Name: generation and culturing of high-grade serous ovarian cancer patient-derived organoids
Uploaded: 2023-01-06T14:50:00
Description: Watch this Scientific Journal Video about Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids at JoVE.com

我们感谢Ron Bose，MD，PhD的指导，以及Barbara Blachut，MD的协助，以建立此协议。我们还要感谢华盛顿大学圣路易斯医学院妇产科和妇科肿瘤科、华盛顿大学院长学者计划和生殖科学家发展计划对该项目的支持。

为研究收集的所有人体组织标本都是根据机构审查委员会（IRB）批准的协议获得的。下面概述的方案是在无菌人体组织培养环境中进行的。从人类受试者那里获得了知情的书面同意。符合条件的患者必须有卵巢癌的诊断或推定诊断，愿意并能够签署知情同意书，并且至少年满18岁。肿瘤组织（恶性原发性肿瘤或转移部位）、腹水和胸腔积液是在手术时从同意的患者那里获得的。这些标本立即被运?...

<ol>
	<li>Bray, 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=Global+cancer+statistics+2018:+GLOBOCAN+estimates+of+incidence+and+mortality+worldwide+for+36+cancers+in+185+countries.">Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries.</a> CA: A Cancer Journal for Clinicians. 68 (6), 394-424 (2018).</li><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>Pauli, 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=Personalized+in+vitro+and+in+vivo+cancer+models+to+guide+precision+medicine.">Personalized in vitro and in vivo cancer models to guide precision medicine.</a> Cancer Discovery. 7 (5), 462-477 (2017).</li><li>Fujii, E., Kato, A., Suzuki, M. <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+xenograft+(PDX)+models:+Characteristics+and+points+to+consider+for+the+process+of+establishment.">Patient-derived xenograft (PDX) models: Characteristics and points to consider for the process of establishment.</a> Journal of Toxicologic Pathology. 33 (3), 153-160 (2020).</li><li>Yang, 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=Application+of+ovarian+cancer+organoids+in+precision+medicine:+Key+challenges+and+current+opportunities.">Application of ovarian cancer organoids in precision medicine: Key challenges and current opportunities.</a> Frontiers in Cell and Developmental Biology. 9, 701429 (2021).</li><li>Yang, 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=Patient-derived+organoids:+A+promising+model+for+personalized+cancer+treatment.">Patient-derived organoids: A promising model for personalized cancer treatment.</a> Gastroenterology Report. 6 (4), 243-245 (2018).</li><li>Karakasheva, T. 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=Generation+and+characterization+of+patient-derived+head+and+neck,+oral,+and+esophageal+cancer+organoids.">Generation and characterization of patient-derived head and neck, oral, and esophageal cancer organoids.</a> Current Protocols in Stem Cell Biology. 53 (1), 109 (2020).</li><li>Madison, B. B., 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=Let-7+represses+carcinogenesis+and+a+stem+cell+phenotype+in+the+intestine+via+regulation+of+Hmga2.">Let-7 represses carcinogenesis and a stem cell phenotype in the intestine via regulation of Hmga2.</a> PLoS Genetics. 11 (8), 1005408 (2015).</li><li>Sato, T., 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=Single+Lgr5+stem+cells+build+crypt-villus+structures+in+vitro+without+a+mesenchymal+niche.">Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche.</a> Nature. 459 (7244), 262-265 (2009).</li><li>Murray, E., 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=HER2+and+APC+mutations+promote+altered+crypt-villus+morphology+and+marked+hyperplasia+in+the+intestinal+epithelium.">HER2 and APC mutations promote altered crypt-villus morphology and marked hyperplasia in the intestinal epithelium.</a> Cellular and Molecular Gastroenterology and Hepatology. 12 (3), 1105-1120 (2021).</li><li>Hill, S. 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=Prediction+of+DNA+repair+inhibitor+response+in+short-term+patient-derived+ovarian+cancer+organoids.">Prediction of DNA repair inhibitor response in short-term patient-derived ovarian cancer organoids.</a> Cancer Discovery. 8 (11), 1404-1421 (2018).</li><li>Passarelli, M. 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=Leucyl-tRNA+synthetase+is+a+tumour+suppressor+in+breast+cancer+and+regulates+codon-dependent+translation+dynamics.">Leucyl-tRNA synthetase is a tumour suppressor in breast cancer and regulates codon-dependent translation dynamics.</a> Nature Cell Biology. 24 (3), 307-315 (2022).</li><li>Pleguezuelos-Manzano, 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=Establishment+and+culture+of+human+intestinal+organoids+derived+from+adult+stem+cells.">Establishment and culture of human intestinal organoids derived from adult stem cells.</a> Current Protocols in Immunology. 130 (1), 106 (2020).</li><li>Stumm, M. 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N., 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+can+predict+response+to+chemotherapy+in+metastatic+colorectal+cancer+patients.">Patient-derived organoids can predict response to chemotherapy in metastatic colorectal cancer patients.</a> Science Translational Medicine. 11 (513), (2019).</li><li>Aisenbrey, E. A., Murphy, W. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthetic+alternatives+to+Matrigel.">Synthetic alternatives to Matrigel.</a> Nature Reviews Materials. 5 (7), 539-551 (2020).</li><li>Nanki, Y., 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+ovarian+cancer+organoids+capture+the+genomic+profiles+of+primary+tumours+applicable+for+drug+sensitivity+and+resistance+testing.">Patient-derived ovarian cancer organoids capture the genomic profiles of primary tumours applicable for drug sensitivity and resistance testing.</a> Scientific Reports. 10, 12581 (2020).</li><li>Mead, B. E., 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=Screening+for+modulators+of+the+cellular+composition+of+gut+epithelia+via+organoid+models+of+intestinal+stem+cell+differentiation.">Screening for modulators of the cellular composition of gut epithelia via organoid models of intestinal stem cell differentiation.</a> Nature Biomedical Engineering. 6 (4), 476-494 (2022).</li></ol>

