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.
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Generating Human Gastric Organoids from Upper Endoscopy Biopsies

<ol>
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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. 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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.

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

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

Our research focuses on hereditary causes of gastric cancer. In particular, we're interested in hereditary diffuse gastric cancer due to disease-causing variants in either the CDH1 or CTNNA1 gene. We also investigate the role of BRCA1 and BRCA2 in gastric cancer risk and carcinogenesis.Organoids are increasingly being utilized in a variety of research domains and gastroenterology is no exception, so with this comes a need for standardization of these techniques to generate these organoids, in particular patient-derived gastric organoids. Our protocol offers a step-by-step method to reliably generate patient-derived gastric organoids from biopsies of both the antral and body regions of the stomach. In particular, we utilize a single-cell digest approach to allow for standardized seeding of cells in an effort to make reliable comparisons amongst organoids generated from different patients.Our findings show that compared to patient-derived gastric organoids from the body region of the stomach, those from the antral region actually grow more numerous and larger in size over the course of 20 days post initial seeding. These findings should be taken into account by researchers looking to utilize gastric organoids derived from different regions of the stomach.

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

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

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

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Research

JoVE Journal

Biology

Isolating Single Cells from Upper Endoscopy Biopsies Derived from Gastric Patients for Organoid Generation

Embedding Gastric Biopsy-Derived Single Cells in a Basement Membrane Matrix Dome for Organoid Generation

Routine Passaging of Patient-Derived Gastric Organoids Through Fragmentation

906 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.

Generating Human Gastric Organoids from Upper Endoscopy Biopsies

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