Name: establishment and analysis of three-dimensional (3d) organoids derived from patient prostate cancer bone metastasis specimens and their xenografts
Uploaded: 2020-02-03T13:00:00
Description: Watch this Scientific Journal Video about Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts at JoVE.com

这项研究得到了利奥和安妮·阿尔伯特慈善基金会和JM基金会的支持。我们感谢加州大学圣地亚哥摩尔斯癌症中心的成员，杨静博士和杨凯博士允许我们使用他们的微体和兰德尔法语，外科系的技术专长。

这项研究是严格按照《加州大学圣地亚哥分校机构审查委员会指南》中的建议进行的。IRB #090401 批准从 UCSD 机构审查委员会 （IRB） 收集手术标本从患者用于研究目的。获得每位患者的知情同意，并取得手术性骨前列腺癌转移标本，从股骨病理性骨折的矫形修复中获得。动物协议是在加州大学圣地亚哥分校（UCSD）动物福利和机构动物护理与使用委员会（IACUC）批准的协议#S10298下执行的。细胞从机?...

<ol>
	<li>Fatehullah, A., Tan, S. H., Barker, N. <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+an+in+vitro+model+of+human+development+and+disease.">Organoids as an in vitro model of human development and disease.</a> Nature Cell Biology. 18 (3), 246-254 (2016).</li><li>Tushir, J. 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=Unregulated+ARF6+activation+in+epithelial+cysts+generates+hyperactive+signaling+endosomes+and+disrupts+morphogenesis.">Unregulated ARF6 activation in epithelial cysts generates hyperactive signaling endosomes and disrupts morphogenesis.</a> Molecular Biology of the Cell. 21 (13), 2355-2366 (2010).</li><li>Karthaus, W. 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=Identification+of+multipotent+luminal+progenitor+cells+in+human+prostate+organoid+cultures.">Identification of multipotent luminal progenitor cells in human prostate organoid cultures.</a> Cell. 159 (1), 163-175 (2014).</li><li>McCray, T., Richards, Z., Marsili, J., Prins, G. S., Nonn, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Handling+and+Assessment+of+Human+Primary+Prostate+Organoid+Culture.">Handling and Assessment of Human Primary Prostate Organoid Culture.</a> Journal of Visualized Experiments. (143), 59051 (2019).</li><li>Drost, 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=Organoid+culture+systems+for+prostate+epithelial+and+cancer+tissue.">Organoid culture systems for prostate epithelial and cancer tissue.</a> Nature Protocols. 11 (2), 347-358 (2016).</li><li>Gao, 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=Organoid+cultures+derived+from+patients+with+advanced+prostate+cancer.">Organoid cultures derived from patients with advanced prostate cancer.</a> Cell. 159 (1), 176-187 (2014).</li><li>Vlachogiannis, G., 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+model+treatment+response+of+metastatic+gastrointestinal+cancers.">Patient-derived organoids model treatment response of metastatic gastrointestinal cancers.</a> Science. 359 (6378), 920-926 (2018).</li><li>Cheung, K. J., Gabrielson, E., Werb, Z., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+invasion+in+breast+cancer+requires+a+conserved+basal+epithelial+program.">Collective invasion in breast cancer requires a conserved basal epithelial program.</a> Cell. 155 (7), 1639-1651 (2013).</li><li>Abou-Kheir, W. G., Hynes, P. G., Martin, P. L., Pierce, R., Kelly, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterizing+the+contribution+of+stem/progenitor+cells+to+tumorigenesis+in+the+Pten-/-TP53-/-+prostate+cancer+model.">Characterizing the contribution of stem/progenitor cells to tumorigenesis in the Pten-/-TP53-/- prostate cancer model.</a> Stem Cells. 28 (12), 2129-2140 (2010).</li><li>Beshiri, M. L., 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+PDX/Organoid+Biobank+of+Advanced+Prostate+Cancers+Captures+Genomic+and+Phenotypic+Heterogeneity+for+Disease+Modeling+and+Therapeutic+Screening.">A PDX/Organoid Biobank of Advanced Prostate Cancers Captures Genomic and Phenotypic Heterogeneity for Disease Modeling and Therapeutic Screening.</a> Clinical Cancer Research. 24 (17), 4332-4345 (2018).</li><li>Debnath, J., Brugge, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+glandular+epithelial+cancers+in+three-dimensional+cultures.">Modelling glandular epithelial cancers in three-dimensional cultures.</a> Nature Reviews Cancer. 5 (9), 675-688 (2005).</li><li>Lee, S. 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=Tumor+Evolution+and+Drug+Response+in+Patient-Derived+Organoid+Models+of+Bladder+Cancer.">Tumor Evolution and Drug Response in Patient-Derived Organoid Models of Bladder Cancer.</a> Cell. 173 (2), 515-528 (2018).</li><li>Puca, L., 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+to+model+rare+prostate+cancer+phenotypes.">Patient derived organoids to model rare prostate cancer phenotypes.</a> Nature Communications. 9 (1), 2404 (2018).</li><li>Murrow, L. M., Weber, R. J., Gartner, Z. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissecting+the+stem+cell+niche+with+organoid+models:+an+engineering-based+approach.">Dissecting the stem cell niche with organoid models: an engineering-based approach.</a> Development. 144 (6), 998-1007 (2017).</li><li>Neal, J. 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=Organoid+Modeling+of+the+Tumor+Immune+Microenvironment.">Organoid Modeling of the Tumor Immune Microenvironment.</a> Cell. 175 (7), 1972-1988 (2018).</li><li>Simmons, J. K., 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=Animal+Models+of+Bone+Metastasis.">Animal Models of Bone Metastasis.</a> Veterinary Pathology. 52 (5), 827-841 (2015).</li><li>Godebu, 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=PCSD1,+a+new+patient-derived+model+of+bone+metastatic+prostate+cancer,+is+castrate-resistant+in+the+bone-niche.">PCSD1, a new patient-derived model of bone metastatic prostate cancer, is castrate-resistant in the bone-niche.</a> Journal of Translational Medicine. 12, 275 (2014).</li><li>. Keyence Fluorescence Microscope Available from: <a target="_blank" href="https://www.keyence.com/ss/products/microscope/bz-x/">https://www.keyence.com/ss/products/microscope/bz-x/</a> (2019)</li></ol>

