>
<li>Smith, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+development+of+ovine+gastric+and+intestinal+organoids+for+studying+ruminant+host-pathogen+interactions%5BTitle%5D)+AND+%22Front+Cell+Infect+Microbiol%22%5BJournal%5D)">The development of ovine gastric and intestinal organoids for studying ruminant host-pathogen interactions.</a> Front Cell Infect Microbiol. 11, 733811(2021).</li>
<li>Nash, T. J., Morris, K. M., Mabbott, N. A., Vervelde, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inside-out+chicken+enteroids+with+leukocyte+component+as+a+model+to+study+host-pathogen+interactions%5BTitle%5D)+AND+%22Commun+Biol%22%5BJournal%5D)">Inside-out chicken enteroids with leukocyte component as a model to study host-pathogen interactions.</a> Commun Biol. 4 (1), 377(2021).</li>
<li>Hanson, K. I., Weiss, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Intestinal+tissue+response+to+Shiga+toxin+exposure%5BTitle%5D)+AND+%22mBio%22%5BJournal%5D)">Intestinal tissue response to Shiga toxin exposure.</a> mBio. 15 (9), e0123224(2024).</li>
<li>Hovhannisyan, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Infection+of+human+organoids+supports+an+intestinal+niche+for+Chlamydia+trachomatis%5BTitle%5D)+AND+%22PLoS+Pathog%22%5BJournal%5D)">Infection of human organoids supports an intestinal niche for Chlamydia trachomatis.</a> PLoS Pathog. 20 (8), e1012144(2024).</li>
</ol>

The authors declare no conflict of interest.

This protocol describes in detail how to induce polarity reversal in standard adult stem cell-derived intestinal organoid cultures. Apical-out organoids serve the purpose of gaining access to the apical cell surface, which is usually oriented towards the organoid lumen. Being able to probe the apical surface can be of high importance for certain applications as this is the portion of the cell membrane that is exposed to all digestive tract contents under physiologic conditions in vivo. Other methods to challenge...

Intestinal organoids are three-dimensional in vitro models recapitulating the intestinal epithelium comprising different cell types. These organoids can be easily established from adult stem cells isolated from intestinal crypts1. Since organoids are much closer to the in vivo epithelium, they are becoming increasingly important in biomedical research. Organoids of the intestine are not only used for the analysis of physiologic mechanisms (e.g., intestinal niche signaling2,3 and cell differentiation4,5) but also for research on infectious diseases6,7. However, growing polarized cells in 3D enclosing a central lumen is challenging, as the apical cell surface ends up inaccessible within the organoid lumen. Examining the differences between apical and basolateral cell surfaces can be important in metabolic studies, as exemplified by differences in fatty acid uptake8 and infectious disease research9,10,11,12.
The generation of so-called apical-out organoids is an easy option to overcome this problem. By removing the extracellular matrix from standard organoid cultures (i.e., basal-out, matrix-embedded organoids) and seeding these organoids in a matrix-free medium, a polarity switch can be induced9.
As we have previously published, most of the organoids invert their polarity within 12 h. However, it takes 48-72 h to obtain a culture with more than 90% apical-organoids. Despite their advantage of enabling access to the apical cell surface, apical-out organoids show significantly decreased proliferation after polarity reversal while rates of cell death increase13. The factor of proliferative activity can represent a confounding variable in various analyses and should be kept in mind when designing an experiment.
Here, we present a detailed protocol for establishing intestinal apical-out organoid cultures and floating basal-out control organoids for downstream analyses. Furthermore, we describe labeling with 5-ethynyl-2&#39;-deoxyuridine (EdU), incorporated into newly synthesized DNA and thus marks actively proliferating cells. We further describe the semiautomatic image analysis of the organoids&#39; proliferation rate using the software arivis Pro (Zeiss) by quantification of EdU+ cells. A schematic of the process is outlined in Figure 1.