卵巢癌由于其诊断时的晚期以及化疗耐药性的普遍发展而极其致命。通过利用癌细胞系和PDX模型，卵巢癌研究取得了许多进展;然而，显然需要一种更具代表性和负担得起的 体外 模型。PDO已被证明可以准确代表肿瘤异质性，肿瘤微环境以及其原发性肿瘤的基因组和转录组学特征，因此是各种研究方法的理想临床前模型，例如在药物治疗中实施类器官模型16。
<p class="jove_...

2021 年，美国约有 21，410 名女性新诊断出患有上皮性卵巢癌，12，940 名女性死于这种疾病1。尽管在手术和化疗方面已经取得了足够的进展，但超过 70% 的晚期疾病患者会出现化疗耐药性，并在诊断后 5 年内死亡2，3。因此，迫切需要治疗这种致命疾病的新策略和具有代表性的、可靠的临床前研究模型。
由原发性卵巢肿瘤产生的癌细胞系和患者来源的异种移植物（PDX）是卵巢癌研究中使用的主要仪器。癌细胞系的一个主要优点是它们的快速扩增。然而，它们的持续培养导致表型和基因型改变，导致癌细胞系偏离原始原发性癌症肿瘤样本。由于癌细胞系和原发肿瘤之间存在差异，在细胞系中具有积极作用的药物测定在临床试验中无法具有相同的效果2。为了克服这些限制，使用了 PDX 模型。这些模型是通过将新鲜的卵巢癌组织植入免疫缺陷小鼠中创建的。由于它们是 体内 模型，它们更准确地类似于人类生物学特征，反过来，更能预测药物结果。然而，这些模型也有很大的局限性，包括生成它们所需的成本、时间和资源4.
PDO为临床前研究提供了一种替代模型，克服了癌细胞系和PDX模型的局限性。PDO概括了患者的肿瘤和肿瘤微环境，因此为临床前研究提供了理想的体外可处理模型2，3，5。这些3D模型具有模拟原发性肿瘤的自组织功能，这是它们的二维（2D）细胞系对应物所不具备的特征。此外，这些模型已被证明在遗传和功能上代表其亲本肿瘤，因此是研究新疗法和生物过程的可靠模型。简而言之，它们提供类似于细胞系的长期扩增和储存能力，但也包括小鼠模型固有的微环境和细胞间相互作用4，6。
本协议描述了从患者来源的肿瘤、腹水和胸腔积液样本中创建 PDO，成功率高于 97%。然后，PDO培养物可以扩增多代，并用于测试药物治疗敏感性和预测生物标志物。这种方法代表了一种可用于根据PDO的治疗反应进行个性化治疗的技术。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1% HEPES</td>
			<td>Life Technologies</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>1% Penicillin-Streptomycin</td>
			<td>Fisher Scientific</td>
			<td>30002CI</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL Eppendorf Tubes&#160;</td>
			<td>Genesee Scientific</td>
			<td>14125</td>
			<td></td>
		</tr>
		<tr>
			<td>10 cm Tissue Culture Dish&#160;</td>
			<td>TPP</td>
			<td>93100</td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL Serological Pipet</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>100 &#181;m Cell Filter</td>
			<td>MidSci</td>
			<td>100ICS</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL centrifuge tubes</td>
			<td>Corning</td>
			<td>430052</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL Cryovial</td>
			<td>Simport Scientific</td>
			<td>T301-2</td>
			<td></td>
		</tr>
		<tr>
			<td>2% Paraformaldehyde Fixative</td>
			<td>Sigma-Aldrich</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>37 &#176;C water bath&#160;</td>
			<td>NEST</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>3dGRO R-Spondin-1 Conditioned Media Supplement</td>
			<td>Millipore Sigma</td>
			<td>SCM104</td>
			<td></td>
		</tr>
		<tr>
			<td>6 well plates</td>
			<td>TPP</td>
			<td>92006</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>R31541GA</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Sigma-Aldrich</td>
			<td>SML0788</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>ThermoFisher</td>
			<td>12634028</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Lamda Biotech</td>
			<td>C121</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cultrex Type 2</td>
			<td>R&#38;D Systems</td>
			<td>3533-010-02</td>
			<td>basement membrane extract</td>
		</tr>
		<tr>
			<td>DNase I</td>
			<td>New England Bio Labs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I Reaction Buffer</td>
			<td>New England Bio Labs</td>
			<td>M0303S</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>PeproTech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-10</td>
			<td>PeproTech</td>
			<td>100-26</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF2</td>
