Name: subculture and cryopreservation of esophageal adenocarcinoma organoids: pros and cons for single cell digestion
Uploaded: 2022-07-06T12:40:00
Description: Watch this Scientific Journal Video about Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion at JoVE.com

这项工作得到了科隆大学科隆财富计划/医学院的支持。我们感谢Susanne Neiss，Michaela Heitmann和Anke Wienand-Dorweiler的技术援助。宁波范由广州市精英奖学金委员会（GESC）资助。作者感谢Joshua D'Rozario博士在语言编辑方面的帮助。

成熟且生长良好的PDO培养物是该协议中描述的成功传代培养和冷冻保存的基础。在这里，EAC PDO是使用Karakasheva T. A.等人17描述的方案从EAC患者的原发肿瘤组织中产生的。EAC组织是在BioMaSOTA的批准下从生物库中收集的（由科隆大学伦理委员会批准，ID：13-091）。
注意：EAC PDO已在37°C和5%CO2的加湿培养箱中使用PDO培养基进行培养（表1）。在以...

<ol>
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H., Lagergren, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+epidemiology+of+esophageal+adenocarcinoma.">The epidemiology of esophageal adenocarcinoma.</a> Gastroenterology. 154 (2), 390-405 (2018).</li><li>Rumgay, 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=International+trends+in+esophageal+squamous+cell+carcinoma+and+adenocarcinoma+incidence.">International trends in esophageal squamous cell carcinoma and adenocarcinoma incidence.</a> The American Journal of Gastroenterology. 116 (5), 1072-1076 (2021).</li><li>Qian, 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=Clinical+characteristics,+prognosis,+and+nomogram+for+esophageal+cancer+based+on+adenosquamous+carcinoma:+a+seer+database+analysis.">Clinical characteristics, prognosis, and nomogram for esophageal cancer based on adenosquamous carcinoma: a seer database analysis.</a> Frontiers in Oncology. 11, 603349 (2021).</li><li>Lagergren, J., Smyth, E., Cunningham, D., Lagergren, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oesophageal+cancer.">Oesophageal cancer.</a> Lancet. 390 (10110), 2383-2396 (2017).</li><li>Rockett, J. 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R., Lee, T. H., Agrawal, D. K., Mittal, S. K. <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+Barrett's+esophagus+and+esophageal+adenocarcinoma-past,+present,+and+future.">Animal models of Barrett's esophagus and esophageal adenocarcinoma-past, present, and future.</a> Clinical and Translational Science. 8 (6), 841-847 (2015).</li><li>Lan, T., Xue, X., Dunmall, L. C., Miao, J., Wang, Y. <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:+a+developing+tool+for+screening+biomarkers+and+potential+therapeutic+targets+for+human+esophageal+cancers.">Patient-derived xenograft: a developing tool for screening biomarkers and potential therapeutic targets for human esophageal cancers.</a> Aging. 13 (8), 12273-12293 (2021).</li><li>Liu, D. S. 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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=Esophageal+adenocarcinoma+cells+and+xenograft+tumors+exposed+to+Erb-b2+receptor+tyrosine+kinase+2+and+3+inhibitors+activate+transforming+growth+factor+beta+signaling,+which+induces+epithelial+to+mesenchymal+transition.">Esophageal adenocarcinoma cells and xenograft tumors exposed to Erb-b2 receptor tyrosine kinase 2 and 3 inhibitors activate transforming growth factor beta signaling, which induces epithelial to mesenchymal transition.</a> Gastroenterology. 153 (1), 63-76 (2017).</li><li>Simian, M., Bissell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organoids:+A+historical+perspective+of+thinking+in+three+dimensions.">Organoids: A historical perspective of thinking in three dimensions.</a> The Journal of Cell Biology. 216 (1), 31-40 (2017).</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>Li, 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=Organoid+cultures+recapitulate+esophageal+adenocarcinoma+heterogeneity+providing+a+model+for+clonality+studies+and+precision+therapeutics.">Organoid cultures recapitulate esophageal adenocarcinoma heterogeneity providing a model for clonality studies and precision therapeutics.</a> Nature Communications. 9, 2983 (2018).</li><li>Ebbing, E. 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=Stromal-derived+interleukin+6+drives+epithelial-to-mesenchymal+transition+and+therapy+resistance+in+esophageal+adenocarcinoma.">Stromal-derived interleukin 6 drives epithelial-to-mesenchymal transition and therapy resistance in esophageal adenocarcinoma.</a> Proceedings of the National Academy of Sciences of the United States of America. 116 (6), 2237-2242 (2019).</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>Ord&#243;&#241;ez, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Broad-spectrum+immunohistochemical+epithelial+markers:+a+review.">Broad-spectrum immunohistochemical epithelial markers: a review.</a> Human Pathology. 44 (7), 1195-1215 (2013).</li><li>Maniar, K. P., Umpires, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokeratin+7+(CK7,+K7).">Cytokeratin 7 (CK7, K7).</a> Pathology Outlines.com website. , (2021).</li><li>Sun, X., Kaufman, P. 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在该协议中，描述了EAC PDO的两种不同的传代培养和冷冻保存方法，即具有和不具有单细胞消化。几项研究建议用单细胞消化15，17传代EAC PDO，这对于大多数需要细胞数量控制，均匀密度和促进大小跟踪的中空结构的实验是有益的。然而，基于单细胞的方法的特征在于从冷冻储备中重新培养后生长较慢，并且在培养期间形态不太紧凑。经验表明，基于单?...

