Name: generation of high-throughput three-dimensional tumor spheroids for drug screening
Uploaded: 2018-09-05T15:30:00.000+00:00
Duration: 5 min 54 s
Description: Watch this Scientific Journal Video about Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening at JoVE.com

作者希望在国家促进转化科学中心 (国家卫生研究院) 上承认他对这些化验的初步发展的支持和指导。此外, 我们要感谢艾莉弗里曼, Mariela Jaskelioff, 迈克尔 Acker, 雅各 Haling, Vesselina 库克作为项目团队成员的科学投入和讨论。

1. 1536年井3D 肿瘤球体的培养
<ol><li>将以下试剂加入500毫升的 Dulbecco 修饰鹰培养基 (DMEM/F12) 中, 制备新鲜的3D 肿瘤球形培养基茎 (细胞培养基 + 挖空血清置换 + 胰岛素-转铁蛋白-硒): 1x 青霉素/链霉素, 10 人/毫升碱性成纤维细胞生长因子 (bFGF), 20 ng/毫升人表皮生长因子 (EGF), 0.4% 牛血清白蛋白 (BSA), 1x 胰岛素-转铁蛋白-硒, 1% 挖空 (高) 血清置换 (不冻融)。</li>
	<li>过滤-通过0.4 µm 瓶顶过滤系统的补充剂消毒整个介质。</li>
	<li>根据推荐的常规培养条件, 分离癌细胞株, 从传统的细胞培养烧瓶中洗涤 3x, 用1x 磷酸盐缓冲盐水 (PBS), 加入适量的0.25% 胰蛋白酶 (1 毫升每5厘米2) 为5分钟, 在单元格上创建一个薄层。将胰蛋白酶与含有血清的培养基的数量5x 中和, 然后进行计数细胞。例如, 使用25毫升培养基5毫升的胰蛋白酶。</li>
	<li>将细胞溶液的浓度调整为 62.5 x 104细胞/毫升, 以将8µL 球形培养基中的500个细胞的总种子, 放入1536井组织培养处理的板材中。</li>
	<li>在一个生物安全柜中, 用一个用蠕动泵为基础的消毒的小的不锈钢尖盒来种子细胞。在不同的细胞线之间, 冲洗的卡带与 1x PBS 和70% 乙醇的导管十年代。</li>
	<li>或者, 使用在高效过滤室内的液体处理器, 使用灭菌的、小的不锈钢尖盒和蠕动泵或附加的注射器泵, 根据所需的每个细胞线的数量来播种细胞。</li>
	<li>使用透气的粘接板密封密封板, 手动或使用板封口机。</li>
	<li>将盘子放在一个纺纱孵化器设置在 10 rpm 和37°c 与5% 二氧化碳 (CO2) 和95% 相对湿度。允许球体形成 3 d。</li>
</ol>2. 培养1536年井2D 癌症线
注: 1536 井2D 癌症线应培养24小时之前, 添加的化合物到3D 板块。
<ol><li>通过将以下试剂添加到500毫升适当的生长培养基中, 制备2D 癌细胞培养基: 1x 青霉素/链霉素和10% 胎牛血清 (血清)。</li>
