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><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&amp;amp;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&amp;amp;D Systems</td><td>DY4120-05</td><td></td></tr><tr><td>Wnt-3a ELISA Kit</td><td>R&amp;amp;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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Biology

1.3K Views.  University of Pennsylvania Perelman School of Medicine. Ons onderzoek richt zich op erfelijke oorzaken van maagkanker. In het bijzonder zijn we geïnteresseerd in erfelijke diffuse maagkanker als gevolg van ziekteverwekkende varianten in het CDH1- of CTNNA1-gen. We onderzoeken ook de rol van BRCA1 en BRCA2 in het risico op maagkanker en carcinogenese.Organoïden worden steeds vaker gebruikt in verschillende onderzoeksdomeinen en gastro-enterologie is daarop geen uitzondering, dus hiermee komt de behoefte aan standaardisatie van deze technie...

Video: Auteur in de schijnwerpers: genereren van en vergelijken tussen van patiënten afgeleide maagorganoïden uit verschillende delen van de maag 

Establishment of Gastric Cancer Patient-derived Xenograft Models and Primary Cell Lines

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. Although there are many established gastric cancer cell lines and many conventional transgenic mouse models for preclinical research, the disadvantages of these in vitro and in vivo models limit their applications. Because the characteristics of these models have changed in culture, they no longer model tumor heterogeneity, and their responses have not been able to predict responses in humans. Thus, alternative models that better represent tumor heterogeneity are being developed. Patient-derived xenograft (PDX) models preserve the histologic appearance of cancer cells, retain intratumoral heterogeneity, and better reflect the relevant human components of the tumor microenvironment. However, it usually takes 4-8 months to develop a PDX model, which is longer than the expected survival of many gastric patients. For this reason, establishing primary cancer cell lines may be an effective complementary method for drug response studies. The current protocol describes methods to establish PDX models and primary cancer cell lines from surgical gastric cancer samples. These methods provide a useful tool for drug development and cancer biology research.

The current protocol describes methods to establish patient-derived xenograft (PDX) models and primary cancer cell lines from surgical gastric cancer samples. The methods provide a useful tool for drug development and cancer biology research.

The use of preclinical models to advance our understanding of tumor biology and investigate the efficacy of therapeutic agents is key to cancer research. ...

Cancer Research

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts from Fresh Tissue

Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. Patient-derived tumor organoids have been used in translational cancer research and can be applied to assess treatment sensitivity and resistance, cell-cell interactions, and tumor cell interactions with the tumor microenvironment. Tumor organoids are complex culture systems that require advanced cell culture techniques and culture media with specific growth factor cocktails and a biological basement membrane that mimics the extracellular environment. The ability to establish primary tumor cultures highly depends on the tissue of origin, the cellularity, and the clinical features of the tumor, such as the tumor grade. Furthermore, tissue sample collection, material quality and quantity, as well as correct biobanking and storage are crucial elements of this procedure. The technical capabilities of the laboratory are also crucial factors to consider. Here, we report a validated SOP/protocol that is technically and economically feasible for the culture of ex vivo tumor organoids from fresh tissue samples of pancreatic adenocarcinoma origin, either from fresh primary resected patient donor tissue or patient-derived xenografts (PDX). The technique described herein can be performed in laboratories with basic tissue culture and mouse facilities and is tailored for wide application in the translational oncology field.

Author Spotlight: Establishment of Pancreatic Cancer-Derived Tumor Organoids and Fibroblasts From Fresh Tissue  

Tumor organoids have revolutionized cancer research and the approach to personalized medicine. They represent a clinically relevant tumor model that allows researchers to stay one step ahead of the tumor in the clinic. This protocol establishes tumor organoids from fresh pancreatic tumor tissue samples and patient-derived xenografts of pancreatic adenocarcinoma origin.

Tumor organoids are three-dimensional (3D) ex vivo tumor models that recapitulate the biological key features of the original primary tumor tissues. ...

