Name: generation of 3d tumor spheroids for drug evaluation studies
Uploaded: 2023-02-24T13:20:00
Description: Watch this Scientific Journal Video about Generation of 3D Tumor Spheroids for Drug Evaluation Studies at JoVE.com

我们感谢实验室的所有成员的重要意见和建议。本研究得到了江苏省卫生健康委员会重点项目（K2019030）的支持。概念化由C.W.和Z.C.进行，方法由W.H.和M.L.执行，调查由W.H.和M.L.执行，数据管理由W.H.，Z.Z.，S.X.和M.L.执行，原始草稿准备由Z.Z.，J.Z.，S.X.，W.H.执行， 和X.L.，审查和编辑由Z.C.执行，项目管理由C.W.和Z.C.执行，资金获取由C.W.进行。所有作者均已阅读并同意该手稿的已出版版本。

1. 球体结构
<ol><li>培养板的抗粘连处理<ol><li>将 100 μL 抗粘附试剂移液到具有 U 形孔底的 48 孔板的每个孔中，并保持 10 分钟。10分钟后，吸出涂层试剂，并用无菌PBS洗涤两次。</li>
		<li>将培养板放入培养箱（37°C在加湿空气中，5%CO2）直至使用。</li>
	</ol></li>
	<li>细胞制备、收集和计数<ol><li>使用细胞特异性培养基在细胞培养瓶中培养细胞（补充表2）。例如，NCI-H...