食管癌 （EC） 是全球第十大常见和第六大癌症死因1.食管腺癌（EAC）是EC的主要组织学亚型之一，主要发生在西方国家2。近十年来，包括德国在内的许多发达国家的 EAC 发病率显著增加3。由于癌症的侵袭性和肿瘤发展早期缺乏症状，EAC患者的整体预后较差，5年生存率约为20%2，4，5。
自二十世纪末以来，已经为EAC的生物医学研究建立了几种模型。20世纪90年代建立的经典人类EAC细胞系6，扩展了我们对EAC肿瘤生物学，肿瘤遗传学以及抗肿瘤策略的知识，并广泛用于EAC研究。此外，一些研究小组通过手术或炎症方法将动物暴露于已知的风险因素（例如胃食管反流）来成功开发了EAC或Barrett食道的动物模型7，8，9。此外，开发了患者源性异种移植（PDX）模型，该模型将EAC原发性癌组织皮下或原位植入免疫缺陷小鼠中，以模拟人EAC肿瘤生物学行为和肿瘤环境10，11，12。然而，尽管这些模型改善了临床应用，并且我们对EAC肿瘤发生和进展背后的分子机制的理解，但将这些研究模型的结果外推到人类仍然存在重大挑战。
患者来源的肿瘤类器官（PDO）在3D培养系统中生长，该系统在体外模仿人类发育和器官再生。PDO由患者的原代组织产生，概括了人类肿瘤的分子和表型特征，并在药物开发和个性化癌症治疗中显示出有希望的应用13，14。通过比较10例EAC PDO与其配对的肿瘤组织，据报道EAC PDO与原发肿瘤具有相似的组织病理学特征和基因组学景观，保留了肿瘤内的异质性并促进了体外有效的药物筛选15。EAC PDO还用于研究EAC肿瘤细胞与患者来源的癌症相关成纤维细胞（CAFs）的相互作用，表明在肿瘤微环境研究领域的强大应用16。不幸的是，可用于开发和传播 EAC PDO 的协议有限。在这里，详细描述了两种不同的传代培养和保存EAC PDO的方法：有和没有单细胞消化。维护EAC PDO及其应用的标准化方法可以支持研究人员在EAC PDO研究中为不同目的选择适当的方法。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>-20&#176;C Freezer</td>
			<td>Bosch</td>
			<td></td>
			<td>Economic</td>
		</tr>
		<tr>
			<td>-80&#176;C Freezer</td>
			<td>Panasonic</td>
			<td></td>
			<td>MDF DU500VH-PE</td>
		</tr>
		<tr>
			<td>Automated Cell counter</td>
			<td>Thermo Fisher</td>
			<td>AMQAX1000</td>
			<td>Countess II</td>
		</tr>
		<tr>
			<td>Biological Safety Cabinet Class II</td>
			<td>Thermo Scientific</td>
			<td>51022482</td>
			<td>Herasafe KS12</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Heraeus</td>
			<td>75003060</td>
			<td>Megafuge 1.0R</td>
		</tr>
		<tr>
			<td>CO2 Incubator</td>
			<td>Thermo Scientific</td>
			<td>50116048</td>
			<td>Heracell 150i</td>
		</tr>
		<tr>
			<td>Inverted automated fluorescence microscope</td>
			<td>Olympus</td>
			<td></td>
			<td>IX83</td>
		</tr>
		<tr>
			<td>Inverted light microscope</td>
			<td>Leica</td>
			<td></td>
			<td>DMIL LED Fluo</td>
		</tr>
		<tr>
			<td>Pipette 1000 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000063</td>
			<td>Research Plus</td>
		</tr>
		<tr>
			<td>Pipette 200 &#181;L</td>
			<td>Eppendorf</td>
			<td>3123000039</td>
			<td>Research Plus</td>
		</tr>
		<tr>
			<td>Rotating Incubator</td>
			<td>Scientific Industries, sc.</td>
			<td>SI-1200</td>
			<td>Enviro-genie</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Eppendorf</td>
			<td>5355 000.011</td>
			<td>Thermomixer Comfort</td>
		</tr>
		<tr>
			<td>Vacuum pump</td>
			<td>Vacuubrand</td>
			<td>20727200</td>
			<td>BVC control</td>
		</tr>
		<tr>
			<td>Waterbath</td>
			<td>Medingen</td>
			<td>p2725</td>
			<td>W22</td>
		</tr>
		<tr>
			<td>Material</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL tube</td>
			<td>Sarstedt</td>
			<td>62.554.502</td>
			<td>Inc&#160;Screw cap tube PP 15 mL</td>
		</tr>
		<tr>
			<td>Cryo vial 2 mL</td>
			<td>Sarstedt</td>
			<td>72.379</td>
			<td>CryoPure 2.0 mL tube</td>
		</tr>
		<tr>
			<td>Low bind tube 1.5 mL</td>
			<td>Sarstedt</td>
			<td>72.706.600</td>
			<td>Micro tube 1.5 mL protein LB</td>
		</tr>
		<tr>
			<td>Low bind tube 5 mL</td>
			<td>Eppendorf</td>
			<td>0030 108.302</td>
			<td>Protein LoBind Tube 5.0 mL</td>
		</tr>
		<tr>
			<td>Pipette tip 200 &#181;L</td>
			<td>Starlab</td>
			<td>E1011-8000</td>
			<td>200 &#181;L Graduated tip, wide orifice</td>
		</tr>
		<tr>
			<td>Pipette tip 1000 &#181;L</td>
			<td>Starlab</td>
			<td>E1011-9000</td>
			<td>1000 &#181;L Graduated tip, wide orifice</td>
		</tr>
		<tr>
			<td>Pipette tip 1000 &#181;L</td>
			<td>Sarstedt</td>
			<td>70.3050</td>
			<td>Pipette tip 1000 &#181;L</td>
