宾夕法尼亚大学基因组医学T32 HG009495（KHB），NCI R21 CA267949（BWK），Basser BRCA中心的男性和BRCA项目（KHB，BWK），DeGregorio家庭基金会资助奖（BWK）。

本协议中使用的所有人体组织都是从通过宾夕法尼亚大学机构审查委员会 （IRB #842961） 批准的胃组织收集研究提供组织收集知情同意的个人收集的。作为常规护理的一部分，本研究的参与者被要求接受上消化道内窥镜检查，至少年满 18 岁，并能够提供知情同意。所有研究均遵循宾夕法尼亚大学制定的指导方针。
1.实验准备
<ol><li>如前所述制备有条件的L-WRN培养基16。虽然不是强制性的，但可以按照制造商的说明使用市售的 ELISA 试剂盒检查条件培养基中所含的 Wnt-3A、R-spondin 和 noggin 的浓度（参见 材料表）。</li>
	<li>制备RPMI-抗生素（RPMI-ABX），PBS-抗生素（PBS-ABX），PBS-二硫苏糖醇（PBS-DTT），消化缓冲液和胃类器官培养基（详细组成见 补充表1 ）。胃类器官培养基在4°C下稳定1周。</li>
	<li>高压灭菌镊、细解剖剪刀和 1.5 mL 管。</li>
	<li>开始在冰上解冻基底膜基质（基质胶）。</li>
	<li>开始在37°C水浴中解冻消化缓冲液和胰蛋白酶-EDTA。</li>
	<li>将培养箱轨道振荡器设置为37°C和200rpm。</li>
</ol>2. 从活检组织中分离单细胞
<ol><li>在上消化道内窥镜检查期间收集胃活检17 遵循机构临床方案，可能涉及使用大镊子。使用钝头针从镊子中提取组织样本，并将其放入含有 RPMI-ABX 培养基的 15 mL 锥形管中。将试管放在冰上，以便快速转移到实验室。 注意：至少应收集 2-4 次活检。</li>
	<li>让活检组织沉淀在锥形管的底部。使用移液管吸出培养基，并用 1 mL PBS-ABX 缓冲液洗涤活检两次。无需离心，因为组织碎片自然沉降在锥形管中。</li>
	<li>将至少 20 mg 活检组织转移到含有 1 mL PBS-DTT 的 1.5 mL 试管中。</li>
	<li>使用精细解剖剪刀将组织切成 1-2 毫米或更小的碎片。</li>
	<li>使用台式微型离心机 15 秒，将组织碎片聚集在试管底部并吸出尽可能多的上清液。少量残留的 PBS-DTT 是可以接受的。</li>
	<li>将 5 mL 新鲜加热的消化缓冲液加入 50 mL 锥形管中。为了在不丢失组织的情况下转移小组织块，取 500 μL 消化缓冲液并将其加入含有组织的 1.5 mL 管中。接下来，用剪刀修改 1000 μL 移液器吸头的吸头，以增加吸头的直径，以便于抽吸并将组织块转移到含有消化缓冲液的 50 mL 锥形管中。</li>
	<li>将消化缓冲液和组织混合物在37°C下孵育30分钟，以200rpm的轨道振荡。</li>
	<li>向消化缓冲液中加入5mL含有0.25%EDTA的温热胰蛋白酶，并在37°C下以200rpm的轨道振荡再孵育10分钟。</li>
	<li>通过加入等体积的高级（高级）DMEM / F12培养基中和消化缓冲液和胰蛋白酶，并将溶液通过70μm细胞过滤器。</li>
	<li>通过在4°C下以1400× g 离心4分钟来沉淀细胞。 根据活检组织的初始大小，小细胞颗粒可能可见，也可能不可见。</li>
	<li>将细胞沉淀重悬于 1 mL Adv. DMEM/F12 培养基中，并使用台盼蓝和血细胞计数器计数活细胞数。</li>
	<li>在4°C下通过1400× g 离心再次沉淀细胞4分钟，并除去上清液。 注： 图1 显示了从活检组织中分离单细胞的过程示意图。</li>
</ol>3. 将单个细胞嵌入基底膜基质“圆顶”
<ol><li>根据计数的活细胞数，计算达到每 50 μL 基底膜基质 105 个活细胞的最终浓度所需的基底膜基质体积。</li>
	<li>在电镀前迅速执行以下操作，以防止基底膜基质聚合。从冰中取出解冻的基底膜基质，将先前计算的基底膜基质体积加入细胞中，并通过上下移液约10秒轻轻混合。 注意：在此步骤中避免产生气泡至关重要。不要稀释基底膜基质。</li>
	<li>将 50 μL 基底膜基质/细胞混合物的等分试样快速移液到 24 孔组织培养板中单个孔的中心。</li>
	<li>立即盖上 24 孔板，然后以一个平稳的动作将板倒置。将倒置的板置于37°C组织培养箱中35分钟，以使基底膜基质聚合。 注意：倒置板对于防止细胞下沉到板底部并使基底膜基质聚合成 3D“圆顶”形状至关重要。</li>
	<li>向每个孔中加入 500 μL 预热的胃类器官培养基，确保每个“圆顶”的顶部完全浸没在培养基中。将介质分配到每个孔的侧面，以免干扰“圆顶”。</li>
	<li>每 2-3 天更换一次介质。</li>
</ol>4. 类器官通过 片段化的常规传代
<ol><li>一旦类器官准备好传代，从每个指定的孔中取出培养基。 注意：要确定适当的传代时间，请参阅代表性结果部分。</li>
	<li>将 1 mL 冰冷的 Adv. DMEM/F12 培养基直接分配到基底膜基质“圆顶”上。这应该很容易引发“穹顶”的解体。继续吸气和分配，直到“圆顶”的所有碎片从板上分离。</li>
	<li>将介质和破碎的基底膜基质运输到下一个孔，对所有计划传代的孔重复此过程。拆卸最终的基底膜基质“圆顶”后，将培养基以及类器官和基底膜基质的混合物分配到 1.5 mL 管中。</li>
	<li>将 1000 μL 吸头连接到 P1000 移液器，然后将该吸头插入 200 μL 吸头中。这将创建一个直径足够小的移液器吸头，以碎片化类器官，同时仍允许吸入更大体积的移液器。</li>
	<li>用力将类器官和基底膜基质的混合物上下移液约25次，以将类器官碎片化成小块。</li>
	<li>使用台式离心机在4°C下以2000× g离心30秒，将碎片化的类器官混合物离心。这将导致从基底膜基质和培养基中分离出碎片类器官的颗粒。使用移液器吸出培养基和基底膜基质上清液。不要使用真空吸出上清液，因为沉淀是松散的。</li>
	<li>计算所需的新解冻基底膜基质的体积，以便以 1：2 的比例（50 μL/圆顶）拆分孔/圆顶的数量。加入新鲜的基底膜基质，轻轻上下移液混合，注意避免产生气泡。</li>
	<li>将 50 μL 类器官片段和基底膜基质的混合物快速分装到 24 孔板的单个孔中。</li>
	<li>盖上板，将其倒置，并将其置于37°C组织培养箱中35分钟，以使基底膜基质聚合。</li>
	<li>向每个孔中加入 500 μL 预热的胃类器官培养基。 注： 图 2 显示了胃患者来源的类器官通过碎片传代的示意图。</li>
</ol>