<ol>
	<li>Carioli, 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=European+cancer+mortality+predictions+for+the+year+2021+with+focus+on+pancreatic+and+female+lung+cancer.">European cancer mortality predictions for the year 2021 with focus on pancreatic and female lung cancer.</a> Annals of Oncology. 32 (4), 478-487 (2021).</li><li>Katti, A., Diaz, B. J., Caragine, C. M., Sanjana, N. E., Dow, L. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR+in+cancer+biology+and+therapy.">CRISPR in cancer biology and therapy.</a> Nature Reviews Cancer. 22 (5), 259-279 (2022).</li><li>Abrantes, R., Duarte, H. O., Gomes, C., Walchili, S., Reis, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CAR-Ts:+New+perspectives+in+cancer+therapy.">CAR-Ts: New perspectives in cancer therapy.</a> FEBS Letter. 596 (4), 403-416 (2022).</li><li>Shokooohi, 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=Effect+of+targeted+therapy+and+immunotherapy+on+advanced+nonsmall-cell+lung+cancer+outcomes+in+the+real+world.">Effect of targeted therapy and immunotherapy on advanced nonsmall-cell lung cancer outcomes in the real world.</a> Cancer Medicine. 11 (1), 86-93 (2022).</li><li>Chen, K., Zhang, Y., Qian, L., Wang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+strategies+to+target+RAS+signaling+in+human+cancer+therapy.">Emerging strategies to target RAS signaling in human cancer therapy.</a> Journal of Hematology &amp; Oncology. 14 (1), 116 (2021).</li><li>Pinto, B., Henriques, A. C., Silva, P. M. A., Bousbaa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+spheroids+as+in+vitro+preclinical+models+for+cancer+research.">Three-dimensional spheroids as in vitro preclinical models for cancer research.</a> Pharmaceutics. 12 (12), 1186 (2020).</li><li>Jensen, C., Teng, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Is+it+time+to+start+transitioning+from+2D+to+3D+cell+culture.">Is it time to start transitioning from 2D to 3D cell culture.</a> Frontiers in Molecular Biosciences. 7, 33 (2020).</li><li>Qin, Y., Hu, X., Fan, W., Yan, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+stretchable+scaffold+with+electrochemical+sensing+for+3D+culture,+mechanical+loading,+and+real-time+monitoring+of+cells.">A stretchable scaffold with electrochemical sensing for 3D culture, mechanical loading, and real-time monitoring of cells.</a> Advanced Science. 8 (13), 2003738 (2021).</li><li>Wartenberg, 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=Regulation+of+the+multidrug+resistance+transporter+P-glycoprotein+in+multicellular+tumor+spheroids+by+hypoxia-inducible+factor+(HIF-1)+ad+reactive+oxygen+species.">Regulation of the multidrug resistance transporter P-glycoprotein in multicellular tumor spheroids by hypoxia-inducible factor (HIF-1) ad reactive oxygen species.</a> FASEB Journal. 17 (3), 503-505 (2003).</li><li>Minchinton, A. I., Tannock, I. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+penetration+in+solid+tumours.">Drug penetration in solid tumours.</a> Nature Reviews Cancer. 6 (8), 583-592 (2006).</li><li>Baker, B. M., Chen, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deconstructing+the+third+dimension:+How+3D+culture+microenvironments+alter+cellular+cues.">Deconstructing the third dimension: How 3D culture microenvironments alter cellular cues.</a> Journal of Cell Science. 125 (13), 3015-3024 (2012).</li><li>Br&#252;ningk, S. C., Rivens, I., Box, C., Oelfke, U., Ter Haar, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumour+spheroids+for+the+prediction+of+the+effects+of+radiation+and+hyperthermia+treatments.">3D tumour spheroids for the prediction of the effects of radiation and hyperthermia treatments.</a> Scientific Reports. 10, 1653 (2020).</li><li>Graves, E. E., Maity, A., Thu Le, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumor+microenvironment+in+non-small-cell+lung+cancer.">The tumor microenvironment in non-small-cell lung cancer.</a> Seminars in Radiation Oncology. 20 (3), 156-163 (2010).