Name: evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates
Uploaded: 2017-03-07T14:00:00
Description: Watch this Scientific Journal Video about Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates at JoVE.com

这项工作是由来自教育，青年，体育和（LO1304）捷克外交部和捷克共和国的技术局（TE01020028）资助。笔者想感谢拉克什曼瓦拉纳西博士采取环保盖的静止图像。

1.琼脂糖包被的平板的制备<ol><li>权衡0.75克低熔点琼脂糖和它的McCoy的5A培养基中的溶液（有或没有酚红）无血清添加到100。加热在微波的溶液，摇动每1-2分钟以完全溶解琼脂糖。高压灭菌的溶液消毒它。 </li><li>冷却该高压灭菌琼脂糖至约70℃，并通过500毫升，0.22微米过滤器顶部通过真空过滤在层流箱过滤。如果不使用整个解决方案立刻分装0.75％的过滤琼脂糖溶液（FAS）为更小的体积。在寒冷?...

<ol>
	<li>Kimlin, L. C., Casagrande, G., Virador, V. M. <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+three-dimensional+(3D)+models+in+cancer+research:+An+update.">In vitro three-dimensional (3D) models in cancer research: An update.</a> Mol Carcinog. 52 (3), 167-182 (2013).</li><li>Das, V., Bruzzese, F., Kone&#269;n&#253;, P., Iannelli, F., Budillon, A., Hajd&#250;ch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathophysiologically+relevant+in+vitro+tumor+models+for+drug+screening.">Pathophysiologically relevant in vitro tumor models for drug screening.</a> Drug Discov. Today. 20 (7), 848-855 (2015).</li><li>Weigelt, B., Ghajar, C. 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=The+need+for+complex+3D+culture+models+to+unravel+novel+pathways+and+identify+accurate+biomarkers+in+breast+cancer.">The need for complex 3D culture models to unravel novel pathways and identify accurate biomarkers in breast cancer.</a> Adv. Drug Deliv. Rev. , 69-70 (2014).</li><li>Fischbach, C., Kong, H. J., Hsiong, S. X., Evangelista, M. B., Yuen, W., Mooney, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+cell+angiogenic+capability+is+regulated+by+3D+culture+and+integrin+engagement.">Cancer cell angiogenic capability is regulated by 3D culture and integrin engagement.</a> Proc Natl Acad Sci U S A. 106 (2), 399-404 (2009).</li><li>Lao, 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=Improved+Methods+to+Generate+Spheroid+Cultures+from+Tumor+Cells,+Tumor+Cells+&amp;+Fibroblasts+or+Tumor-Fragments:+Microenvironment,+Microvesicles+and+MiRNA.">Improved Methods to Generate Spheroid Cultures from Tumor Cells, Tumor Cells &amp; Fibroblasts or Tumor-Fragments: Microenvironment, Microvesicles and MiRNA.</a> PLoS ONE. 10 (7), e0133895 (2015).</li><li>Wenzel, 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+high-content+screening+for+the+identification+of+compounds+that+target+cells+in+dormant+tumor+spheroid+regions.">3D high-content screening for the identification of compounds that target cells in dormant tumor spheroid regions.</a> Exp. Cell Res. 323 (1), 131-143 (2014).</li><li>Li, Q., 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+Models+of+Epithelial-Mesenchymal+Transition+in+Breast+Cancer+Metastasis:+High-Throughput+Screening+Assay+Development,+Validation,+and+Pilot+Screen.">3D Models of Epithelial-Mesenchymal Transition in Breast Cancer Metastasis: High-Throughput Screening Assay Development, Validation, and Pilot Screen.</a> J. Biomol. Screen. 16 (2), 141-154 (2011).</li><li>Das, V., F&#252;rst, T., Gursk&#225;, S., D&#382;ub&#225;k, P., Hajd&#250;ch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reproducibility+of+Uniform+Spheroid+Formation+in+384-Well+Plates:+The+Effect+of+Medium+Evaporation.">Reproducibility of Uniform Spheroid Formation in 384-Well Plates: The Effect of Medium Evaporation.