University of Pennsylvania Genomic Medicine T32 HG009495 (KHB), NCI R21 CA267949 (BWK), Men &#38; BRCA Program at the Basser Center for BRCA (KHB, BWK), DeGregorio Family Foundation Grant Award (BWK).

All human tissue utilized in this protocol was collected from individuals who provided informed consent for tissue collection through a gastric tissue collection study approved by the University of Pennsylvania Institutional Review Board (IRB #842961). Participants in this study were required to undergo an upper endoscopy as part of their routine care, be at least 18 years old, and be able to provide informed consent. All research conducted adhered to the guidelines set forth by the University of Pennsylvania.
1. Experimental preparation
<ol>
	<li>Prepare conditioned L-WRN media as previously described16. Although not mandatory, the concentrations of Wnt-3A, R-spondin, and noggin contained in the conditioned media can be checked using a commercially available ELISA kits following the manufacturer&#39;s instructions (see Table of Materials).</li>
	<li>Prepare RPMI-Antibiotics (RPMI-ABX), PBS-Antibiotics (PBS-ABX), PBS-Dithiothreitol (PBS-DTT), digestion buffer, and gastric organoid media (see Supplementary Table 1 for detail composition). Gastric organoid media is stable for 1 week at 4 &#176;C.</li>
	<li>Autoclave forceps, fine dissection scissors, and 1.5 mL tubes.</li>
	<li>Begin thawing the basement membrane matrix (Matrigel) on ice.</li>
	<li>Begin thawing the digestion buffer and Trypsin-EDTA in a 37 &#176;C water bath.</li>
	<li>Set an incubator orbital shaker to 37 &#176;C and 200 rpm.</li>
</ol>
2. Isolating single cells from the biopsy tissue
<ol>
	<li>Collect gastric biopsies during an upper endoscopy17 following the institutional clinical protocol, likely involving the use of jumbo forceps. Utilize a blunt-tipped needle to extract tissue samples from the forceps and place them in a 15 mL conical tube containing RPMI-ABX media. Keep the tube on ice for rapid transfer to the laboratory. 
	NOTE: At minimum, 2-4 biopsies should be collected.</li>
	<li>Allow the biopsy tissue to settle at the bottom of the conical tube. Use a pipette to aspirate the media and wash the biopsies twice with 1 mL of PBS-ABX buffer. There is no need for centrifugation, as the tissue pieces settle naturally in the conical tube.</li>
	<li>Transfer a minimum of 20 mg of biopsy tissue to a 1.5 mL tube containing 1 mL of PBS-DTT.</li>
	<li>Use fine dissection scissors to cut the tissue into pieces that are 1-2 mm or smaller.</li>
	<li>Utilize a tabletop minicentrifuge for 15 s to aggregate tissue pieces at the tube&#39;s bottom and aspirate as much supernatant as possible. A small residual volume of PBS-DTT is acceptable.</li>
	<li>Add 5 mL of freshly warmed digestion buffer to a 50 mL conical tube. To transfer the small tissue pieces without losing tissue, take 500 &#181;L of the digestion buffer and add it to the 1.5 mL tube containing the tissue. Next, modify the tip of a 1000 &#181;L pipette tip with scissors to increase the tip&#39;s diameter, allowing for easy aspiration and transfer of tissue pieces to the 50 mL conical tube containing the digestion buffer.</li>
	<li>Incubate the digestion buffer and tissue mix at 37 &#176;C for 30 min with orbital shaking at 200 rpm.</li>
	<li>Add 5 mL of warmed trypsin with 0.25% EDTA to the digestion buffer and incubate for an additional 10 min at 37 &#176;C with orbital shaking at 200 rpm.</li>
	<li>Neutralize the digestion buffer and trypsin by adding an equal volume of Advanced (Adv.) DMEM/F12 media and pass the solution through a 70 &#181;m cell strainer.</li>
	<li>Pellet the cells by centrifugation at 1400 x g for 4 min at 4 &#176;C. Depending on the initial size of the biopsy tissue, a small cell pellet may or may not be visible.</li>
	<li>Resuspend the cell pellet in 1 mL of Adv. DMEM/F12 media and count the number of viable cells using Trypan Blue and a hemocytometer.</li>
	<li>Pellet the cells again through centrifugation at 1400 x g for 4 min at 4 &#176;C and remove the supernatant. 