</li><li>Kunz-Schughart, L. A., Frreyer, J. P., Ebner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+3-D+cultures+for+high-throughput+screening:+The+multicellular+spheroid+model.">The use of 3-D cultures for high-throughput screening: The multicellular spheroid model.</a> Journal of Biomolecular Screening. 9 (4), 273-285 (2004).</li><li>Carragher, 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=Concerns,+challenges+and+promises+of+high-content+analysis+of+3D+cellular+models.">Concerns, challenges and promises of high-content analysis of 3D cellular models.</a> Nature Review Drug Discovery. 17 (8), 606 (2018).</li><li>Huang, 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=Longitudinal+morphological+and+physiological+monitoring+of+three-dimensional+tumor+spheroids+using+optical+coherence+tomography.">Longitudinal morphological and physiological monitoring of three-dimensional tumor spheroids using optical coherence tomography.</a> Journal of Visualized Experiments. (144), e59020 (2019).</li><li>Yazdanfar, 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=Simple+and+robust+image-baed+autofocusing+for+digital+microscopy.">Simple and robust image-baed autofocusing for digital microscopy.</a> Optics Express. 16 (12), 8670-8677 (2008).</li><li>Chen, 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=Automated+evaluation+of+tumor+spheroid+behavior+in+3D+culture+using+deep+learning-based+recognition.">Automated evaluation of tumor spheroid behavior in 3D culture using deep learning-based recognition.</a> Biomaterials. 22 (272), 120770 (2021).</li><li>Boucherit, N., Gorvel, L., Olive, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+tumor+models+and+their+use+for+the+testing+of+immunotherapies.">3D tumor models and their use for the testing of immunotherapies.</a> Frontiers in Immunology. 11, 603640 (2020).</li><li>Anastasiou, 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=Microenvironment+factors+shaping+the+cancer+metabolism+landscape.">Microenvironment factors shaping the cancer metabolism landscape.</a> British Journal of Cancer. 116 (3), 277-286 (2017).</li><li>Zhou, 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=Functions+and+clinical+significance+of+mechanical+tumor+microenvironment:+Cancer+cell+sensing,+mechanobiology+and+metastasis.">Functions and clinical significance of mechanical tumor microenvironment: Cancer cell sensing, mechanobiology and metastasis.</a> Cancer Communications. 43 (5), 374-400 (2022).</li><li>Zhu, G. 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=Targeting+KRAS+cancers:+From+druggable+therapy+to+druggable+resistance.">Targeting KRAS cancers: From druggable therapy to druggable resistance.</a> Molecular Cancer. 21 (1), 159 (2022).</li><li>Ando, Y., Mariano, C., Shen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineered+in+vitro+tumor+models+for+cell-based+immunotherapy.">Engineered in vitro tumor models for cell-based immunotherapy.</a> Acta Biomaterialia. 132, 345-359 (2021).</li><li>Timmins, N. E., Dietmair, S., Nielsen, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hanging-drop+multicellular+spheroids+as+a+model+of+tumor+angiogenesis.">Hanging-drop multicellular spheroids as a model of tumor angiogenesis.</a> Angiogenesis. 7 (2), 97-103 (2004).</li><li>Costa, E. 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=3D+tumor+spheroids:+An+overview+on+the+tools+and+techniques+used+for+their+analysis.">3D tumor spheroids: An overview on the tools and techniques used for their analysis.</a> Biotechnology Advances. 34 (8), 1427-1441 (2016).</li><li>Sant, S., Johnston, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+production+of+3D+tumor+spheroids+for+cancer+drug+discovery.">The production of 3D tumor spheroids for cancer drug discovery.</a> Drug Discovery Today. Technologies. 23, 27-36 (2017).</li><li>Zanoni, 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=3D+tumor+spheroid+models+for+in+vitro+therapeutic+screening:+A+systematic+approach+to+enhance+the+biological+relevance+of+data+obtained.">3D tumor spheroid models for in vitro therapeutic screening: A systematic approach to enhance the biological relevance of data obtained.</a> Scientific Reports. 6, 19103 (2016).</li></ol>