</a> J. Biomol. Screen. , (2016).</li><li>Celli, J. 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=An+imaging-based+platform+for+high-content,+quantitative+evaluation+of+therapeutic+response+in+3D+tumour+models.">An imaging-based platform for high-content, quantitative evaluation of therapeutic response in 3D tumour models.</a> Sci. Rep. 17 (4), 3751 (2014).</li><li>Solomon, M. A., Lemera, J., D'Souza, G. G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+in+vitro+tumor+spheroid+culture+model+amenable+to+high-throughput+testing+of+potential+anticancer+nanotherapeutics.">Development of an in vitro tumor spheroid culture model amenable to high-throughput testing of potential anticancer nanotherapeutics.</a> J. Liposome Res. 26 (3), 246-260 (2016).</li><li>Costa, E. C., Gaspar, V. M., Coutinho, P., Correia, I. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+liquid+overlay+technique+to+formulate+heterogenic+3D+co-cultures+models.">Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models.</a> Biotechnol. Bioeng. 111 (8), 1672-1685 (2014).</li><li>Walzl, 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=A+Simple+and+Cost+Efficient+Method+to+Avoid+Unequal+Evaporation+in+Cellular+Screening+Assays,+Which+Restores+Cellular+Metabolic+Activity.">A Simple and Cost Efficient Method to Avoid Unequal Evaporation in Cellular Screening Assays, Which Restores Cellular Metabolic Activity.</a> Int. J. Appl. Sci. Technol. 2 (6), 17-25 (2012).</li><li>Berthier, E., Warrick, J., Yu, H., Beebe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Managing+evaporation+for+more+robust+microscale+assays.+Part+1.+Volume+loss+in+high+throughput+assays.">Managing evaporation for more robust microscale assays. Part 1. Volume loss in high throughput assays.</a> Lab Chip. 8 (6), 852-859 (2008).</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> Sci. Rep. 6, 19103 (2016).</li><li>Zimmermann, H. F., John, G. T., Trauthwein, H., Dingerdissen, U., Huthmacher, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+Evaluation+of+Oxygen+and+Water+Permeation+through+Microplate+Sealing+Tapes.">Rapid Evaluation of Oxygen and Water Permeation through Microplate Sealing Tapes.</a> Biotechnol. Prog. 19 (3), 1061-1063 (2003).</li><li>Sirenko, O., Mitlo, T., Hesley, J., Luke, S., Owens, W., Cromwell, E. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Content+Assays+for+Characterizing+the+Viability+and+Morphology+of+3D+Cancer+Spheroid+Cultures.">High-Content Assays for Characterizing the Viability and Morphology of 3D Cancer Spheroid Cultures.</a> Assay Drug Dev. Technol. 13 (7), 402-414 (2015).</li><li>Chen, W., Wong, C., Vosburgh, E., Levine, A. J., Foran, D. J., Xu, E. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+Image+Analysis+of+Tumor+Spheroids:+A+User-friendly+Software+Application+to+Measure+the+Size+of+Spheroids+Automatically.">High-throughput Image Analysis of Tumor Spheroids: A User-friendly Software Application to Measure the Size of Spheroids Automatically.</a> J. Vis. Exp. (89), e51639 (2014).</li></ol>