	<li>过滤-通过0.4 µm 瓶顶过滤系统的补充剂消毒整个介质。</li>
	<li>根据推荐的常规培养条件, 分离癌细胞株, 从传统的细胞培养烧瓶中冲洗3x 的 1x PBS, 并添加适量的0.25% 胰蛋白酶 (1 毫升每5厘米2), 以5分钟在单元格上创建一个薄层。将胰蛋白酶与含有血清的培养基的数量5x 中和, 然后进行计数细胞。例如, 使用25毫升培养基5毫升的胰蛋白酶。</li>
	<li>将细胞溶液的浓度调整为 62.5 x 104细胞/毫升, 以便在8µL 的完全培养基中, 将500个细胞的总粒数种到 1536-良好的组织培养处理板中。</li>
	<li>在一个生物安全柜中, 用一个用蠕动泵为基础的消毒的小的不锈钢尖盒来种子细胞。在不同的细胞线之间, 冲洗导管的卡带与1x 无菌 PBS 和70% 乙醇十年代。</li>
	<li>或者, 使用在高效过滤的机器人室内的液体处理器, 使用灭菌的、小的不锈钢尖盒和蠕动泵或所附的注射器泵, 根据每个单元格的板量来种子细胞线需要。</li>
	<li>使用透气的粘接板密封密封板, 手动或使用板封口机。</li>
	<li>将板块放在一个纺纱孵化器设置在 10 rpm 和37°c 24 小时之前, 复合添加, 与 5% CO2和95% 相对湿度。</li>
</ol>3. 复合加法
<ol><li>在100% 二甲基亚砜 (亚砜) 上制备8点3倍的 MAPK 抑制剂序列稀释, 其浓度为10毫米。蛋白酶体抑制剂佐米被用来作为一个完整的细胞杀死的积极控制, 以确定的动态范围的检测。</li>
	<li>快速旋转所有的化验板和复合板在 100 x g 收集任何冷凝。除去每个盘子上的粘合剂密封, 并将一个盘子放在一个音响分配器上, 以增加每一个混合物的 8 nL, 代表最终浓度为0.1% 亚砜每井和一个最高的复合剂量10µM。</li>
	<li>在化合物的添加完成后, 放置一个定制的, 不锈钢细胞培养盖子, 防止在板上的蒸发, 并将板块放入一个旋转孵化器在37°c 为 5 d, 与 5% CO2和95% 相对湿度。</li>
</ol>4. 检测试剂添加和原始数据的获取
<ol><li>预热适量的细胞裂解试剂, 含有荧光素, 以检测三磷酸腺苷 (ATP) 的变化, 从而改变细胞在室温或37°c 水浴中的增殖。使用蠕动泵在每个井中添加3µL 的检测试剂, 并在室温下孵化出至少15分钟的平板。</li>
	<li>捕捉紫外线板上的发光。</li>
</ol>5. 数据处理
<ol><li>从仪器中提取原始数据, 并将包含测试化合物的所有字段正常化为仅含有亚砜的所有井的平均值作为中性控制。从这个值计算每个化合物的生长抑制百分比。这是在 Microsoft Excel 中使用公式函数完成的, 其中f (x) = {(示例值相对光单位 (RLUs)/(平均亚砜值 RLUs) * 100)}。</li>
	<li>通过图形程序将步骤5.1 中规范化数据的值与复合浓度进行绘制, 生成剂量响应曲线和抑制 IC50s。 注: 我们使用了一个内部 NIBR 软件工具分析了数据。为了完成这一功能, 我们选择了非参数曲线分析工具。</li>
</ol>