从上消化道内窥镜活检生成人胃类器官

<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>

作者没有相关披露。

本文概述了从胃体和胃窦的良性上皮活检中分离出的单细胞可靠地产生人胃类器官的详细方案。该协议中的关键步骤围绕着定时以及处理基底膜基质。为了保持活力，必须在获得活检组织后尽快启动方案。目的是在进行活检后 30 分钟内开始消化活检组织。处理基底膜基质也可能具有挑战性。在冰上解冻时，它仍然是液体;然而，在高于4°C的温度下，它会聚合。因此，必须立即将基底膜基质从冰中?...

类器官是微型三维 （3D） 细胞结构，类似于它们衍生的器官的结构和功能 1,2。这些实验室培养的模型是通过在受控环境中培养干细胞或组织特异性细胞而创建的，该环境允许这些细胞自组织并分化成各种细胞类型1,2,3。类器官的主要优势之一是它们能够比传统的二维 （2D） 细胞培养物更接近地概括人类生物学 1,2,3。特别是，人类类器官已被证明可以维持其起源组织的遗传多样性3,4,5。类器官提供了一个独特的机会，可以在受控的实验室环境中研究人体器官发育、模拟疾病和测试潜在的治疗方法。此外，类器官可以从个体患者样本中衍生出来，从而实现个性化医疗方法和个性化治疗的潜在发展3,6,7。
研究人员使用人类胃类器官来研究胃生物学和病理学的各个方面。突出的例子包括使用患者来源的类器官 （PDO） 来预测胃癌化疗反应 8,9,10 和模拟对幽门螺杆菌感染的上皮反应 11,12,13。人胃类器官由胃中发现的各种细胞类型组成，包括颈部细胞、凹陷细胞和其他支持细胞11,14。胃类器官可以由诱导多能干细胞（iPSCs）产生，也可以从通过活检获得的胃组织或胃切除标本中直接分离的干细胞产生11,14。从胃组织中分离胃干细胞通常通过分离和培养胃腺或酶消化组织样本以释放单个细胞来完成 9,13,15。重要的是，使用这些技术中的任何一种产生的胃类器官内细胞的分化已被证明是相似的13。本文描述的方案侧重于单细胞消化物。
类器官代表了一种科学创新，它弥合了传统细胞培养和整个器官之间的差距。随着该领域研究的不断进展，类器官有望为开发更有效的治疗方法和疗法做出贡献，以实现广泛的应用。鉴于胃PDO的使用率不断提高，及时需要一种标准化的方法来产生它们。在这里，描述了从上消化道内窥镜检查期间获得的良性胃活检组织中分离的单个细胞中产生人胃PDO的方案。重要且独特的是，确定标准化数量的单细胞进行接种，以可靠地产生胃PDO并允许随后的表征。使用这种技术，证明了胃体或胃窦活检产生的类器官形成和生长的可靠差异。