		</tr>
		<tr>
			<td>Sterile filter 0.2 &#181;m</td>
			<td>Sarstedt</td>
			<td>83.1826.001</td>
			<td>Filtropur 0.2 &#181;m sterile filter</td>
		</tr>
		<tr>
			<td>Tissue culture plate</td>
			<td>Sarstedt</td>
			<td>83.3921</td>
			<td>12 well-plate</td>
		</tr>
		<tr>
			<td>Reagent/Chemical</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>A83-01</td>
			<td>Tocris</td>
			<td>2939</td>
			<td></td>
		</tr>
		<tr>
			<td>Advanced DMEM/F-12</td>
			<td>Thermo Fisher Scientific</td>
			<td>12634010</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Thermo Fisher Scientific</td>
			<td>15290026</td>
			<td></td>
		</tr>
		<tr>
			<td>B-27</td>
			<td>Thermo Fisher Scientific</td>
			<td>17504001</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Recovery Solution</td>
			<td>Corning</td>
			<td>354253</td>
			<td></td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>MedChemExpress</td>
			<td>HY-10182/CS-0181</td>
			<td></td>
		</tr>
		<tr>
			<td>DNase I grade II, from bovine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>10104159001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate-buffered saline (DPBS)</td>
			<td>Thermo Fisher Scientific</td>
			<td>14190094</td>
			<td></td>
		</tr>
		<tr>
			<td>Extracellular matrix (ECM) gel: Matrigel Growth Factor Reduced (GFR) Basement Membrane Matrix</td>
			<td>Corning</td>
			<td>356231</td>
			<td></td>
		</tr>
		<tr>
			<td>FGF-10a</td>
			<td>Peprotech</td>
			<td>100-26-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Freezing medium: Recovery Cell Freezing Medium</td>
			<td>Thermo Fisher Scientific</td>
			<td>12648010</td>
			<td></td>
		</tr>
		<tr>
			<td>Gastrin</td>
			<td>Sigma</td>
			<td>G9020</td>
			<td></td>
		</tr>
		<tr>
			<td>Gentamicin-25 (25 mg/ 500 &#181;L)</td>
			<td>PromoCell</td>
			<td>C-36030</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES (1 M)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15630080</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamine 200 mM (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>25030024</td>
			<td></td>
		</tr>
		<tr>
			<td>N-2</td>
			<td>Thermo Fisher Scientific</td>
			<td>17502-048</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165</td>
			<td></td>
		</tr>
		<tr>
			<td>Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636-100</td>
			<td></td>
		</tr>
		<tr>
			<td>Noggin</td>
			<td>Peprotech</td>
			<td>120-10C-50</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin 10,000 U/ mL (100X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant human epidermal growth factor (EGF)</td>
			<td>Peprotech</td>
			<td>AF-100-15</td>
			<td></td>
		</tr>
		<tr>
			<td>R-Spondin1 conditioned medium from Cultrex R-Spondin Cells</td>
			<td>Biotechne</td>
			<td>3710-001-01</td>
			<td></td>
		</tr>
		<tr>
			<td>SB202190</td>
			<td>MedChemExpress</td>
			<td>152121-30-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin inhibitor from Glycine max (soybean)</td>
			<td>Sigma-Aldrich</td>
			<td>93620-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25 %), phenol red</td>
			<td>Thermo Fisher Scientific</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>Wnt-3A conditioned medium</td>
			<td>Wnt-3A expressing cell line was kindly provided by Prof. Hans Clevers&#39; group</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Y-27632</td>
			<td>Sigma</td>
			<td>Y0503</td>
			<td></td>
		</tr>
	</tbody>
</table>