<ol>
	<li>Lee, M., Vasioukhin, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+polarity+and+cancer--cell+and+tissue+polarity+as+a+non-canonical+tumor+suppressor.">Cell polarity and cancer--cell and tissue polarity as a non-canonical tumor suppressor.</a> Journal of Cell Science. 121 (Pt 8), 1141-1150 (2008).</li><li>Nath, S., Devi, G. R. <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+culture+systems+in+cancer+research:+Focus+on+tumor+spheroid+model.">Three-dimensional culture systems in cancer research: Focus on tumor spheroid model.</a> Pharmacology &amp; Therapeutics. 163, 94-108 (2016).</li><li>Weigelt, B., Ghajar, C. M. <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> Advanced Drug Delivery Reviews. 69, 42-51 (2014).</li><li>Li, Q., Chow, A. B., Mattingly, R. R. <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+overlay+culture+models+of+human+breast+cancer+reveal+a+critical+sensitivity+to+mitogen-activated+protein+kinase+kinase+inhibitors.">Three-dimensional overlay culture models of human breast cancer reveal a critical sensitivity to mitogen-activated protein kinase kinase inhibitors.</a> The Journal of Pharmacology and Experimental Therapeutics. 332 (3), 821-828 (2010).</li><li>Li, L., Lu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimizing+a+3D+Culture+System+to+Study+the+Interaction+between+Epithelial+Breast+Cancer+and+Its+Surrounding+Fibroblasts.">Optimizing a 3D Culture System to Study the Interaction between Epithelial Breast Cancer and Its Surrounding Fibroblasts.</a> Journal of Cancer. 2, 458-466 (2011).</li><li>Pickl, M., Ries, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+3D+and+2D+tumor+models+reveals+enhanced+HER2+activation+in+3D+associated+with+an+increased+response+to+trastuzumab.">Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab.</a> Oncogene. 28 (3), 461-468 (2009).</li><li>Nyga, A., Cheema, U., Loizidou, M. <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+models:+novel+in+vitro+approaches+to+cancer+studies.">3D tumour models: novel in vitro approaches to cancer studies.</a> Journal of Cell Communication and Signaling. 5 (3), 239-248 (2011).</li><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> Molecular Carcinogenesis. 52 (3), 167-182 (2013).</li><li>Mathews Griner, L. 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=Large-scale+pharmacological+profiling+of+3D+tumor+models+of+cancer+cells.">Large-scale pharmacological profiling of 3D tumor models of cancer cells.</a> Cell Death &amp; Disease. 7 (12), e2492 (2016).</li><li>Polo, 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=Responsiveness+to+PI3K+and+MEK+inhibitors+in+breast+cancer.+Use+of+a+3D+culture+system+to+study+pathways+related+to+hormone+independence+in+mice.">Responsiveness to PI3K and MEK inhibitors in breast cancer. Use of a 3D culture system to study pathways related to hormone independence in mice.</a> PLoS One. 5 (5), e10786 (2010).</li><li>Hatzivassiliou, 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=RAF+inhibitors+prime+wild-type+RAF+to+activate+the+MAPK+pathway+and+enhance+growth.">RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.</a> Nature. 464 (7287), 431-435 (2010).</li><li>Bhargava, 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=Registered+report:+RAF+inhibitors+prime+wild-type+RAF+to+activate+the+MAPK+pathway+and+enhance+growth.">Registered report: RAF inhibitors prime wild-type RAF to activate the MAPK pathway and enhance growth.</a> Elife. 5, e09976 (2016).</li><li>Mathews, L. A., Hurt, E. M., Zhang, X., Farrar, 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=Epigenetic+regulation+of+CpG+promoter+methylation+in+invasive+prostate+cancer+cells.">Epigenetic regulation of CpG promoter methylation in invasive prostate cancer cells.</a> Molecular Cancer. 9, 267 (2010).</li><li>Mathews, L. A., Crea, F., Farrar, 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=Epigenetic+gene+regulation+in+stem+cells+and+correlation+to+cancer.">Epigenetic gene regulation in stem cells and correlation to cancer.</a> Differentiation. 78 (1), 1-17 (2009).</li><li>Mathews, L. 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=Increased+expression+of+DNA+repair+genes+in+invasive+human+pancreatic+cancer+cells.">Increased expression of DNA repair genes in invasive human pancreatic cancer cells.</a> Pancreas. 40 (5), 730-739 (2011).</li><li>Mathews, L. 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+1536-well+quantitative+high-throughput+screen+to+identify+compounds+targeting+cancer+stem+cells.">A 1536-well quantitative high-throughput screen to identify compounds targeting cancer stem cells.</a> Journal of Biomolecular Screening. 17 (9), 1231-1242 (2012).</li><li>Horii, T., Nagao, Y., Tokunaga, T., Imai, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serum-free+culture+of+murine+primordial+germ+cells+and+embryonic+germ+cells.">Serum-free culture of murine primordial germ cells and embryonic germ cells.</a> Theriogenology. 59 (5-6), 1257-1264 (2003).</li><li>Sun, 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=Epigenetic+regulation+of+SOX9+by+the+NF-kappaB+signaling+pathway+in+pancreatic+cancer+stem+cells.">Epigenetic regulation of SOX9 by the NF-kappaB signaling pathway in pancreatic cancer stem cells.</a> Stem Cells. 31 (8), 1454-1466 (2013).</li><li>Mathews, L. 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+1536-well+quantitative+high-throughput+screen+to+identify+compounds+targeting+cancer+stem+cells.">A 1536-well quantitative high-throughput screen to identify compounds targeting cancer stem cells.</a> Journal of Biomolecular Screening. 17 (9), 1231-1242 (2012).</li><li>Mathews Griner, L. 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=High-throughput+combinatorial+screening+identifies+drugs+that+cooperate+with+ibrutinib+to+kill+activated+B-cell-like+diffuse+large+B-cell+lymphoma+cells.">High-throughput combinatorial screening identifies drugs that cooperate with ibrutinib to kill activated B-cell-like diffuse large B-cell lymphoma cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 111 (6), 2349-2354 (2014).</li><li>Herter, 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=A+novel+three-dimensional+heterotypic+spheroid+model+for+the+assessment+of+the+activity+of+cancer+immunotherapy+agents.">A novel three-dimensional heterotypic spheroid model for the assessment of the activity of cancer immunotherapy agents.</a> Cancer Immunology, Immunotherapy. 66 (1), 129-140 (2017).</li></ol>