从患者骨转移性前列腺癌细胞中提取的3D器官仍然相对少见。在这里，我们描述了成功建立BMPC串行3D患者衍生器官（PDO）的策略和进一步优化的协议。此外，还描述了用于保护具有较低细胞密度的样品中的有机物的协议，用于IFC和IHC分析。囊肿、球形和更复杂的有机体形式的差分表型表明，该协议提供了培养条件，允许患者的异体肿瘤细胞群保持其细胞表型和结构。因此，本文描述的培养条件，?...

三维培养的有机物已经引起注意，因为他们有潜力重述其原始组织1，2，3，4，5的体内结构，细胞功能和遗传特征。最重要的是，从患者肿瘤组织或患者衍生异种移植（PDX）模型建立的3D类有机体提供了宝贵的机会，以了解细胞信号机制对肿瘤发生，并确定药物治疗对每个细胞群的影响，每个细胞群6，7，8，9，10，11，12，13。Drost等人为建立人类和小鼠前列腺器官制定了标准规程，在泌尿学领域得到了广泛的应用。此外，已作出重大努力，以进一步表征3D有机体，并了解肿瘤发生和转移4，12，14，15的详细机制。除了以前为 3D 有机体培养建立且被广泛接受的协议之外，我们在这里还介绍了 PDO 的 3D 区域性的分步协议，在优化的培养条件下使用三种不同的点胶方法。
在这份手稿中，3D有机体被确立为骨转移性前列腺癌（BMPC）的体外模型。用于这些培养的细胞来自前列腺癌圣地亚哥（PCSD）系列，直接来自患者前列腺癌骨转移肿瘤组织（PCSD18和PCSD22）或患者衍生异种移植（PDX）肿瘤模型（样本命名为PCSD1、PCSD13和PCSD17）。由于前列腺癌细胞的自发性骨转移在基因工程小鼠模型16中很少见，我们利用直接将人类肿瘤细胞注射到雄性Rag2-/----/-小鼠中建立了骨转移性前列腺癌的PDX模型17。
一旦从异质患者肿瘤细胞或患者衍生的异种移植物中建立3D有机体，确认其作为前列腺肿瘤细胞的身份并确定其在3D器官培养物中的表型至关重要。免疫荧光化学（IFC）允许在每个细胞中原位显示蛋白质表达，通常指示特定细胞群2、4的潜在功能。一般来说，包括组织和细胞在内的绝大多数样品的IFC协议是简单明了的，并且完全优化。然而，细胞密度和器官的数量可以明显低于传统培养物。因此，国际有机物的国际金融委员会协议要求采取额外步骤，以确保样品中所有有机物的正确处理和嵌入石蜡。我们描述了腺苷预嵌入工艺的其他步骤，并介绍了在幻灯片上标记切片有机体的位置的提示，提高了IFC在有机物上的成功率，特别是当有机体样本的细胞密度低于预期时。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1 mL Pipettman</td>
			<td>Gilson</td>
			<td>F123602</td>
			<td></td>
		</tr>
		<tr>
			<td>1 mL Syringe</td>
			<td>BD Syringe</td>
			<td>329654</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5 mL tube</td>
			<td>Spectrum Lab Products</td>
			<td>941-11326-ATP083</td>
			<td></td>
		</tr>
		<tr>
			<td>25G Needle</td>
			<td>BD PrecisionGlide Needle</td>
			<td>305122</td>
			<td></td>
		</tr>
		<tr>
			<td>4% Paraformaldehyde (PFA)</td>
			<td>Alfa Aesar</td>
			<td>J61899</td>
			<td></td>
		</tr>
		<tr>
			<td>70% Ethanol (EtOH)</td>
			<td>VWR</td>
			<td>BDH1164-4LP</td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris Bioscience</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Accumax</td>
			<td>Innovative Cell Technologies, Inc.</td>
			<td>AM105</td>
			<td></td>
		</tr>
		<tr>
			<td>adDMEM</td>
			<td>Life Technologies</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Lonza</td>
			<td>50000</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody -for Cytokeratin 5</td>
			<td>Biolegend</td>
			<td>905901</td>
			<td></td>
		</tr>
		<tr>
			<td>Antibody for Cytokeratin 8</td>
			<td>Biolegend</td>
			<td>904801</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Life Technologies</td>
			<td>17504044</td>
			<td></td>
		</tr>
		<tr>
			<td>Bioluminescence imaging system, IVIS 200</td>
			<td>Perkin Elmer Inc</td>
			<td>IVIS 200</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Plate - 24 well</td>
			<td>Costar</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Plate - 48 well</td>
			<td>Costar</td>
			<td>3548</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Culture Plate - 6 well</td>
			<td>Costar</td>
			<td>3516</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Dissociation Solution, Accumax</td>
			<td>Innovative Cell Technologies, Inc.</td>
			<td>AM105</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Scraper</td>
			<td>Sarstedt</td>
			<td>83.180</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer</td>
			<td>Falcon (Corning)</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>Fisher Scientific</td>