subculture and cryopreservation of esophageal adenocarcinoma organoids: pros and cons for single cell digestion

缺乏反映原发病的合适的转化研究模型来探索肿瘤发生和治疗策略是食管腺癌（EAC）的主要障碍。患者来源的类器官（PDO）最近已成为各种癌症中一种非凡的临床前模型。但是，可用于开发 EAC PDO 的协议仍然有限。一旦建立了PDO，繁殖和低温保存对于进一步的下游分析至关重要。在这里，两种不同的方法已经标准化为EAC PDO传代培养和冷冻保存，即有和没有单细胞消化。这两种方法都可以可靠地获得适当的细胞活力，并适用于不同的实验设置。目前的研究表明，用单细胞消化传代培养EAC PDO适用于大多数需要细胞数量控制，均匀密度和有利于尺寸跟踪的空心结构的实验。然而，基于单细胞的方法在培养物中以及在从冷冻库存重新培养后显示出较慢的生长。此外，单细胞消化的传代培养的特点是形成具有空心核心的中空结构。相反，在不进行单细胞消化的情况下处理EAC PDO有利于冷冻保存，扩增和组织学表征。在该协议中，描述了具有和不具有单细胞消化的EAC PDO的传代培养和冷冻保存的优缺点，以使研究人员能够选择适当的方法来处理和研究其类器官。

该协议介绍了包括具有和不具有单细胞消化的EAC PDO的传代培养和冷冻保存的程序。
图1 显示了两种不同亚文化策略的代表性相差图片。EAC PDO达到适当的传代培养密度（图1，左）。没有单细胞消化的传代培养需要更少的时间即可达到可比的密度，并且主要导致结构紧凑（图1，顶行）。相比之下，单细胞消化?...

Watch this Scientific Journal Video about Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion at JoVE.com

Subculture and Cryopreservation of Esophageal Adenocarcinoma Organoids: Pros and Cons for Single Cell Digestion

该协议描述了食管腺癌类器官的传代培养和冷冻保存方法，有和没有单细胞消化，使研究人员能够根据其实验设计选择适当的策略。

食管腺癌类器官的传代培养和冷冻保存：单细胞消化的利弊

The lack of suitable translational research models reflecting primary disease to explore tumorigenesis and therapeutic strategies is a major obstacle in ...