			<td>PeproTech</td>
			<td>100-18B</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS C Tubes</td>
			<td>Miltenyi BioTech</td>
			<td>130-096-334</td>
			<td></td>
		</tr>
		<tr>
			<td>gentleMACS Octo Dissociator with Heaters</td>
			<td>Miltenyi BioTech</td>
			<td>130-096-427</td>
			<td>We use the manufacturers protocol.</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>Life Technologies</td>
			<td>35050061</td>
			<td>dipeptide, L-alanyl-L-glutamine</td>
		</tr>
		<tr>
			<td>Hematoxylin and Eosin Staining Kit</td>
			<td>Fisher Scientific</td>
			<td>NC1470670</td>
			<td></td>
		</tr>
		<tr>
			<td>Histoplast Paraffin Wax</td>
			<td>Fisher Scientific</td>
			<td>22900700</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mr. Frosty Freezing Container</td>
			<td>Fisher Scientific</td>
			<td>07202363S</td>
			<td></td>
		</tr>
		<tr>
			<td>N-acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636</td>
			<td></td>
		</tr>
		<tr>
			<td>p1000 Pipette with Tips&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>p200 Pipette with Tips&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur Pipettes 9&#34;</td>
			<td>Fisher Scientific</td>
			<td>1367820D</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Fisher Scientific</td>
			<td>MT21031CM</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipet Controller</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prostaglandin E2</td>
			<td>R&#38;D Systems</td>
			<td>2296</td>
			<td></td>
		</tr>
		<tr>
			<td>Puromycin&#160;</td>
			<td>ThermoFisher</td>
			<td>A1113802</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Murine Noggin</td>
			<td>PeproTech</td>
			<td>250-38</td>
			<td></td>
		</tr>
		<tr>
			<td>Recovery Cell Culture Freezing Medium</td>
			<td>Invitrogen</td>
			<td>12648010</td>
			<td></td>
		</tr>
		<tr>
			<td>Red Blood Cell Lysis Buffer</td>
			<td>BioLegend</td>
			<td>420301</td>
			<td></td>
		</tr>
		<tr>
			<td>ROCK Inhibitor (Y-27632)</td>
			<td>R&#38;D Systems</td>
			<td>1254/1</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>Sigma-Aldrich</td>
			<td>S7076</td>
			<td></td>
		</tr>
		<tr>
			<td>T75 Flask</td>
			<td>MidSci</td>
			<td>TP90076</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Culture Hood&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Embedding Cassette</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE Express</td>
			<td>Invitrogen</td>
			<td>12604013</td>
			<td>animal origin-free, recombinant enzyme</td>
		</tr>
		<tr>
			<td>Type II Collagenase</td>
			<td>Life Technologies</td>
			<td>17101015</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

generation and culturing of high-grade serous ovarian cancer patient-derived organoids

类器官是3D动态肿瘤模型，可以从患者来源的卵巢肿瘤组织，腹水或胸腔积液中成功生长，并有助于发现卵巢癌的新疗法和预测性生物标志物。 这些模型概括了克隆异质性、肿瘤微环境以及细胞间和细胞间基质相互作用。此外，它们已被证明在形态学、细胞学、免疫组织化学和遗传学上与原发肿瘤相匹配。因此，类器官有助于肿瘤细胞和肿瘤微环境的研究，并且优于细胞系。本协议描述了从患者肿瘤，腹水和胸腔积液样本中生成患者来源的卵巢癌类器官的不同方法，成功率高于97%。患者样品通过机械和酶消化分离成细胞悬浮液。然后使用基底膜提取物（BME）对细胞进行铺板，并用含有特异性用于培养高级别浆液性卵巢癌（HGSOC）的补充剂的优化生长培养基支持。在形成初始类器官后，PDO可以维持长期培养，包括传代以扩增以进行后续实验。