	&#8203;NOTE: Figure 1 presents a schematic representation of the process for isolating single cells from the biopsy tissue.</li>
</ol>
3. Embedding single cells in a basement membrane matrix &#34;dome&#34;
<ol>
	<li>Based on the counted number of viable cells, calculate the volume of basement membrane matrix required to achieve a final concentration of 105 viable cells per 50 &#181;L of basement membrane matrix.</li>
	<li>Perform the following swiftly to prevent the basement membrane matrix from polymerizing before plating. Remove the thawed basement membrane matrix from ice, add the previously calculated volume of basement membrane matrix to the cells, and gently mix by pipetting up and down for approximately 10 s. 
	NOTE: It is crucial to avoid creating bubbles during this step. Do not dilute the basement membrane matrix.</li>
	<li>Rapidly pipette 50 &#181;L aliquots of the basement membrane matrix/cell mixture into the center of individual wells in a 24-well tissue culture plate.</li>
	<li>Immediately cover the 24-well plate and, in a single smooth motion, flip the plate upside-down. Place the inverted plate in a 37 &#176;C tissue culture incubator for 35 min to allow the basement membrane matrix to polymerize. 
	NOTE: Inverting the plate is essential to prevent the cells from sinking to the bottom of the plate and to enable the basement membrane matrix to polymerize into a 3D &#34;dome&#34; shape.</li>
	<li>Add 500 &#181;L of pre-warmed gastric organoid media to each well, ensuring that the top of each &#34;dome&#34; is fully submerged in the media. Dispense the media down the side of each well to avoid disturbing the &#34;dome.&#34;</li>
	<li>Change the media every 2-3 days.</li>
</ol>
4. Routine passaging of organoids&#160;&#160;via fragmentation
<ol>
	<li>Once the organoids are ready for passaging, remove the media from each designated well. 
	NOTE: To determine the appropriate time for passaging, please refer to the Representative Results section.</li>
	<li>Dispense 1 mL of ice-cold Adv. DMEM/F12 media directly onto a basement membrane matrix &#34;dome.&#34; This should readily initiate the breakup of the &#34;dome.&#34; Continue aspirating and dispensing until all fragments of the &#34;dome&#34; detach from the plate.</li>
	<li>Transport both the media and the fragmented basement membrane matrix to the next well, repeating this process for all wells slated for passaging. After disassembling the final basement membrane matrix &#34;dome,&#34; dispense the media and the mixture of organoids and basement membrane matrix into a 1.5 mL tube.</li>
	<li>Attach a 1000 &#181;L tip to a P1000 pipette, and then insert this tip into a 200 &#181;L tip. This will create a pipette tip with a small enough diameter to fragment the organoids while still allowing the aspiration of a larger volume.</li>
	<li>Vigorously pipette the mixture of organoids and basement membrane matrix up and down approximately 25 times to fragment the organoids into small pieces.</li>
	<li>Centrifuge the fragmented organoid mixture using a tabletop centrifuge at 4 &#176;C for 30 s at 2000 x g. This will result in a pellet of fragmented organoids separated from the basement membrane matrix and media. Use a pipette to aspirate the media and basement membrane matrix supernatant. Do not use a vacuum to aspirate the supernatant, as the pellet is loose.</li>
	<li>Calculate the volume of freshly thawed basement membrane matrix required so that the number of wells/domes is split at a 1:2 ratio (50 &#181;L/dome). Add the fresh basement membrane matrix and gently pipette up and down to mix, taking care to avoid creating bubbles.</li>
	<li>Swiftly aliquot 50 &#181;L of the mixture of organoid fragments and basement membrane matrix into individual wells of a 24-well plate.</li>
	<li>Cover the plate, flip it upside down, and place it in a 37 &#176;C tissue culture incubator for 35 min to allow the basement membrane matrix to polymerize.</li>
	<li>Add 500 &#181;L of pre-warmed gastric organoid media to each well. 
	NOTE: Figure 2 presents a schematic overview of the passaging of gastric patient-derived organoids through fragmentation.</li>
</ol>