涂层384孔TC板用过滤琼脂糖在很多的标准做法是使用1-1.5％低熔点琼脂糖涂覆板，这需要琼脂糖和/或分配单元也可以保持加热，以防止琼脂糖6的胶凝。琼脂糖的凝胶化是可能引起关注的，而使用具有小管的孔的范围直径在0.2和0.4之间的液体分配盒制备多块板。为了克服堵塞分配盒的潜在问题，0.75％的FAS被使用，因为它在分配期间不需要额外的加热。顺便说一?...

在肿瘤癌细胞生理学布置在一个复杂的，三维（3D）细胞外基质和相互作用的细胞所包围的结构。作为近组织中的所有细胞驻留在3D环境中，有必要在该模拟肿瘤性状体外肿瘤模型更生理学相关导致的几个三维培养技术1，2，3的发展。这些模型正在成为基本的研究工具用于研究在三维2转移和细胞响应于治疗肿瘤微环境的作用。此外，相对于2维（2D）细胞培养物4，3D模型允许更好地了解肿瘤-基质的相互作用，从而影响细胞信号通路的。 的肿瘤细胞株多细胞肿瘤球状体（MCTSes）在三维细胞c的常用由于其相对接近体内肿瘤ulture车型。出的使用中的多种技术，MCTS代的琼脂糖包被的平板的液体覆盖技术（LOT）已经获得了铅的优化和靶标确认5，6，7，8，9，10，11显著兴趣。这是从最近的研究已成功能在使用LOT 6,7 MCTS文化运行化合物文库的导频屏幕明显。然而，由于介质的蒸发引起的不均匀损耗油井到井变异MCTS形态学和生长是伴随使用微量滴定板（MPS）的LOT常见障碍。因此，非均匀MCTSes形成危及意义和相关从药理学试验8，12，13的数据。除了再现性的问题，使用自动液体分配单元时影响基于LOT-高通量测定另一实际问题是在MP的涂布用琼脂糖。虽然分配单元可以保持加热，以防止琼脂糖的凝胶化，分配盒和管道的堵塞是机器人系统6的电位的关注。 为了克服这些挑战，我们最近在很多关于MCTS文化8设计了一些修改。这些修改主要是根据可能的方法来防止用户使用该工具在高通量筛查实验室中常见的国会议员中损失不均匀。修改后的很多跨3大小均匀，重现性MCTSes生成的详细过程84孔板（WPS）这里提出。手稿还提出了MCTS大小的评价半自动程序，特别是在不具有横截面面积的测量一个明确定义的边界部分破碎，药物处理MCTSes。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9414</td>
			<td>Low-melting</td>
		</tr>
		<tr>
			<td>McCOY&#39;s 5A Medium</td>
			<td>Sigma-Aldrich</td>
			<td>M8403</td>
			<td></td>
		</tr>
		<tr>
			<td>&#8220;rapid&#8221; Filtermax filter</td>
			<td>TPP</td>
			<td>99505</td>
			<td>0.22 &#956;m, 500 mL</td>
		</tr>
		<tr>
			<td>Multidrop&#8482; Combi Reagent Dispenser&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>5840300</td>
			<td></td>
		</tr>
		<tr>
			<td>Small Tube Dispensing cassette&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td>24073295</td>
			<td>Metal tip&#160;</td>
		</tr>
		<tr>
			<td>384-well TC plate&#160;</td>
			<td>PerkinElmer</td>
			<td>6057308</td>
			<td>Plate type- CellCarrier</td>
		</tr>
		<tr>
			<td>Standard Tube Dispensing Cassette</td>
			<td>Thermo Fisher Scientific</td>
			<td>24072670</td>
			<td></td>
		</tr>
		<tr>
			<td>MicroClime Environmental Lid</td>
			<td>Labcyte</td>
			<td>LLS-0310</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma</td>
			<td>D4540</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary Incubator (SteriStore )</td>
			<td>HighRes Biosolutions</td>
			<td>23641</td>
			<td>Serial No.: D00384</td>
		</tr>
		<tr>
			<td>Microplate Washer Dispenser&#160;</td>
			<td>BioTek</td>
			<td>Unspecified</td>
			<td>Model: EL406&#160;</td>
		</tr>
		<tr>
			<td>High-Content Imaging System (CellVoyager )</td>
			<td>Yokogawa Electric Corporation</td>
			<td>Unspecified</td>
			<td>Model: CV7000</td>
		</tr>
		<tr>
			<td>Orange G</td>
			<td>New England Biolabs</td>
			<td>B7022S</td>
			<td></td>
		</tr>
		<tr>
			<td>TrypLE&#8482; Express recombinant cell dissociation reagent</td>
			<td>Thermo Fisher Scientific</td>
			<td>12604021</td>
			<td>Phenol red free</td>
		</tr>
	</tbody>
</table>

evaporation-reducing culture condition increases the reproducibility of multicellular spheroid formation in microtiter plates

肿瘤模型， 在体内条件密切模仿正成为药物发现和开发的潜在的抗癌药物的筛选日益流行。多细胞肿瘤球状体（MCTSes）有效模仿实体瘤的生理条件下，使它们在体外模型优良铅优化和目标验证。从可供MCTS文化的各种技术，在琼脂糖液体覆盖法是对MCTS一代最便宜的方法之一。然而，使用液体的覆盖为高通量筛选MCTS培养物的可靠传输可能由许多限制，包括微量滴定板（MPS）琼脂糖的涂层并在井均匀MCTS地层的不可再现性受到损害。国会议员是显著容易发生边缘从培养基从板的外部过度蒸发造成的影响，防止药物使用整个板块的试验。这个手稿提供到液体覆盖技术来增加均匀MCTS形成的可扩展性和可重复性的详细技术的改进。此外，药物治疗提出后，对MCTS的评价简​​单，半自动，和普遍适用的软件工具的细节特征。

不均匀的损失中，尤其是来自周边井，是小培养体积议员们经常遇到的问题。大大改善培养条件，如与良好控制的温度/加湿系统和蒸发减少国会议员和板盖孵化器，降低介质的整个井8的显著损失。测量相对蒸发，橙G（OG）等体积加入到每孔中，并用3天OG吸光度变化被记录在与在标准和旋转孵化定期和环境盖板。板孔被分成基于从板（ 图2A）...

Watch this Scientific Journal Video about Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates at JoVE.com

Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates

培养基从微量滴定板的凹凸损失影响均匀的多细胞肿瘤球体形成的再现性。改善培养条件，以减少显著介质损耗将提高球体形成的再现和基于球状体的测定的使用液体覆盖技术的结果。

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