微环境在肿瘤生长中起着重要作用。它可能影响细胞外基质、氧梯度、营养和机械相互作用的提供，从而影响肿瘤细胞的基因表达、信号通路和许多功能19,20,21。在许多情况下，2D细胞不会产生这种效果，甚至不会产生相反的效果，从而影响药物治疗的评估。然而，3D模型的出现解决了这个问题。例如，我们使用3D方法完成了对A...

癌症是人类最害怕的疾病之一，尤其是因为它的高死亡率1。近年来，随着新疗法的引入，治疗癌症的可能性有所增加2,3,4,5。二维（2D）和三维（3D）体外模型用于在实验室环境中研究癌症。然而，2D模型不能立即准确地评估指示抗肿瘤敏感性的所有重要参数;因此，它们无法完全代表药物治疗测试中的体内相互作用6。
自2020年以来，全球三维（3D）文化市场得到了极大的推动。根据纳斯达克OMX的一份报告，到2025年底，3D细胞培养市场的全球价值将超过27亿美元。与2D培养方法相比，3D细胞培养表现出有利的特性，不仅可以优化增殖和分化，还可以优化长期存活7,8。通过这种方法，可以模拟体内细胞微环境以获得更准确的肿瘤表征，以及代谢分析，从而可以更好地了解基因组和蛋白质的改变。因此，3D测试系统现在应该被纳入主流药物开发业务，特别是那些专注于筛选和评估新型抗肿瘤药物的操作。永生化已建立的细胞系或原代细胞培养物在球状结构中的三维生长具有肿瘤的缺氧和药物渗透等体内特征，以及细胞相互作用、反应和耐药性，可被视为进行体外药物筛选的严格和代表性模型9,10,11。
然而，这些3D文化模型也存在一些可能需要一些时间才能解决的问题。可以使用这些方案形成细胞球状体，但它们在某些细节上有所不同，例如培养时间或包埋凝胶12，因此这些构建的细胞球体不能在有限的尺寸范围内得到很好的控制。微球的大小可能会影响活力测试和成像分析的一致性。生长微环境和生长因子也不同，由于细胞间分化的差异，可能导致不同的形态13。现在显然需要一种标准、简单且具有成本效益的方法来构建具有受控大小的所有类型的肿瘤。
从另一个角度来看，尽管已经开发了均质测定和高内涵成像方法来评估形态、活力和生长速率，但由于文献中报道的各种原因，例如肿瘤球体的位置、大小和形态缺乏均匀性，3D 模型的高通量筛选仍然是一个挑战14,15,16。
在这里介绍的协议中，我们列出了构建3D肿瘤微球的每个步骤，并描述了一种使用高通量，高内涵成像系统的球状体观察和分析方法，该系统涉及自动聚焦，自动成像和分析，以及其他有利特征。我们展示了这种方法如何产生适用于高通量成像的均匀大小的3D肿瘤球体。这些微球还表现出对癌症药物治疗的高敏感性，并且可以使用高内涵成像监测微球的形态变化。总之，我们证明了这种方法作为生成用于药物评估目的的3D肿瘤构建体的手段的稳健性。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5-10 &#956;L Pipette tips</td>
			<td>AXYGEN</td>
			<td>T-300</td>
			<td></td>
		</tr>
		<tr>
			<td>1.5&#160;mL Boil proof microtubes</td>
			<td>Axygen</td>
			<td>MCT-150-C</td>
			<td></td>
		</tr>
		<tr>
			<td>100-1000&#956;L Pipette tips</td>
			<td>KIRGEN</td>
			<td>KG1313</td>
			<td></td>
		</tr>
		<tr>
			<td>15 mL Centrifuge Tube</td>
			<td>Nest</td>
			<td>601052</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#956;L Pipette tips</td>
			<td>AXYGEN</td>
			<td>T-200-Y</td>
			<td></td>
		</tr>
		<tr>
			<td>3D gel</td>
			<td>Avatarget</td>
			<td>MA02</td>
			<td></td>
		</tr>
		<tr>
			<td>48-well U bottom Plate</td>
			<td>Avatarget</td>
			<td>P02-48UWP</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL Centrifuge Tube</td>
			<td>Nest</td>
			<td>602052</td>
			<td></td>
		</tr>
		<tr>
			<td>Alamar Blue</td>
			<td>Thermo</td>
			<td>&#160;DAL1100</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Adherence Rinsing Solution</td>
			<td>STEMCELL</td>
			<td>#07010</td>
			<td></td>
		</tr>
		<tr>
			<td>Certified FBS</td>
			<td>BI</td>
			<td>04-001-1ACS</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>aladdin</td>
			<td>W433884-500ml</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM (Dulbecco&#39;s Modified Eagle Medium)</td>
			<td>Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>sigma</td>
			<td>D2650-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel sofware&#160;</td>
			<td>Microsoft office</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Graphpad prism sofware&#160;</td>
			<td>GraphPad software&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>High Content Microscope and SMART system</td>
			<td>Avatarget</td>
			<td>1-I01</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J software</td>
			<td>National Institutes of Health</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Insulin-Transferrin-Selenium-A Supplement (100X)</td>
			<td>Gibco</td>
			<td>51300-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Parafilm</td>
			<td>Bemis</td>
			<td>PM-996</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Solarbio</td>
			<td>P1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin Sol</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>Scientific Fluoroskan Ascent</td>
			<td>Thermo</td>
			<td>Fluoroskan Ascent</td>
			<td></td>
		</tr>
		<tr>
			<td>T25 Flask</td>
			<td>JET Biofil</td>
			<td>TCF012050</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin, 0.25% (1X)</td>
			<td>Hyclone</td>
			<td>SH30042.01</td>
			<td></td>
		</tr>
	</tbody>
</table>

generation of 3d tumor spheroids for drug evaluation studies

近几十年来，除了单层培养细胞外，三维肿瘤球状体已被开发为评估抗癌药物的潜在强大工具。然而，传统的培养方法缺乏在三维水平上以均匀方式操纵肿瘤球状体的能力。为了解决这一局限性，在本文中，我们提出了一种构建平均大小的肿瘤球体的方便有效的方法。此外，我们描述了一种使用基于人工智能的分析软件进行基于图像的分析方法，该软件可以扫描整个板并获得三维球体的数据。研究了几个参数。通过使用肿瘤球体构建的标准方法和高通量成像和分析系统，可以显着提高对三维球状体进行的药物测试的有效性和准确性。

图1A，B显示了本研究中用于构建肿瘤球体的过程。 我们首先将细胞接种在48孔U底板中。此步骤与2D细胞培养中使用的步骤几乎相同。我们将板保存在一个公共培养箱中，孔周围有水，以便沉积的细胞在自组装过程中开始形成球状体。在正常操作条件下，大多数类型的肿瘤球状体在使用靶向培养基5天后完全形成（补充表2）。我们在5天内用数码显微镜...