这是一种简单且可视化的方案，可演示在两种不同的亚培养过程中处理食管腺癌类器官，伴有和不伴有单细胞消化。以两种不同的方法传代培养EAC类器官的标准化方案可以支持研究人员在基于EAC PDO的实验中为不同的下游应用选择合适的技术。在处理PDO时，必须注意每个步骤中的细节。基于团队的 SOP 对于 POD 的质量至关重要。演示该程序的将是我们的博士生Ning Bo Fan和实验室的技术助理Lisa Raatz。使用二氧化碳在37摄氏度下过夜孵育12孔板。如果可用，使用平板中的空孔和患者来源的类器官培养物。将适当体积的ECM凝胶在冰上孵育一小时以液化。将细胞回收溶液放在冰上。从培养箱中取出装有生长PDO的板。使用真空泵吸出旧介质。将每圆顶冰冷细胞回收溶液加入500微升到孔中。通过上下移液几次来分解ECM凝胶，使用具有宽孔的1， 000微升吸头将ECM凝胶圆顶破碎成小块。从最多两个孔中混合PDO，ECM凝胶和细胞回收溶液，并将其转移到五毫升低结合管中。将此管在冰上孵育20分钟。通过倒置管子每五分钟混合一次。以500倍G的速度在四摄氏度下离心管四分钟。如果没有可见的沉淀，用真空泵小心地除去上清液，直到达到含有ECM凝胶PDO溶液的相，并加入三毫升冰冷的细胞回收溶液。将管子倒置几次，然后在冰上孵育10分钟。在4摄氏度下以500倍G离心4分钟。使用真空泵或1， 000微升移液器小心地丢弃上清液。尽量去除上清液。将PDO颗粒储存在冰上。从零下20摄氏度的冰箱中取出预冷的201， 000微升宽孔头，并将其放在干净的工作台上。计算每个计划用于播种的圆顶50微升的ECM凝胶，加上额外的50微升。然后使用预冷的1， 000微升吸头将沉淀重悬于ECM凝胶中，并通过上下移液约10次混合。在播种圆顶之前，从培养箱中取出预热的12孔板。将含有50微升ECM凝胶的圆顶播种到温暖的板中。将板在37摄氏度和5%二氧化碳下孵育20至30分钟，以固化ECM凝胶。小心地添加预热的PDO介质，而不会干扰圆顶。在显微镜下监测播种的PDO。培养PDO7至14天，直到达到所需的密度和形态。通过将两毫升0.25%胰蛋白酶EDTA和20微升DNAase I混合以消化从两个孔中收获的PDO来制备消化培养基。将先前制备的沉淀重悬于适当体积的预热的0.25%胰蛋白酶EDTA和DNAase I中，并使用1，000微升移液管上下移液将其混合约10次。在转速至少为 28 RPM 的旋转培养箱中以 37 摄氏度孵育 10 分钟。制备含有6毫升大豆胰蛋白酶抑制剂溶液的15毫升管。消化后，用1， 000微升移液管将消化的PDO彻底混合几次，以破坏PDO。将消化的PDO转移到含有SDI溶液的15毫升管中以停止消化。在4摄氏度下以500倍G离心4分钟。使用真空泵或1， 000微升移液器小心地丢弃上清液。将沉淀重悬于一毫升基础培养基中。使用自动细胞计数器或血细胞计数器确定细胞浓度和活力。根据计划用于播种的圆顶计算细胞数量，加上一个额外的圆顶，并将它们转移到新鲜的1.5毫升低结合管中。在4摄氏度下以500倍G离心4分钟。使用1， 000微升移液管小心地丢弃上清液。使用预冷的1， 000微升宽孔头向沉淀物中加入适当体积的ECM凝胶。通过上下移液约10次混合。如前所述，将消化的PDO播种到12孔板中。用先前制备的沉淀物开始未消化PDO的冷冻保存过程。使用500微升冷冷冻介质从两个圆顶重悬沉淀并将其转移到低温小瓶中。使用适当的细胞冷冻容器在冰箱中冷冻PDO过夜。将冷冻的PDO转移到零下150摄氏度的冰箱中。对于消化的PDO的冷冻保存，将细胞转移到新鲜的1.5毫升低结合管中。在4摄氏度下以500倍G离心4分钟。使用1，000微升移液器小心地丢弃上清液。将沉淀重悬于500微升冷冻介质中，并将其转移到低温小瓶中。使用适当的细胞冷冻容器在零下80摄氏度的冰箱中冷冻PDO过夜，并将其转移到零下150摄氏度的冰箱中长期储存。没有单细胞消化的传代培养需要更少的时间即可达到可比的密度，并且主要导致结构紧凑。相比之下，单细胞消化的PDO显示出具有空心核心的结构。该图显示了具有紧凑和中空结构的石蜡包埋EAC PDO的苏木精曙红染色和免疫组织化学染色。全血细胞蛋白能够识别上皮肿瘤细胞。细胞角蛋白7突出了腺体分化肿瘤细胞。致密结构主要存在于未消化的培养物中，而空心结构在经过单细胞消化的培养物中占主导地位。Ki67突出了具有更高细胞增殖的细胞群。红色的Ki67和绿色的PanCK在EAC初级组织，EAC PDO致密结构和EAC PDO中空结构中分布相似。该图显示了基于单细胞的冷冻保存和基于未消化的基于PDO的冷冻保存从冷冻储备中恢复的第一天的EAC PDO的形态学特征。我们建议提前制定明确的亚文化计划。在传代培养PDO时，温度控制和温和移液对于正确处理ECM凝胶至关重要。我们的方案为研究人员提供了根据其研究目的维护其EAC PDO的大量资金，用于下游分析，例如药物筛选测定，流式细胞术或组织学分析。我们希望我们的工作可以帮助类器官研究人员快速轻松地决定类器官的储存和亚培养，特别是在转化研究中患者衍生材料的稳健结果。