为了产生PDO，将样品以机械和酶促方式消化成单细胞悬浮液。然后将细胞重悬于BME中，并补充专门设计的培养基（图3）。类器官通常在 10 天的时间范围内建立，之后它们在培养物中展示离散的类器官（图 4）。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64878/64878fig03.jpg"> 图3</stron...

Watch this Scientific Journal Video about Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids at JoVE.com

Generation and Culturing of High-Grade Serous Ovarian Cancer Patient-Derived Organoids

患者来源类器官（PDO）是一种三维（3D）培养物，可以在 体外模拟肿瘤环境。在高级别浆液性卵巢癌中，PDO代表了研究新型生物标志物和治疗方法的模型。

高级别浆液性卵巢癌患者来源类器官的生成和培养

Organoids are 3D dynamic tumor models that can be grown successfully from patient-derived ovarian tumor tissue, ascites, or pleural fluid and aid in the ...

患者驱动的类器官模型在遗传和功能上代表其亲本肿瘤，因此是研究卵巢癌新疗法和复杂生物学过程的可靠模型。通过概括克隆异质性、肿瘤微环境和体外细胞间相互作用，这些模型克服了癌细胞系和患者来源的异种移植物的局限性。患者来源的类器官提供长期扩增和储存能力，并模拟肿瘤生理学。因此，它们是研究新型疗法和预测性生物标志物的理想模型。通过将肿瘤样品放入 10 厘米的组织培养皿中并使用一次性手术刀切碎组织以创建均匀的混合物来开始收获类器官。使用镊子将均匀的组织混合物放入解离管中。对于每1至2毫升均质组织，在类器官基础培养基中加入7至8毫升每毫升1毫克的二型胶原酶溶液和一毫升DNase One溶液。使用解离机在胶原酶溶液中液化组织时以458G.液化溶液。运行程序 37 C 下划线 H 下划线 TDK 三个一小时，直到它是单细胞悬浮液。完成后，将均质化的混合物转移到新的50毫升锥形管中。加入20至40毫升类器官基培养基，并以458G涡旋溶液，然后通过100微米细胞过滤器将溶液过滤到新标记的50毫升锥形管中。接下来，将过滤后的混合物在 1， 650 G 下在 4 摄氏度下离心五分钟，并使用玻璃牧场移液管吸出上清液。准备每毫升100微克的DNA酶一，并通过添加逐滴的DNA酶一溶液将细胞重悬于一毫升的Dnase One溶液中。在室温下孵育15分钟后，向细胞中加入25毫升类器官碱基并倒置混合。然后将细胞在1， 650 G下在4摄氏度下离心5分钟，并使用玻璃牧场移液管小心地吸出上清液。将新形成的细胞沉淀重悬于五毫升预热的红细胞或红细胞裂解缓冲液中。以458G涡旋每个锥形管中的溶液并孵育五分钟。再次，离心悬浮在RBC裂解缓冲液中的沉淀，并使用玻璃牧场移液管吸出上清液。用10毫升PBS洗涤沉淀并在离心细胞之前涡旋细胞。对于大细胞沉淀，吸出PBS并在沉淀顶部添加一毫升类器官基质培养基。涡旋溶液后，将300至400微升细胞悬液转移到微量离心管中。离心五分钟后，吸出上清液并使用冷吸头将其重悬于基底膜提取物或BME和类器官碱基中。将重悬的细胞溶液以40微升等分试样接种到六孔板中。每孔最多铺五等分试样。立即将孔板放入培养箱中20分钟，孵育后，轻轻地向每个孔中加入两毫升完整的类器官培养基。向每个孔中加入一毫升类器官碱基，并将培养基直接上下移液到类器官标签上进行解离。将所有含有重悬颗粒的培养基收集在15毫升锥形管中。然后，在吸出上清液之前，将含有混合物的15毫升锥形管离心五分钟。向细胞沉淀中加入一毫升动物源性游离重组酶。通过涡旋混合并将悬浮液转移到1.5毫升微量离心管中。在37摄氏度的水浴中孵育15分钟并离心5分钟后，吸出上清液。将沉淀重悬于BME和类器官基础培养基中，并将重悬的细胞溶液以40微升等分试样接种到六孔板中。每孔最多铺五等分试样。孵育20分钟后，将两毫升完整的类器官培养基轻轻加入每个孔中。将传代类器官细胞沉淀重悬于0.5至1毫升的回收细胞培养冷冻培养基中，并将1毫升转移到每个冷冻瓶中，然后将其转移到零下80度和液氮罐中长期储存。通过将储存在液氮中的冷冻瓶转移到37摄氏度的水浴中来解冻类器官。解冻后，将类器官转移到15毫升锥形管中，并在吸出上清液之前离心。接下来，向沉淀中加入一毫升PBS，并将重悬的沉淀转移到1.5毫升微量离心管中后，在4摄氏度下以1，650G离心5分钟后，使用玻璃牧场移液管小心地吸出上清液。将沉淀重悬于BME和类器官基础培养基中，并将重悬的细胞以40微升等分试样铺到六孔板上。每孔最多放置五个等分试样。立即将板放入培养箱中20分钟，然后将两毫升完整的类器官培养基加入孔中。在10天内建立类器官，之后它们在培养物中显示出离散的类器官。通过将患者来源的类器官培养物嵌入琼脂糖中并用苏木精和伊红染色进行评估来评估。重要的是要考虑与BME合作的挑战。细胞沉淀和BME的重悬必须在冰上进行，确保在此过程中不会形成气泡。由于接受过新辅助化疗的患者遇到的问题，该技术允许进一步研究化疗对类器官生成和发育的影响。