Generating Human Gastric Organoids from Upper Endoscopy Biopsies

<ol>
	<li>Drost, J., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+in+cancer+research.">Organoids in cancer research.</a> Nature Reviews Cancer. 18 (7), 407-418 (2018).</li><li>Corr&#242;, C., Novellasdemunt, L., Li, V. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+brief+history+of+organoids.">A brief history of organoids.</a> American Journal of Physiology-Cell Physiology. 319 (1), C151-C165 (2020).</li><li>Zhao, Z., 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=Organoids.">Organoids.</a> Nature Reviews Methods Primers. 2 (1), 94 (2022).</li><li>Weeber, F., 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=Preserved+genetic+diversity+in+organoids+cultured+from+biopsies+of+human+colorectal+cancer+metastases.">Preserved genetic diversity in organoids cultured from biopsies of human colorectal cancer metastases.</a> Proceedings of the National Academy of Sciences. 112 (43), 13308-13311 (2015).</li><li>Boretto, 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=Patient-derived+organoids+from+endometrial+disease+capture+clinical+heterogeneity+and+are+amenable+to+drug+screening.">Patient-derived organoids from endometrial disease capture clinical heterogeneity and are amenable to drug screening.</a> Nature Cell Biology. 21 (8), 1041-1051 (2019).</li><li>Lo, Y. H., Karlsson, K., Kuo, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Applications+of+organoids+for+cancer+biology+and+precision+medicine.">Applications of organoids for cancer biology and precision medicine.</a> Nature Cancer. 1 (8), 761-773 (2020).</li><li>Gr&#246;nholm, 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=Patient-derived+organoids+for+precision+cancer+immunotherapy.">Patient-derived organoids for precision cancer immunotherapy.</a> Cancer research. 81 (12), 3149-3155 (2021).</li><li>Yan, H. 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=A+comprehensive+human+gastric+cancer+organoid+biobank+captures+tumor+subtype+heterogeneity+and+enables+therapeutic+screening.">A comprehensive human gastric cancer organoid biobank captures tumor subtype heterogeneity and enables therapeutic screening.</a> Cell Stem Cell. 23 (6), 882-897 (2018).</li><li>Yoon, C., 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=Patient-derived+organoids+from+locally+advanced+gastric+adenocarcinomas+can+predict+resistance+to+neoadjuvant+chemotherapy.">Patient-derived organoids from locally advanced gastric adenocarcinomas can predict resistance to neoadjuvant chemotherapy.</a> Journal of Gastrointestinal Surgery. 27 (4), 666-676 (2023).</li><li>Miao, X., 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=Establishment+of+gastric+cancer+organoid+and+its+application+in+individualized+therapy.">Establishment of gastric cancer organoid and its application in individualized therapy.</a> Oncology Letters. 24 (6), 1-8 (2022).</li><li>Pompaiah, M., Bartfeld, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+organoids:+an+emerging+model+system+to+study+Helicobacter+pylori+pathogenesis.">Gastric organoids: an emerging model system to study Helicobacter pylori pathogenesis.</a> Molecular Pathogenesis and Signal Transduction by Helicobacter pylori. 400, 149-168 (2017).</li><li>Schlaermann, 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=A+novel+human+gastric+primary+cell+culture+system+for+modelling+Helicobacter+pylori+infection+in+vitro.">A novel human gastric primary cell culture system for modelling Helicobacter pylori infection in vitro.</a> Gut. 65 (2), 202-213 (2016).</li><li>Bartfeld, 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=In+vitro+expansion+of+human+gastric+epithelial+stem+cells+and+their+responses+to+bacterial+infection.">In vitro expansion of human gastric epithelial stem cells and their responses to bacterial infection.