Watch this Scientific Journal Video about Generation of 3D Tumor Spheroids for Drug Evaluation Studies at JoVE.com

Generation of 3D Tumor Spheroids for Drug Evaluation Studies

本文演示了一种构建三维肿瘤球体的标准化方法。还描述了使用自动成像系统进行球体观察和基于图像的深度学习分析的策略。

生成用于药物评估研究的 3D 肿瘤微球

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the ...

该协议展示了构建球状体三维肿瘤的标准化方法。这种方法提供了3D肿瘤构建体的高回路和高内涵分析。通过使用肿瘤球体构建的标准方法和高通量成像和分析系统，可以显着提高在三维球体上形成的药物测试的有效性和准确性。在该协议中，我们使用AMG510处理NCI-H23球体为例。从实验中，我们可以观察到癌靶药物对肿瘤球状体的显着影响。首先，将100微升抗粘附试剂移液到具有U形孔底的48孔板的每个孔中并保持10分钟。10分钟后，吸出涂层试剂并用无菌PBS洗涤两次。将培养板置于37摄氏度的培养箱中，在含有5%二氧化碳的潮湿空气中直至使用。在显微镜下观察细胞。然后，用PBS洗涤在T25烧瓶中培养的细胞两次以除去培养基，并在37摄氏度，5%二氧化碳的培养箱中用一毫升0.25%胰蛋白酶EDTA处理扩增的细胞一至两分钟。在显微镜下确认细胞形状。然后通过在T25烧瓶中吸入用过的胰蛋白酶EDTA悬浮液并用4毫升新鲜培养基洗涤细胞来停止胰蛋白酶处理。将所有悬浮液转移到15毫升管中，并使用一毫升新鲜培养基冲洗残留细胞并将其添加到管中。在室温下以186.48G离心细胞五分钟，弃去上清液。向细胞沉淀中加入10毫升新鲜培养基，轻轻移液，直到细胞处于均匀的悬浮液中。将0.1毫升细胞悬液吸入新的离心管中。加入0.9毫升新鲜培养基，然后充分移液悬浮液。提取10微升细胞悬液进行细胞计数。重复此过程一次或两次并取平均值。稀释悬浮液以达到每毫升50， 000个细胞的最终接种密度。接下来，将 200 微升细胞悬液添加到 48 孔 U 底板的每个孔中。将密封膜缠绕在板上，并在室温下以119.35G离心五分钟。离心后，拉下保护膜，将五至八毫升灭菌水加入井周围的水道中。将板在 37 摄氏度下孵育五天。在此期间不要向水通道更换或补充任何水，并在接下来的五天内观察细胞聚集。对于凝胶包埋，从零下20摄氏度的冰箱中取出冷冻凝胶后，在实验过程中将其整个时间放在冰盒上。在显微镜下观察细胞球状体。在凝胶包埋开始之前，应再次检查微球的状态。小心地除去150微升培养基，并通过从孔壁侧缓慢添加液体凝胶，同时将预冷移液器吸头在孔周围和内部移动，将每个球体嵌入凝胶中。等待五分钟，如果凝胶没有均匀扩散，请用 10 微升移液器吸头轻轻移液凝胶。每个孔包含一个肿瘤球体、25微升3.5毫克/毫升凝胶和50微升完全培养基。向对照中也加入 75 微升培养基。将板在37摄氏度下孵育30分钟，直到水凝胶完全完成。在显微镜下确认凝胶状态。在每个样品上覆盖125微升新鲜培养基，并将球状体再培养7至10天。准备每组有四到六个孔的球体，并选择至少三个进行分析。根据制造商的说明溶解药物，并用DMSO制备100倍工作溶液。使用0.1%DMSO作为阳性对照，并向每个孔中加入125微升药物治疗培养基。将板放回培养箱中，在含有5%二氧化碳的潮湿空气中，温度为37摄氏度。在此阶段，每个孔包含一个肿瘤球状体，25微升3.5毫克/毫升凝胶和175微升培养基。对照包含 200 微升培养基。根据制造商的指南，使用alamarBlue检测试剂盒测量球体活力。从每个孔中吸取100微升上清液培养基到新的测试板中。然后，使用微孔板光度计测量活性。在将微球嵌入凝胶后的第1天，第4天，第7天和第10天进行测量。向培养板的每个孔中加入80微升新鲜培养基。然后更换另外100微升药物治疗的培养基。确保井中没有阿拉马尔蓝的残留物。成像前吸出100微升培养基，并将板放在载物台上。使用具有十倍物镜的自动显微镜获得微球的数字图像。显微镜可以自动聚焦和集中这些球体。等待自动成像。为每个微球获取四个图像。使用连接到高内涵成像系统的软件形成和处理集成图像。单击映像修补过程按钮，然后在软件中选择集成映像。选择U净模型并输入转化率。单击屏幕底部以开始图像处理。将直径、周长和粗糙度数据保存在电子表格软件中。最后，用药物加入100微升新鲜培养基，并将板放回含有5%二氧化碳的潮湿空气中的37摄氏度培养箱中。用不同浓度的AMG510处理并由高内涵显微镜自动捕获的NCI-H23细胞球体的明场图像如图所示。列表示不同的日期，行表示不同的药物浓度。结果包括每个条件的三个球体。在第1天，第4天，第7天和第10天测量AMG510处理样品组的肿瘤球状体活力。具有浓度梯度的样品的终细胞活力如图所示。在第1天，第4天，第7天和第10天测量肿瘤球体直径。球体生长比定义为相对于原始体积的终端体积，并使用球体直径计算。相对于体积定义球体生长抑制比，并使用球体直径计算。在第1天、第4天、第7天和第10天用软件测量肿瘤粗糙度，表明肿瘤球状体的侵袭性。球体的周长由软件使用深度学习算法定位和绘制。然后通过图像J测量焦平面上的球体区域，并根据这些数据计算多余的周长指数。尽管此消息很容易遵循，但仍然需要仔细处理示例。