			<td>3546</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Vector Vectashield</td>
			<td>H-1200</td>
			<td></td>
		</tr>
		<tr>
			<td>DHT</td>
			<td>Sigma-Aldrich</td>
			<td>D-073-1ML</td>
			<td></td>
		</tr>
		<tr>
			<td>dPBS</td>
			<td>Corning/Cellgro</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>PeproTech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Gemini Bio-Products</td>
			<td>100-106</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF10</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>Forceps</td>
			<td>Denville Scientific</td>
			<td>S728696</td>
			<td></td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Gibco</td>
			<td>15630-080</td>
			<td></td>
		</tr>
		<tr>
			<td>LS Columns</td>
			<td>Miltenyi</td>
			<td>130-0420401</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnetic Column Seperator: QuadroMACS Separator</td>
			<td>Miltenyi</td>
			<td>130-090-976</td>
			<td></td>
		</tr>
		<tr>
			<td>Marker</td>
			<td>VWR</td>
			<td>52877-355</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (Growth Factor Reduced)</td>
			<td>Mediatech Inc. (Corning)</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel (High Concentration)</td>
			<td>BD (Fisher Scientific)</td>
			<td>CB354248</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope Imaging Software, Keyence</td>
			<td>BZ-X800 (newest software) BZ-X700 (old software)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope, Keyence</td>
			<td>BZ-X700 (model 2016-2017)/BZ-X710 (model 2018-2019)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse Cell Depletion Kit</td>
			<td>Miltenyi</td>
			<td>130-104-694</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma-Aldrich</td>
			<td>A9165-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma-Aldrich</td>
			<td>N0636-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>PeproTech</td>
			<td>120-10C</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT Compound</td>
			<td>Tissue-Tek</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>American National Can</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Mediatech Inc. (Corning)</td>
			<td>30-002-CI-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tipes for 1 mL (Blue Tips)</td>
			<td>Fisherbrand Redi-Tip</td>
			<td>21-197-85</td>
			<td></td>
		</tr>
		<tr>
			<td>Plunger (from 3 mL syringe)</td>
			<td>BD Syringe</td>
			<td>309657</td>
			<td></td>
		</tr>
		<tr>
			<td>Prostaglandin E2</td>
			<td>Tocris Bioscience</td>
			<td>2296</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Spondin 1</td>
			<td>Trevigen</td>
			<td>3710-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SB2021190</td>
			<td>Sigma-Aldrich</td>
			<td>S7076-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Table Top Centrifuge</td>
			<td>ThermoFisher Scientific</td>
			<td>75002426</td>
			<td></td>
		</tr>
		<tr>
			<td>Water Bath</td>
			<td>Fisher Sci</td>
			<td>2320</td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632 Dihydrochloride</td>
			<td>Abmole Bioscience</td>
			<td>M1817</td>
			<td></td>
		</tr>
	</tbody>
</table>

establishment and analysis of three-dimensional (3d) organoids derived from patient prostate cancer bone metastasis specimens and their xenografts