</a> Gastroenterology. 148 (1), 126-136 (2015).</li><li>Seidlitz, T., Koo, B. K., Stange, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gastric+organoids-an+in+vitro+model+system+for+the+study+of+gastric+development+and+road+to+personalized+medicine.">Gastric organoids-an in vitro model system for the study of gastric development and road to personalized medicine.</a> Cell Death &amp; Differentiation. 28 (1), 68-83 (2021).</li><li>Bartfeld, S., Clevers, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids+as+model+for+infectious+diseases:+culture+of+human+and+murine+stomach+organoids+and+microinjection+of+Helicobacter+pylori.">Organoids as model for infectious diseases: culture of human and murine stomach organoids and microinjection of Helicobacter pylori.</a> Journal of Visualized Experiments. 105, e53359 (2015).</li><li>Miyoshi, H., Stappenbeck, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+expansion+and+genetic+modification+of+gastrointestinal+stem+cells+in+spheroid+culture.">In vitro expansion and genetic modification of gastrointestinal stem cells in spheroid culture.</a> Nature Protocols. 8 (12), 2471-2482 (2013).</li><li>Yang, H. 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=Sample+collection+methods+in+upper+gastrointestinal+research.">Sample collection methods in upper gastrointestinal research.</a> Journal of Korean Medical Science. 38 (32), e255 (2023).</li><li>Kim, 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=Comparison+of+cell+and+organoid-level+analysis+of+patient-derived+3D+organoids+to+evaluate+tumor+cell+growth+dynamics+and+drug+response.">Comparison of cell and organoid-level analysis of patient-derived 3D organoids to evaluate tumor cell growth dynamics and drug response.</a> SLAS DISCOVERY: Advancing the Science of Drug Discovery. 25 (7), 744-754 (2020).</li><li>Maru, Y., Tanaka, N., Itami, M., Hippo, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+use+of+patient-derived+organoids+as+a+preclinical+model+for+gynecologic+tumors.">Efficient use of patient-derived organoids as a preclinical model for gynecologic tumors.</a> Gynecologic Oncology. 154 (1), 189-198 (2019).</li><li>McGowan, K. P., Delgado, E., Hibdon, E. S., Samuelson, L. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+sensitivity+to+Wnt+signaling+gradients+in+human+gastric+organoids+derived+from+corpus+and+antrum.">Differential sensitivity to Wnt signaling gradients in human gastric organoids derived from corpus and antrum.</a> American Journal of Physiology-Gastrointestinal and Liver Physiology. 325 (2), G158-G173 (2023).</li><li>Busslinger, G. 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=Human+gastrointestinal+epithelia+of+the+esophagus,+stomach,+and+duodenum+resolved+at+single-cell+resolution.">Human gastrointestinal epithelia of the esophagus, stomach, and duodenum resolved at single-cell resolution.</a> Cell Reports. 34 (10), 108819 (2021).</li><li>Yang, 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=A+quick+and+reliable+image-based+AI+algorithm+for+evaluating+cellular+senescence+of+gastric+organoids.">A quick and reliable image-based AI algorithm for evaluating cellular senescence of gastric organoids.</a> Cancer Biology &amp; Medicine. 20 (7), 519 (2023).</li><li>Skubleny, 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=Murine+and+Human+gastric+tissue+establishes+organoids+after+48+hours+of+cold+ischemia+time+during+shipment.">Murine and Human gastric tissue establishes organoids after 48 hours of cold ischemia time during shipment.</a> Biomedicines. 11 (1), 151 (2023).</li></ol>