Organoids as Model for Infectious Diseases: Culture of Human and Murine Stomach Organoids and Microinjection of Helicobacter Pylori

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. Three cellular sources have been used: Embryonic stem cells (ESC), induced pluripotent stem cells (iPSC) or adult stem cells. Here, culture of mouse and human gastric organoids derived from adult stem cells is described and used for infection with the gastric pathogen Helicobacter pylori. Human gastric glands are isolated from resection material, seeded in a basement matrix and embedded in medium containing growth factors epidermal growth factor (EGF), R-spondin, Noggin, Wnt, fibroblast growth factor (FGF) 10, gastrin and transforming growth factor (TGF) beta inhibitor. In these conditions, gastric glands grow into 3-dimensional organoids containing 4 lineages of the stomach. The organoids expand indefinitely and can be frozen and thawed similarly as cell lines. For infection studies, bacteria are microinjected into the lumen of the organoids. Infected organoids are processed for imaging. The described methods can be adapted to other organoids and infections with other bacteria, viruses or parasites. This allows the study of infection-induced changes in primary cells.

Stem cell derived cultures harbor tremendous potential to model infectious diseases. Here, the culture of mouse and human gastric organoids derived from adult stem cells is described. The organoids are microinjected with the gastric pathogen Helicobacter pylori.

Recently infection biologists have employed stem cell derived cultures to answer the need for new and better models to study host-pathogen interactions. ...

Immunology and Infection

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Medicine

Mouse- and Human-derived Primary Gastric Epithelial Monolayer Culture for the Study of Regeneration

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and migration. One critical limitation of such assays, however, is their homogenous assortment of cellular types. Repair is a complex process which demands the interaction of several cell types. Therefore, to study a culture devoid of cellular subtypes, is a concern that must be overcome if we are to understand the repair process. The gastric organoid model may alleviate this issue whereby the heterogeneous collection of cell types closely reflects that of the gastric epithelium or other native tissues in vivo. Demonstrated here is a novel, in vitro scratch-wound assay derived from human or mouse 3-dimensional organoids which can then be transferred to a gastric epithelial monolayer as either intact organoids or as a single cell suspension of dissociated organoids. The goal of the protocol is to establish organoid-derived gastric epithelial monolayers that can be used in a novel scratch-wound assay system to study gastric regeneration.

Here we describe a protocol for establishing and culturing human- and mouse-derived 3-dimensional (3D) gastric organoids, and the method for the transfer of 3D organoids to a 2-dimensional monolayer. The use of the gastric epithelial monolayer as a novel scratch-wound assay for regeneration studies is also described.

In vitro studies of gastric wound repair typically involves the use of gastric cancer cell lines in a scratch-wound assay of cellular proliferation and ...

A Preclinical Human-Derived Autologous Gastric Cancer Organoid/Immune Cell Co-Culture Model to Predict the Efficacy of Targeted Therapies

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to evade immune surveillance leading to the inhibition of CTL proliferation, survival, and effector function, and subsequently cancer persistence. Approximately 40% of gastric cancers express PD-L1, yet the response rate to immunotherapy is only 30%. We present the use of human-derived autologous gastric cancer organoid/immune cell co-culture as a preclinical model that may predict the efficacy of targeted therapies to improve the outcome of cancer patients. Although cancer organoid co-cultures with immune cells have been reported, this co-culture approach uses tumor antigen to pulse the antigen-presenting dendritic cells. Dendritic cells (DCs) are then cultured with the patient's CD8+ T cells to expand the cytolytic activity and proliferation of these T lymphocytes before co-culture. In addition, the differentiation and immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in culture are investigated within this co-culture system. This organoid approach may be of broad interest and appropriate to predict the efficacy of therapy and patient outcome in other cancers, including pancreatic cancer.

The goals of the protocol are to use this approach to 1) understand the role of the immunosuppressive gastric tumor microenvironment and 2) predict the efficacy of patient response, thus increasing the survival rate of patients.

Tumors expressing programmed cell death-ligand 1 (PD-L1) interact with programmed cell death protein 1 (PD-1) on CD8+ cytotoxic T lymphocytes (CTLs) to ...