<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

胃患者来源的类器官 （PDO） 为研究胃生物学和病理学提供了一种独特的工具。因此，这些 PDO 在广泛的研究应用中得到越来越多的应用。然而，在保持标准化初始细胞接种密度的同时，从单细胞消化物中生产胃 PDO 的已发表方法存在短缺。在该协议中，重点是从分离的单细胞中启动胃类器官，并提供一种通过片段传代类器官的方法。重要的是，该方案表明，初始细胞接种密度的标准化方法始终如一地从良性活检组织中产生胃类器官，并允许类器官生长的标准化定量。最后，证据支持新的观察结果，即胃PDOs显示出不同的形成和生长速率，这取决于类器官是来自身体的活检还是胃的窦区。具体来说，与胃体活检产生的类器官相比，使用窦活检组织进行类器官启动会导致在 20 天内形成更多的类器官和更快的类器官生长。本文描述的方案为研究人员提供了一种及时且可重复的方法，用于成功生成和使用胃 PDO。

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.

作者聚焦：来自胃不同区域的患者来源的胃类器官的生成和比较 

良性胃体和胃窦上皮活检患者来源的胃类器官的建立和表征

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.

随后的代表性结果来自接受上消化道内窥镜检查的五名不同患者的胃体和胃窦区域的良性上皮的活检。每位患者铺上两到四个“圆顶”/孔，并分析胃体和胃窦活检。从所有五名患者的胃体和胃窦活检组织中成功生成类器官。平均而言，每个“圆顶”/孔分析了 41 个类器官。所有图像都是使用共聚焦显微镜采集的 z 投影，并使用市售的图像分析软件进行类器官大小和球形度的定量（参见 材料?...

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

胃患者来源的类器官在研究中得到了越来越多的应用，但缺乏从具有标准化接种密度的单细胞消化物中生成人胃类器官的正式方案。该协议提出了一种从上消化道内窥镜检查期间获得的活检组织可靠地创建胃类器官的详细方法。

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

我们的研究重点是胃癌的遗传原因。特别是，我们对遗传性弥漫性胃癌感兴趣，这是由于CDH1或CTNNA1基因的致病变异引起的。我们还研究了 BRCA1 和 BRCA2 在胃癌风险和致癌中的作用。类器官越来越多地被用于各种研究领域，胃肠病学也不例外，因此需要对这些技术进行标准化以生成这些类器官，特别是患者来源的胃类器官。我们的方案提供了一种循序渐进的方法，可以从胃的窦和身体区域的活检中可靠地生成患者来源的胃类器官。特别是，我们利用单细胞消化方法允许细胞的标准化接种，以努力在不同患者产生的类器官之间进行可靠的比较。我们的研究结果表明，与来自胃体区域的患者来源的胃类器官相比，来自胃窦区域的类器官实际上在初始播种后的 20 天内变得更多、更大。希望利用来自胃不同区域的胃类器官的研究人员应该考虑这些发现。

establishment-characterization-patient-derived-gastric-organoids-from

Research

JoVE Journal

Biology

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

953 次观看.  University of Pennsylvania Perelman School of Medicine. 我们的研究重点是胃癌的遗传原因。特别是，我们对遗传性弥漫性胃癌感兴趣，这是由于CDH1或CTNNA1基因的致病变异引起的。我们还研究了 BRCA1 和 BRCA2 在胃癌风险和致癌中的作用。类器官越来越多地被用于各种研究领域，胃肠病学也不例外，因此需要对这些技术进行标准化以生成这些类器官，特别是患者来源的胃类器官。我们的方案提供了一种循序渐进的方法，可以从?...

视频: 作者聚焦：来自胃不同区域的患者来源的胃类器官的生成和比较 

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.

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.