人类患者肿瘤标本的三维（3D）培养和前列腺癌患者衍生异种移植（PDX）模型，称为患者衍生器官（PDO），是研究前列腺癌的肿瘤发生和转移。它们的主要优点是，与不传统的细胞系相比，它们保持了原始组织独特的基因组和功能异质性。此外，PDO的3D培养物可用于预测药物治疗对个别患者的影响，是向个性化医学迈出的一步。尽管有这些优点，但少数组通常使用此方法，部分原因是 PDO 培养条件的广泛优化，这可能需要不同的患者样本。我们之前已经证明，我们的前列腺癌骨转移PDX模型，PCSD1，重述了供体患者的骨转移对抗雄激素治疗的抵抗力。我们使用 PCSD1 3D 有机体进一步描述抗雄激素抵抗机制。在概述当前发布的 PDX 和 PDO 模型研究之后，我们描述了在优化培养条件下使用圆顶或浮动基底膜（例如 Matrigel）球体（例如 Matrigel）的 PDO 3D 培养的分步协议。介绍了本体缝合成像和细胞处理，用于本体。该协议可进一步针对其他应用进行优化，包括西印、共培养等，并可用于探索与耐药性、肿瘤发生、转移和治疗相关的3D培养PDO特性。

3D有机体成功地建立了从患者衍生异种移植（PDX）模型骨转移前列腺癌（BMPC）以及直接从患者骨转移前列腺癌组织（图4）。简单地说，我们的BMPCPDX模型是通过将肿瘤细胞注射到雄性Rag2-/----c-/-小鼠中建立的，然后PDX肿瘤被采集和处理，如本手稿所述。如图4所示，PCSD系列的PDX肿瘤组织产生了具有差异表型的3D类有机体，这些表型表现为?...

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Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

患者BMPC标本和骨转移性前列腺癌异种移植物的三维培养，保持其原始肿瘤的功能异质性，导致囊肿、球形和复杂的肿瘤状有机体。本手稿为异质患者衍生样本的 3D 培养及其使用 IFC 进行分析提供了优化策略和方案。