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

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations as the former do not faithfully represent original tissue physiology, and the availability of the latter is limited. Hence, their application hampers fundamental and drug development research. Adult stem-cell-based organoids (henceforth referred to as organoids) are miniatures of normal or diseased epithelial tissue from which they are derived. They can be established very efficiently from different gastrointestinal (GI) tract regions, have long-term expandability, and simulate tissue- and patient-specific responses to treatments in vitro. Here, the establishment of intestinal organoid-derived epithelial monolayers has been demonstrated along with methods to measure epithelial barrier integrity, permeability and transport,&#160;antimicrobial protein secretion, as well as histology. Moreover, intestinal organoid-derived monolayers can be enriched with proliferating stem and transit-amplifying cells as well as with key differentiated epithelial cells. Therefore, they represent a model system that can be tailored to study the effects of compounds on target cells and their mode of action. Although organoid cultures are technically more demanding than cell lines, once established, they can reduce failures in the later stages of drug development as they truly represent in vivo epithelium complexity and interpatient heterogeneity.

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.

In the past, intestinal epithelial model systems were limited to transformed cell lines and primary tissue. These model systems have inherent limitations ...

Precise Luminal pH Measurement in 3D Gastric Organoids with Microelectrodes

Profiling Luminal pH in Three-Dimensional Gastrointestinal Organoids Using Microelectrodes

The optimization and detailed characterization of gastrointestinal organoid models require advanced methods for analyzing their luminal environments. This paper presents a highly reproducible method for the precise measurement of pH within the lumina of 3D human gastric organoids via micromanipulator-controlled microelectrodes. The pH microelectrodes are commercially available and consist of beveled glass tips of 25 &#181;m in diameter. For measurements, the pH microelectrode is advanced into the lumen of an organoid (&gt;200 &#181;m) that is suspended in Matrigel, while a reference electrode rests submerged in the surrounding medium in the culture plate. 
Using such microelectrodes to profile organoids derived from the human gastric body, we demonstrate that luminal pH is relatively consistent within each culture well at ~7.7 &plusmn; 0.037 and that continuous measurements can be obtained for a minimum of 15 min. In some larger organoids, the measurements revealed a pH gradient between the epithelial surface and the lumen, suggesting that pH measurements in organoids can be achieved with high spatial resolution. In a previous study, microelectrodes were successfully used to measure luminal oxygen concentrations in organoids, demonstrating the versatility of this method for organoid analyses. In summary, this protocol describes an important tool for the functional characterization of the complex luminal space within 3D organoids.

Author Spotlight: Gastric Epithelial Cell Responses in Helicobacter pylori infection

The present protocol describes pH measurements in human tissue-derived gastric organoids using microelectrodes for spatiotemporal characterization of intraluminal physiology.

The optimization and detailed characterization of gastrointestinal organoid models require advanced methods for analyzing their luminal environments. This ...

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

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of the human intestinal epithelium, in which they exhibit crypt/villus-like structures. Here, we describe the generation of hiPSC-derived intestinal organoids by the stepwise differentiation of hiPSCs into definitive endoderm, which is then posteriorized to form hindgut epithelium before being transferred into 3D culture conditions. The 3D culture environment consists of extracellular matrix (ECM) (e.g., Matrigel or other compatible ECM) supplemented with SB202190, A83-01, Gastrin, Noggin, EGF, R-spondin-1 and CHIR99021. Organoids undergo passaging every 7 days, where they are mechanically disrupted before transfer to fresh extracellular matrix and allowed to expand. QPCR and immunocytochemistry confirm that hiPSC-derived intestinal organoids contain mature intestinal epithelial cell types including goblet cells, Paneth cells and enterocytes. Additionally, organoids show evidence of polarization by expression of villin localized on the apical surface of epithelial cells.
The resulting organoids can be used to model human intestinal development as well as numerous human intestinal diseases including inflammatory bowel disease. To model intestinal inflammation, organoids can be exposed to inflammatory mediators such as TNF-&#945;, TGF-&#946;, and bacterial LPS. Organoids exposed to proinflammatory cytokines display an inflammatory and fibrotic phenotype in response. Pairing of healthy versus hiPSCs derived from patients with IBD may be useful in understanding mechanisms driving IBD. This may reveal novel therapeutic targets and novel biomarkers to assist in early disease diagnosis.

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.

hiPSC-derived intestinal organoids are epithelial structures that self-assemble from differentiated cells into complex 3D structures, representative of ...

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

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