Research

JoVE Journal

Cancer Research

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

建立胃癌患者衍生异种移植模型和原细胞系

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

科研

癌症研究

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

High-Throughput In Vitro Assay using Patient-Derived Tumor Organoids

使用患者源性肿瘤类器官的高通量 体外 检测

Patient-derived tumor organoids (PDOs) are expected to be a preclinical cancer model with better reproducibility of disease than traditional cell culture ...

Profiling Sensitivity to Targeted Therapies in EGFR-Mutant NSCLC Patient-Derived Organoids

分析EGFR突变型NSCLC患者衍生类器官靶向治疗的敏感性

Novel 3D cancer organoid cultures derived from clinical patient specimens represent an important model system to evaluate intratumor heterogeneity and ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

小鼠患者来源的癌细胞原位植入概括了晚期结直肠癌

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...

Establishment and Culture of Patient-Derived Breast Organoids

患者来源乳腺类器官的建立和培养

Breast cancer is a complex disease that has been classified into several different histological and molecular subtypes. Patient-derived breast tumor ...

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

Visualizing DNA Damage Repair Proteins in Patient-Derived Ovarian Cancer Organoids via Immunofluorescence Assays

通过免疫荧光测定 可视化 患者来源的卵巢癌类器官中的 DNA 损伤修复蛋白

Immunofluorescence is one of the most widely used techniques to visualize target antigens with high sensitivity and specificity, allowing for the accurate ...

Ovarian Cancer Patient-Derived Organoid Models for Pre-Clinical Drug Testing

Ovarian cancer is a fatal gynecologic cancer and the fifth leading cause of cancer death among women in the United States. Developing new drug treatments ...

Automated Kinetic Imaging for Drug Assessment in Patient-Derived Tumor Organoids

Multiplexed Live-Cell Imaging for Drug Responses in Patient-Derived Organoid Models of Cancer

Author Spotlight: Developing Multiplexed Kinetic Assays for Organoid-Based Drug Response Analysis

Patient-derived organoid (PDO) models of cancer are a multifunctional research system that better recapitulates human disease as compared to cancer cell ...

Derivation of 3D Glioma Organoids from Chopper-Processed Patient Tumors

Cultivating Ex Vivo Patient-Derived Glioma Organoids Using a Tissue Chopper

Author Spotlight: Enhanced Generation of Patient-Derived 3D Organoids for Glioblastoma and Glioma

Glioblastoma, IDH-wild type, CNS WHO grade 4 (GBM) is a primary brain tumor associated with poor patient survival despite aggressive treatment. Developing ...

Standardizing Ovarian Cancer Organoid Biobanking

Strategy for Biobanking of Ovarian Cancer Organoids: Addressing the Interpatient Heterogeneity across Histological Subtypes and Disease Stages

Author Spotlight: Advancing Personalized Medicine in Ovarian Cancer 

卵巢癌类器官的生物样本库策略：解决跨组织学亚型和疾病阶段的患者间异质性

While the establishment of an ovarian cancer biobank from patient-derived organoids along with their clinical background information promises advances in ...