Research

JoVE Journal

Cancer Research

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

In Vivo Model for Testing Effect of Hypoxia on Tumor Metastasis

体内模型对缺氧对肿瘤转移的影响测试

Hypoxia has been implicated in the metastasis of Ewing sarcoma (ES) by clinical observations and in vitro data, yet direct evidence for its pro-metastatic ...

科研

癌症研究

Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening

高通量三维肿瘤球体药物筛选的生成

Cancer cells have routinely been cultured in two dimensions (2D) on a plastic surface. This technique, however, lacks the true environment a tumor mass is ...

Paramyxoviruses for Tumor-targeted Immunomodulation: Design and Evaluation Ex Vivo

肿瘤靶向免疫调节副病毒的设计与评价

Successful cancer immunotherapy has the potential to achieve long-term tumor control. Despite recent clinical successes, there remains an urgent need for ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Longitudinal Morphological and Physiological Monitoring of Three-dimensional Tumor Spheroids Using Optical Coherence Tomography

三维肿瘤球体的纵向形态和生理监测

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, ...

Physiologic Patient Derived 3D Spheroids for Anti-neoplastic Drug Screening to Target Cancer Stem Cells

生理患者衍生的3D类血球体用于抗肿瘤药物筛选,以靶向癌症干细胞

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of ...

Using Human Induced Pluripotent Stem Cells for the Generation of Tumor Antigen-specific T Cells

利用人类诱导多能干细胞生成肿瘤抗原特异性T细胞

The generation and expansion of functional T cells in vitro can lead to a broad range of clinical applications. One such use is for the treatment of ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

小肠神经内分泌肿瘤类球体的建立与特征

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

正交胰腺肿瘤的产生与肿瘤渗透T细胞细胞毒性的外体特征

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

A Novel Stromal Fibroblast-Modulated 3D Tumor Spheroid Model for Studying Tumor-Stroma Interaction and Drug Discovery

用于研究肿瘤-斯特罗马相互作用和药物发现的新型斯特罗姆纤维细胞-模组3D肿瘤球体模型

Tumor-stroma interactions play an important role in cancer progression. Three-dimensional (3D) tumor spheroid models are the most widely used in vitro ...