Research

JoVE Journal

Cancer Research

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

单细胞下游特征的靶细胞预富集与全基因组扩增

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

科研

癌症研究

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

合成肺腺癌细胞原位移植研究 pd-l1 表达

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Phospho Flow Cytometry with Fluorescent Cell Barcoding for Single Cell Signaling Analysis and Biomarker Discovery

磷流式细胞仪与荧光细胞条形码用于单细胞信号分析和生物标志物发现

Aberrant cell signaling plays a central role in cancer development and progression. Most novel targeted therapies are indeed directed at proteins and ...

Establishment and Characterization of Three Afatinib-resistant Lung Adenocarcinoma PC-9 Cell Lines Developed with Increasing Doses of Afatinib

建立和表征三个抗阿法替尼的肺腺癌PC-9细胞系与增加剂量的阿法替尼开发

Acquired resistance to molecular target inhibitors is a severe problem in cancer therapy. Lung cancer remains the leading cause of cancer-related death in ...

Isolation of Proximal Fluids to Investigate the Tumor Microenvironment of Pancreatic Adenocarcinoma

分离近端液研究胰腺腺癌肿瘤微环境

Pancreatic adenocarcinoma (PDAC) is the fourth leading cause of cancer-related death, and soon to become the second. There is an urgent need of variables ...

Generating and Imaging Mouse and Human Epithelial Organoids from Normal and Tumor Mammary Tissue Without Passaging

从正常和肿瘤乳腺组织中生成和成像小鼠和人上皮类器官，无需传代

Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after ...

Modeling Oral-Esophageal Squamous Cell Carcinoma in 3D Organoids

在 3D 类器官中模拟口腔-食管鳞状细胞癌

Esophageal squamous cell carcinoma (ESCC) is prevalent worldwide, accounting for 90% of all esophageal cancer cases each year, and is the deadliest of all ...

Enhancing Targeted Cancer Diagnosis by Quantifying Single-Cell Mechanics

Shear Assay Protocol for the Determination of Single-Cell Material Properties

Shear Assay Protocol for the Determination of Single-Cell Material Properties (Video) | JoVE 

用于测定单细胞材料特性的剪切测定方案

Irregular biomechanics are a hallmark of cancer biology subject to extensive study. The mechanical properties of a cell are similar to those of a ...

TMEM200A: A Novel Oncogene Regulating EMT and PI3K/AKT in Gastric Cancer

Multiomics Analysis of TMEM200A as a Pan-Cancer Biomarker

Unveiling Transmembrane Protein Family-Related Markers in Gastric Cancer and Implications for Targeted Therapies (Video) | JoVE

TMEM200A作为泛癌生物标志物的多组学分析

The transmembrane protein, TMEM200A, is known to be associated with human cancers and immune infiltration. Here, we assessed the function of TMEM200A in ...