作者是诺华的雇员, 一些员工也是股东。

本文介绍了如何在1536年的大型复合筛板上生产肿瘤球体的详细协议。这些方法最初是根据国家癌症研究所的工作改编的, 那里的肿瘤球体生长在6井和96井板块的低吞吐量化验中, 询问有关遗传依赖性和复合敏感性13的问题,14,15. 本议定书的一个关键和独特的特点是, 球体是由传统的2D 组织培养的细胞组成的 1536年-板块使用定义的?...

在过去的十年中, 越来越多的研究已经实施了3D 细胞培养模型, 以了解不完全概括的概念, 在2D 的塑料上生长的细胞。这些概念的例子包括正常上皮细胞极性1的交替, 在那里细胞的顶端和基底层的空间方向丢失, 以及细胞外基质在调节生存和细胞命运方面的作用。特别是肿瘤学研究已经使用3D 模型来了解癌细胞的基本生物学和2D 和3D 细胞培养系统之间的差异3,4。更复杂的细胞培养技术的发展以及它们对多井格式的进一步适应使得在3D 环境中寻找新药。与2D 条件下生长的细胞相比, 3D 模型的肿瘤范围复杂, 从分层细胞系统5到单细胞型球体不同大小, 到复杂的多细胞型球体6,7,8. 因此, 发现完全诱导这些3D 模型系统中的细胞死亡的新型化合物或生物制品, 对药物发展运动有很大的兴趣。这些化验的终点通常与在2D 文化中进行的实验的端点相同, 以评估细胞增殖的变化, 但是当在生理上相关的环境中进行时, 它们可能揭示目标基因或通路的真正依赖性水平。审问。
如上所述, 已经开发了各种模型系统来研究3D 培养系统中的药物反应, 但是大多数使用96或384井微量滴定板, 并且不容易适应经常使用的高通量筛选 (高温超导) 格式。药物发现筛选运动。这类系统包括使用悬浮液滴技术、球形培养、带磁性微粒的脉动细胞来诱导漂浮, 以及含有天然或合成凝胶的培养物, 如胶原蛋白、胶或聚乙二醇 (PEG)2.在这里, 我们提出了一个以前开发的技术的详细方法, 以生产3D 球状文化从已建立的癌细胞系1536井板格式。在本协议中, 使用高度定义的介质来防止附着正常附着的细胞线9。这个系统有局限性 (也就是说, 它不能完全重述一个复杂的癌症模型系统), 但尽管如此, 这些化验能够对大量小分子集合进行高通量筛选, 并对药物反应进行横向比较。在2D 和3D 之间的文化对各种细胞系和化合物。
所选的细胞系用于演示本文中的方法, 所有与 MAPK 信号通路相关的基因的海港突变, 一种在癌症中高度失调的通路, 并且有许多疗法可用。许多线有激活致癌突变在柯尔多大鼠肉瘤病毒也称为 KRAS, 神经母细胞瘤 RAS 或 NRAS, 哈维大鼠肉瘤病毒癌基因或 HRAS, 和相关的激酶迅速加速纤维肉瘤, 也称为RAFs。最近的文献表明, 在3D 条件9,10的情况下, 这种通路的不同节点抑制剂在细胞系的子集中具有独特的功效。一项研究发现, 当有活性 RAS 的癌细胞在3D 培养时, 它们显示出对 MAPK 抑制剂的敏感性增强, 而且这种方法可以识别传统2D 中可能遗漏的通路和靶向抑制剂。设置。本研究的目的是介绍用于培养这些细胞系的方法, 并进一步证明对这些抑制剂的差异反应, 只有在使用3D 细胞培养系统时才能观察到。