The authors have no relevant disclosures.

Herein, a detailed protocol for reliably generating human gastric organoids from single cells isolated from biopsies of benign epithelium from the gastric body and antrum is outlined. Critical steps in the protocol revolve around timing as well as handling the basement membrane matrix. To preserve viability, it is essential to initiate the protocol as soon as possible after acquiring the biopsy tissue. The aim is to start digesting the biopsy tissue within 30 min of the biopsy being performed. Handling the basement membr...

Organoids are miniature three-dimensional (3D) cellular structures that resemble the architecture and functionality of the organs from which they were derived1,2. These lab-grown models are created by cultivating stem cells or tissue-specific cells in a controlled environment that allows these cells to self-organize and differentiate into various cell types1,2,3. One of the key advantages of organoids is their ability to recapitulate human biology more closely than traditional two-dimensional (2D) cell cultures1,2,3. In particular, human organoids have been shown to maintain the genetic diversity of their tissue of origin3,4,5. Organoids offer a unique opportunity to study human organ development, model diseases, and test potential therapeutics in a controlled laboratory setting. Furthermore, organoids can be derived from individual patient samples, enabling personalized medicine approaches and the potential development of individualized treatments3,6,7.
Researchers have used human gastric organoids to investigate various aspects of gastric biology and pathology. Prominent examples include the use of patient-derived organoids (PDOs) to predict gastric cancer chemotherapy responses8,9,10 and model the epithelial response to Helicobacter pylori infection11,12,13. Human gastric organoids consist of various cell types found in the stomach, including neck cells, pit cells, and other supporting cells11,14. Gastric organoids can either be generated from induced pluripotent stem cells (iPSCs) or stem cells directly isolated from gastric tissue obtained via biopsies or from gastric resection specimens11,14. The isolation of gastric stem cells from gastric tissue is commonly done by isolating and culturing gastric glands or enzymatically digesting tissue samples to liberate single cells9,13,15. Importantly, the differentiation of cells within gastric organoids generated using either of these techniques has been shown to be similar13. The protocol described herein focuses on a single-cell digest.
Organoids represent a scientific innovation that bridges the gap between traditional cell culture and whole organs. As research in the field continues to progress, organoids are poised to contribute to the development of more effective treatments and therapies for a wide range of applications. Given the rising utilization of gastric PDOs, there is a timely need for a standardized approach to their generation. Here, the protocol for generating human gastric PDOs from single cells isolated from benign gastric biopsy tissue acquired during upper endoscopy is described. Importantly and uniquely, a standardized number of single cells is determined for seeding to reliably generate gastric PDOs and allow subsequent characterization. Using this technique, reliable differences in the formation and growth of organoids generated from biopsies of either the gastric body or gastric antrum are demonstrated.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>R&#38;D Systems</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Gibco</td>
			<td>12634-010</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Invitrogen</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Invitrogen</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>BZ-X710</td>
			<td>Keyence</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>cellSens</td>
			<td>Olympus</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase III</td>
			<td>Worthington</td>
			<td>LS004182</td>
			<td></td>
		</tr>
		<tr>
			<td>Dispase II</td>
			<td>Sigma</td>
			<td>D4693-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithiothreitol (DTT)</td>
			<td>EMSCO/Fisher</td>
			<td>BP1725</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14200-075</td>
			<td></td>
		</tr>
		<tr>
			<td>Fungin</td>
			<td>InvivoGen</td>
			<td>NC9326704</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin I</td>
			<td>Sigma Aldrich</td>
			<td>G9145</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Invitrogen</td>
			<td>1570060</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>hEGF</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Invitrogen</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>hFGF-10</td>
			<td>Peprotech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>L-WRN Cell Line</td>
			<td>ATCC</td>
			<td>CRL-3276</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Corning</td>
			<td>47743-715</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole</td>
			<td>MP Biomedicals</td>
			<td>155710</td>
			<td></td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502048</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin ELISA Kit</td>
			<td>Novus Biologicals</td>
			<td>NBP2-80296</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen Strep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>11875-085</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Spondin ELISA Kit</td>
			<td>R&#38;D Systems</td>
			<td>DY4120-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Wnt-3a ELISA Kit</td>
			<td>R&#38;D Systems</td>
			<td>DY1324B-05</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma Aldrich</td>
			<td>Y0503</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

The subsequent representative results are derived from biopsies taken from the benign epithelium of both the gastric body and gastric antrum regions of the stomachs of five different patients undergoing upper endoscopy. Two to four &#34;domes&#34;/wells were plated and analyzed per patient for both gastric body and antrum biopsies. Organoids were successfully generated from the gastric body and gastric antrum biopsy tissue from all five patients. On average, 41 organoids were analyzed per &#34;dome&#34;/well. All images ...

Watch this Scientific Journal Video about Generation of and Comparison Between Patient-Derived Gastric Organoids from Different Regions of the Stomach at JoVE.com

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.

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 ...

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JoVE Journal

Biology

1.3K Views.  University of Pennsylvania Perelman School of Medicine. 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.

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

MicroRNA Expression Profiles of Human iPS Cells, Retinal Pigment Epithelium Derived From iPS, and Fetal Retinal Pigment Epithelium

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human iPS cells (iPS-RPE), and fetal RPE. The protocols include collection of RNA for analysis by microarray, and the analysis of microarray data to identify miRNAs that are differentially expressed among three cell types. The methods for culture of iPS cells and fetal RPE are explained. The protocol used for differentiation of RPE from human iPS is also described. The RNA extraction technique we describe was selected to allow maximal recovery of very small RNA for use in a miRNA microarray. Finally, cellular pathway and network analysis of microarray data is explained. These techniques will facilitate the comparison of the miRNA profiles of three different cell types.

The microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, retinal pigment epithelium (RPE) derived from human induced-pluripotent stem (iPS) cells (iPS-RPE), and fetal RPE, were compared.

The objective of this report is to describe the protocols for comparing the microRNA (miRNA) profiles of human induced-pluripotent stem (iPS) cells, ...

Isolation and Characterization of Microvesicles from Peripheral Blood

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived microvesicles (MVs, diameter &#62; 100 nm) is a fundamental cellular process that occurs in all living cells. These vesicles transport proteins, lipids and nucleic acids specific for their cell of origin and in vitro studies have highlighted their importance as mediators of intercellular communication. EVs have been successfully isolated from various body fluids and especially EVs in blood have been identified as promising biomarkers for cancer or infectious diseases. In order to allow the study of MV subpopulations in blood, we present a protocol for the standardized isolation and characterization of MVs from peripheral blood samples. MVs are pelleted from EDTA-anticoagulated plasma samples by differential centrifugation and typically possess a diameter of 100 - 600 nm. Due to their larger size, they can easily be studied by flow cytometry, a technique that is routinely used in clinical diagnostics and available in most laboratories. Several examples for quality control assays of the isolated MVs will be given and markers that can be used for the discrimination of different MV subpopulations in blood will be presented.

Extracellular vesicles present in blood have been suggested as novel biomarkers for various diseases. Here, we present a protocol for the isolation of large plasma membrane-derived microvesicles from peripheral blood samples and their subsequent analysis by conventional flow cytometry and Western Blotting.

The release of extracellular vesicles (EVs) including small endosomal-derived exosomes (Exos, diameter &#60; 100 nm) and large plasma membrane-derived ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle biopsies are often collected for these endeavors. However, relatively few technical descriptions of biopsy procedures, outside of the commonly used musculus vastus lateralis, are available. Although the biopsy techniques are often adjusted to accommodate the characteristics of each muscle under study, few technical reports share these changes to the greater community. Thus, muscle tissue from human participants is often wasted as the operator reinvents the wheel. Expanding the available material on biopsies from a variety of muscles can reduce the incident of failed biopsies. This technical report describes a variation of the modified Bergstr&#246;m technique on the musculus tibialis anterior that limits fiber damage and provides fiber lengths adequate for mechanical evaluation. The surgery is an outpatient procedure that can be completed in an hour. The recovery period for this procedure is immediate for light activity (i.e., walking), up to three days for the resumption of normal physical activity, and about one week for wound care. The extracted tissue can be used for mechanical force experiments and here we present representative activation data. This protocol is appropriate for most collection purposes, potentially adaptable to other skeletal muscles, and may be improved by modifications to the collection needle.

This technical report describes a variation of the modified Bergstr&#246;m technique for the biopsy of the musculus tibialis anterior that limits fiber damage.

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...

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 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 ...

Establishment and Genetic Manipulation of Murine Hepatocyte Organoids

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the executor for most of the liver functions, primary hepatocytes have limited proliferating capacity. This requires the establishment of ex vivo hepatocyte expansion models for liver physiological and pathological research. Here, we isolated murine hepatocytes by two steps of collagenase perfusion and established a 3D organoid culture as the 'mini-liver' to recapitulate cell-cell interactions and physical functions. The organoids consist of heterogeneous cell populations including progenitors and mature hepatocytes. We introduce the process in detailed to isolate and culture the murine hepatocytes or fetal hepatocyte to form organoids within 2-3 weeks and show how to passage them by mechanically pipetting up and down. In addition, we will also introduce how to digest the organoids into single cells for lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering. The organoids can be used for drug screens, disease modelling, and basic liver research by modeling liver biology and pathobiology.

A long-term ex vitro 3D organoid culture system was established from mouse hepatocytes. These organoids can be passaged and genetically manipulated by lentivirus infection of shRNA/ectopic construction, siRNA transfection and CRISPR-Cas9 engineering.

The liver is the largest organ in mammals. It plays an important role in glucose storage, protein secretion, metabolism and detoxification. As the ...