<table><tbody><tr><td>24 well plates</td><td>Biologix</td><td>07-6024</td><td></td></tr><tr><td>[Leu15]-Gastrin I human, &amp;ge;95% (HPLC)</td><td>Sigma-Aldrich</td><td>G-9145</td><td></td></tr><tr><td>&amp;micro;-Slide 18 Well Glass Bottom</td><td>ibidi</td><td>81817</td><td></td></tr><tr><td>100X Penicillin-Streptomycin supplement for Media</td><td>Gibco/Thermo</td><td>15140122</td><td></td></tr><tr><td>A 83-01&amp;nbsp;</td><td>Tocris Bioscience</td><td>2939/10&amp;nbsp;</td><td></td></tr><tr><td>Anti-Adherence Rinsing Solution</td><td>StemCell Technologies</td><td>7010</td><td></td></tr><tr><td>arivis Pro&amp;nbsp;</td><td>Zeiss</td><td>Version 4.2.2</td><td></td></tr><tr><td>B27 SerumFree Supplement (50X), liquid</td><td>Gibco/Thermo</td><td>17504044</td><td></td></tr><tr><td>Bisbenzimide H 33342 (Hoechst 33342)</td><td>Abcam</td><td>ab145597</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Sigma-Aldrich</td><td>A7906-100G</td><td></td></tr><tr><td>Click-iT EdU Cell Proliferation Kit for Imaging, Alexa Fluor 647 dye</td><td>Thermo Scientific</td><td>C10340</td><td></td></tr><tr><td>DMEM-F12</td><td>Gibco/Thermo</td><td>11320033</td><td></td></tr><tr><td>Geltrex</td><td>Gibco/Thermo</td><td>A1413202</td><td></td></tr><tr><td>GlutaMAX Supplement&amp;nbsp;</td><td>Gibco/Thermo</td><td>35050061</td><td></td></tr><tr><td>HEPES Buffer Solution 1 M, liquid</td><td>Gibco/Thermo</td><td>15630056</td><td></td></tr><tr><td>Human FGF-basic</td><td>PeproTech</td><td>100-18B-100&amp;micro;G</td><td></td></tr><tr><td>Human HGF&amp;nbsp;</td><td>PeproTech</td><td>100-39-100&amp;micro;G&amp;nbsp;</td><td></td></tr><tr><td>Human IGF-I&amp;nbsp;</td><td>PeproTech</td><td>100-11-100&amp;micro;G</td><td></td></tr><tr><td>Human NOGGIN (Mammalian)&amp;nbsp;</td><td>PeproTech</td><td>120-10C-200&amp;micro;G&amp;nbsp;</td><td></td></tr><tr><td>N-Acetyl-L-cysteine,cell culture tested, BioReagent&amp;nbsp;</td><td>Sigma-Aldrich</td><td>A9165-5G&amp;nbsp;</td><td></td></tr><tr><td>Organoid Harvesting Solution</td><td>Thermo</td><td>C10340</td><td></td></tr><tr><td>Triton(TM) X-100,for molecular biology</td><td>Sigma-Aldrich</td><td>T8787-250ML</td><td></td></tr><tr><td>Trypsin-EDTA (0.05%), phenol red</td><td>Gibco/Thermo</td><td>25300054</td><td></td></tr></tbody></table>

generation of apical-out intestinal organoids and assessment of organoid proliferation rate using edu-labeling

Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

Here, we describe the generation of floating cultures of apical-out intestinal organoids from hydrogel-embedded intestinal organoid cultures. Concurrently, floating basal-out organoid cultures are established for direct comparison between apical-out and basal-out organoids. Apical-out and basal-out organoids are subsequently subjected to the thymidine analog 5-ethynyl-2&#39;-deoxyuridine (EdU), which is integrated into the newly synthesized DNA during the S-phase of cell division. This incorporation into DNA can be&#160;visualized&#160;in morphologically intact organoids using laser scanning confocal microscopy. Cells labeled with Hoechst33342 and EdU are then quantified in a semiautomatic manner using image analysis software. Calculation of the percentage of EdU-positive cells of the total number of cells allows for the analysis of cell proliferation in three-dimensional (3D) organoids. Despite being used here for the analysis of proliferation in intestinal organoids, the protocol is applicable to the analysis of nucleus-specific stainings of various sorts in other organoids or two-dimensional cell cultures as well.

Organoids grown for 3 days after trypsinization should be ideally between 50-250 µm, as depicted in Figure 2A. Organoids that are considerably larger than this may not reverse their polarity efficiently. Larger organoids may also start budding, and we have noticed that these organoids can have problems with efficient polarity reversal as well. Basal-out and apical-out organoids present obvious morphological differences already in brightfield imaging. While basal-out orga...

Here, we describe a protocol for generating apical-out intestinal organoids from standard matrix-embedded organoid cultures. It also outlines the subsequent incorporation of EdU into actively proliferating cells and the semiautomatic quantification of EdU-positive cells.

Here, we describe the generation of floating cultures of apical-out intestinal organoids from hydrogel-embedded intestinal organoid cultures. ...

generation-apical-out-intestinal-organoids-assessment-organoid

Research

JoVE Journal

Biology

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In vitro Organoid Culture of Primary Mouse Colon Tumors

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.

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

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

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

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

Generation of Mosaic Mammary Organoids by Differential Trypsinization

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

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

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

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

Here we present two protocols that allow the modeling of intestinal apical-specific interactions. Organoid-derived intestinal monolayers and Air-Liquid Interface (ALI) cultures facilitate the generation of well-differentiated epithelia accessible from both luminal and basolateral sides, whereas polarity-inverted intestinal organoids expose their apical side and are amenable to high throughput assays.

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

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.

Generation and Manipulation of Rat Intestinal Organoids

This study outlines a protocol for generating 2D monolayers of porcine organoids derived from the small and large intestines. The growth of these monolayers is marked by increasing TEER values, indicating robust epithelial integrity. Additionally, these monolayers exhibit physiological secretory responses in Ussing chamber experiments following the application of forskolin.

Two-dimensional Porcine Intestinal Organoids Reflecting the Physiological Properties of Native Gut

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.

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Learn to generate apical-out intestinal organoids from adult dog stem cells and assess proliferation using EdU-labeling. This method enables accessible apical surface analysis while preserving 3D structure.

Generation of Apical-Out Intestinal Organoids and Assessment of Organoid Proliferation Rate Using EdU-Labeling

Explore More Videos