<table><tbody><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>DMEM Dulbecco`s Modified Eagle Media (DMEM)/F12</td><td>ATCC</td><td>30-2006</td><td>Purchased as a prepared solution. This reciepe was found to be preferred compared to other vendors.</td></tr><tr><td>DMEM Dulbecco`s Modified Eagle Media (DMEM)/F12</td><td>Lonza</td><td>12-604F</td><td>Purchased as a prepared solution.</td></tr><tr><td>Roswell Park Memorial Institute (RPMI)</td><td>Lonza</td><td>12-115Q</td><td>Purchased as a prepared solution.</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Seradigm</td><td>1500-500</td><td>Purchased as a prepared solution.</td></tr><tr><td>1x 0.25% Trypsin with Ethylenediaminetetraacetic acid (EDTA)</td><td>Hyclone</td><td>SH30042.01</td><td>Purchased as a prepared solution.</td></tr><tr><td>1x Penicillin/Streptomycin</td><td>Gibco</td><td>15140</td><td>Purchased as a prepared solution.</td></tr><tr><td>10 ng/mL Human basic Fibrobast Growth Factor (bFGF)</td><td>Sigma</td><td>F0291</td><td>Resuspend in sterile water.</td></tr><tr><td>20 ng/mL Human Epidermal Growth Factor (EGF)</td><td>Sigma</td><td>E9644</td><td>Resuspend in sterile water.</td></tr><tr><td>0.4% Bovine Serum Albumin (BSA)</td><td>Sigma</td><td>A9418</td><td>Resuspend in sterile PBS and filter.</td></tr><tr><td>1x Insulin Transferrin Selenium (ITS)</td><td>Gibco</td><td>51500056</td><td></td></tr><tr><td>1% KnockOut (KO) Serum Replacement</td><td>Gibco</td><td>10828-028</td><td>Add fresh right before use.</td></tr><tr><td>100% Dimethyl sulfoxide (DMSO)</td><td>Sigma</td><td>276855-100ML</td><td></td></tr><tr><td>CellTiter-Glo</td><td>Promega</td><td>G7573</td><td>Purchased as a prepared solution.</td></tr><tr><td>&lt;strong&gt;Consumables&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Small Combi Cassette</td><td>ThermoFisher</td><td>24073295</td><td></td></tr><tr><td>Small Multiflow Cassette</td><td>BioTek</td><td>294085</td><td></td></tr><tr><td>1536-well sterile TC plates (white)</td><td>Corning</td><td>3727</td><td></td></tr><tr><td>1536-well sterile TC plates (black)</td><td>Corning</td><td>3893</td><td></td></tr><tr><td>Scivax Plates</td><td>MBL International</td><td>NCP-LH384-10</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Adhesives seals</td><td>ThermoFisher</td><td>AB0718</td><td></td></tr><tr><td>Spinning Incubator</td><td>LiCONiC</td><td></td><td></td></tr><tr><td>Stainless Steel Lids</td><td>The Genomics Institute of Novartis Research Foundation (GNF)</td><td></td><td></td></tr><tr><td>ATS Acoustic Dispensor</td><td>EDS Biosystems</td><td></td><td></td></tr><tr><td>Echo Acoustic Dispensor</td><td>Labcyte</td><td></td><td></td></tr><tr><td>Luminometer</td><td>Envision-Perkin Elmer</td><td></td><td></td></tr><tr><td>Peristaltic Pump- Multidrop Combi</td><td>ThermoFisher</td><td></td><td></td></tr><tr><td>Liquid Handler-Multiflow FX</td><td>BioTek</td><td></td><td></td></tr></tbody></table>