Efficient and Consistent Generation of Retinal Pigment Epithelium/Choroid Flatmounts from Human Eyes for Histological Analysis

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and vision. Proteins on the RPE apical surface are tightly associated with proteins on the photoreceptor outer segment surface, making it difficult to consistently separate the RPE from the photoreceptors/retina. We developed a method to efficiently separate the retina from the RPE of human eyes to generate complete RPE/choroid and retina flatmounts for separate cellular analysis of the photoreceptors and RPE cells. An intravitreal injection of a high-osmolarity solution of D-mannitol, a sugar not transported by the RPE, induced the separation of the RPE and retina across the entire posterior chamber without causing damage to the RPE cell junctions. No RPE patches were observed attached to the retina. Phalloidin labeling of actin showed RPE shape preservation and allowed morphometric analysis of the entire epithelium. An artificial intelligence (AI)-based software was developed to accurately recognize and segment the RPE cell borders and quantify 30 different shape metrics. This dissection method is highly reproducible and can be easily extended to other animal models.

We describe a method to efficiently separate retinal pigment epithelium (RPE) from the retina in human eyes and generate whole RPE/choroid flatmounts for histological and morphometric analyses of the RPE.

The retinal pigment epithelium (RPE) and retina are functionally and structurally connected tissues that work together to regulate light perception and ...

Extraction, Labeling, and Purification of Lineage-Specific Cells from Human Antral Follicles

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary but also across multiple points of control within the hypothalamic/pituitary/ovarian circuit. Within the ovary, multiple specialized cell types grow in close association with the oocyte within the ovarian follicles. The biology of these cells has been well described at the later stages, when they are easily recovered as byproducts of assisted reproductive treatments. However, the in-depth analysis of small antral follicles isolated directly from the ovary is not commonly carried out due to the scarcity of human ovarian tissue and the limited access to the ovary in patients undergoing assisted reproductive treatments. 
These methods for processing whole ovaries for the cryopreservation of cortical strips with the concurrent identification/isolation of ovary resident cells enable the high-resolution analysis of the early stages of antral follicle development. We demonstrate protocols for isolating discrete cell types by treating antral follicles enzymatically and separating the granulosa, theca, endothelial, hematopoietic, and stromal cells. The isolation of cells from the antral follicles at various sizes and developmental stages enables the comprehensive analysis of the cellular and molecular mechanisms that drive follicle growth and ovarian physiology and provides a source of viable cells that can be cultured in vitro to recapitulate the follicle microenvironment.

Here, we present protocols for the identification and purification of ovarian cells from antral follicles. We elaborate on methods for processing whole ovaries for the cryopreservation of cortical strips while also harvesting intact antral follicles that are treated enzymatically to liberate multiple follicle resident cell types, including granulosa, theca, endothelial, hematopoietic, and stromal cells.

The activation, growth, development, and maturation of oocytes is a complex process that is coordinated not just between multiple cell types of the ovary ...

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 ...

Establishment, Maintenance, Differentiation, Genetic Manipulation, and Transplantation of Mouse and Human Lacrimal Gland Organoids

The lacrimal gland is an essential organ for ocular surface homeostasis. By producing the aqueous part of the tear film, it protects the eye from desiccation stress and external insults. Little is known about lacrimal gland (patho)physiology because of the lack of adequate in vitro models. Organoid technology has proven itself as a useful experimental platform for multiple organs. Here, we share a protocol to establish and maintain mouse and human lacrimal gland organoids starting from lacrimal gland biopsies. By modifying the culture conditions, we enhance lacrimal gland organoid functionality. Organoid functionality can be probed through a "crying" assay, which involves exposing the lacrimal gland organoids to selected neurotransmitters to trigger tear release in their lumen. We explain how to image and quantify this phenomenon. To investigate the role of genes of interest in lacrimal gland homeostasis, these can be genetically modified. We thoroughly describe how to genetically modify lacrimal gland organoids using base editors-from guide RNA design to organoid clone genotyping. Lastly, we show how to probe the regenerative potential of human lacrimal gland organoids by orthotopic implantation in the mouse. Together, this comprehensive toolset provides resources to use mouse and human lacrimal gland organoids to study lacrimal gland (patho)physiology.

This protocol describes how to establish, maintain, genetically modify, differentiate, functionally characterize, and transplant lacrimal gland organoids derived from primary mouse and human tissue.