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

我们描述了建立骨转移性前列腺癌的连续3D患者衍生器官的策略和分步方案。我们优化的协议是实用的，用于直接从患者或患者衍生的异种移植肿瘤组织使用有限的起始材料进行实验的 3D 培养物。该协议的3D器官可以用作外体模型，以了解骨转移性前列腺癌病理学的基本机制和测试治疗。该协议中的3D器官培养基是特定于前列腺衍生细胞。我们协议的其他部分适用于不同类型的肿瘤组织和转移位点。多明技术可能是这个过程最困难的部分。实践多明技术将确保有机体、介质和 Matrigel 的圆顶混合物的一致尺寸，并最大限度地减少样品悬浮过程中气泡的形成。视觉演示此方法提供了更好的理解如何处理粘性 Matrigel 成功地培养低密度器官从低数量的细胞。首先将细胞颗粒重新在适当体积的地下室膜中重新吸收，进行 24 孔板设置。轻柔地向上和向下移液，以确保细胞被重新悬浮，然后移液将适当的细胞悬浮量移入预热组织培养板的中心。倒置板，并立即将它倒置在细胞培养培养箱设置在37摄氏度和5%二氧化碳15分钟。将含有10微摩尔Y-27632二氢氯的适当体积预加热介质放入每个井中。要从连接的圆形圆顶上形成浮动圆顶，请使用电池刮刀将圆顶从板上拆下。切一块两四英寸的石蜡薄膜，从一毫升塑料移液器尖端盒中放在空尖端架的外壳上。使用戴手套的食指轻轻按下石蜡膜，以追踪注意不要突破膜的神器。用 70% 乙醇喷洒石蜡膜，并打开细胞培养罩中的 UV 灯，对准备好的薄膜进行至少 30 分钟的消毒。同时，在20微升的地下室膜中重新吸收预处理的细胞。然后移液将悬浮液放入石蜡膜中形成的分管中。将凝固的珠子和石蜡膜放入六井板和移液器中，将含有10微摩尔Y-27632二氢的预加热介质放入每个井中，同时轻轻刷掉石蜡膜上的珠子。要处理组织学的器官，请从井中去除现有介质，注意不要吸气地下室膜圆顶。加入等量的细胞恢复溶液，在4摄氏度下孵育板60分钟。孵育后，使用移液器将地下室膜圆顶移出，用移液器尖端压碎。将分离的圆顶和细胞恢复溶液收集到 1.5 毫升管中。在4摄氏度内将管以300倍g离心5分钟，然后取出上一代，放在一边。保存所有的超级纳，直到最后一步，当器官的存在确认在所需的体积的冷PBS和轻轻地移液器上下机械地移液颗粒，而不会破坏器官。重复离心并去除上经剂。在室温下将颗粒以 4%PFA 的匹配体积固定 60 分钟。固定后，重复离心步骤和 PBS 洗涤。然后慢慢加入200微升热2%agarose，并立即，但轻轻地从管壁分离细胞颗粒，而不中断它使用25量针连接到一毫升注射器。对于气糖自旋方法，使用 25 量表针添加气糖后，将细胞颗粒从管壁上分离出至关重要。一旦阿加罗斯完全凝固，用一根毫升注射器上连接25针将其从管子上分离，并将其转移到新的1.5毫升管中。用70%乙醇填充管子，并继续执行传统的组织脱水和石蜡嵌入方案。该协议被成功地用于建立3D器官从患者衍生的异种移植模型的骨转移前列腺癌，以及直接从癌症组织。器官显示不同的表型，表现为囊肿，球状体，和更高复杂性的器官。从 25 个高分辨率、10 倍放大图像的拼接图像中可以看到整个地下室膜和器官圆顶。为了进一步研究肿瘤组织，对针对基底上皮细胞标记细胞蛋白-5、发光上皮细胞标记细胞蛋白-8和DAPI的五微米厚的器官石蜡部分进行了免疫荧光细胞化学。该协议可以针对其他应用（如西方印迹、共培养和流式细胞学）进行优化，以探索 3D 器官的特性和药物治疗的效果。这些实验可以单独或结合当前疗法，解决耐药性机制和新型疗法的疗效。该技术的3D器官保留了主体间和主体内异质性，因此是患者疾病的更准确模型。这项技术为研究人员探索耐药性、肿瘤生成、转移和治疗机制铺平了道路，这些机制可以更预测患者的疾病进展及其对治疗的反应。

Research

JoVE Journal

Cancer Research

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Labeling of Breast Cancer Patient-derived Xenografts with Traceable Reporters for Tumor Growth and Metastasis Studies

可追踪记者乳腺癌患者来源的移植瘤贴标肿瘤生长和转移的研究

The use of preclinical models to study tumor biology and response to treatment is central to cancer research. Long-established human cell lines, and many ...

科研

癌症研究

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

病人衍生软组织肉瘤原代培养的建立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Isolation and Characterization of Tumor-initiating Cells from Sarcoma Patient-derived Xenografts

来自沙柯玛患者衍生异种移植物的肿瘤启动细胞的分离和表征

The existence and importance of tumor-initiating cells (TICs) have been supported by increasing evidence during the past decade. These TICs have been ...

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. ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

使用结构DNA改变分析和患者衍生异种移植测试癌症的靶向疗法

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Enhanced Viability for Ex vivo 3D Hydrogel Cultures of Patient-Derived Xenografts in a Perfused Microfluidic Platform

在全融合微流体平台中增强患者衍生异种移植物的外体 3D 水凝胶培养物的生存能力

Patient-derived xenografts (PDX), generated when resected patient tumor tissue is engrafted directly into immunocompromised mice, remain biologically ...

Drug Screening of Primary Patient Derived Tumor Xenografts in Zebrafish

斑马鱼原发性患者衍生肿瘤异种移植物的药物筛选

Patient derived xenograft models are critical in defining how different cancers respond to drug treatment in an in vivo system. Mouse models are the ...

The Establishment and Utilization of Patient Derived Xenograft Models of Central Nervous System Metastasis

患者衍生的中枢神经系统转移异种移植模型的建立与利用

The development of novel therapies for central nervous system (CNS) metastasis has been hindered by the lack of preclinical models that accurately ...

Establishing 3-Dimensional Spheroids from Patient-Derived Tumor Samples and Evaluating their Sensitivity to Drugs

从患者来源的肿瘤样本中建立三维微球并评估其对药物的敏感性

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a ...

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

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 ...