generation of high-throughput three-dimensional tumor spheroids for drug screening

癌细胞通常在塑料表面的两个维度 (2D) 培养。然而, 这种技术缺乏肿瘤肿块在体内暴露的真实环境。实体肿瘤生长不像附着在塑料上的一张纸, 而是作为一个三维 (3D) 空间中的克隆细胞的集合, 与邻居相互作用, 并具有不同的空间特性, 如正常细胞极性的破坏。这些相互作用导致 3 d-培养细胞获得形态学和细胞特征, 这是更相关的体内肿瘤。此外, 肿瘤肿块与其他细胞类型 (如基质和免疫细胞) 直接接触, 以及其他所有细胞类型的细胞外基质。沉积的基质由大分子如胶原蛋白和纤连蛋白组成。
为了增加从长凳到床边的肿瘤学研究结果的翻译, 许多团体开始研究3D 模型系统在药物开发策略中的应用。这些系统被认为在生理上是相关的, 因为它们试图重述肿瘤的复杂和异构环境。然而, 这些系统可能相当复杂, 尽管可以在96井格式中增长, 而有些现在甚至在 384, 但它们对于大规模的增长和筛选提供了很少的选择。这一观察到的差距导致了这里详细描述的方法的发展, 以培养肿瘤球体在高吞吐量能力在 1536年-井板。这些方法是对高度复杂的基于矩阵的系统的一种折衷, 它们很难筛选, 而且是常规的2D 检测。各种癌细胞系窝藏不同的基因突变被成功筛选, 检查复合功效通过使用一个精心策划的化合物库, 针对丝裂原活化蛋白激酶或 MAPK 通路。然后将球状培养反应与2D 生长的细胞反应进行比较, 并报告差异活动。这些方法为在高通量3D 设置中测试复合活动提供了唯一的协议。

在2D 的培养条件下, 各种已知生长良好的癌细胞系使用此处概述的方法进行了测试。图 1中有来自各种 MAPK 突变癌细胞系 (Calu-6:KRAS、NCI-H1299:NRAS、SK-MEL-30:NRAS 和 KNS-62:HRAS) 的代表性图像。这些图像表明, 虽然细胞系有不同的形貌, 每一个形成了3D 结构在1536井的化验板块。图 2表明, 当用于大规模筛选时, 可以观察2D 和3D 之间的差异?...

Watch this Scientific Journal Video about Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening at JoVE.com

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

在各种疾病征兆中, 越来越多的生理相关模型被开发并实施到药物发现计划中。这里描述的新的模型系统展示了三维肿瘤球体如何在高通量1536井板系统中进行培养和筛选, 以寻找新的肿瘤药物。

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 ...

generation-high-throughput-three-dimensional-tumor-spheroids-for-drug

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

8.5K 次观看.  Novartis Institutes for Biomedical Research. 在各种疾病征兆中, 越来越多的生理相关模型被开发并实施到药物发现计划中。这里描述的新的模型系统展示了三维肿瘤球体如何在高通量1536井板系统中进行培养和筛选, 以寻找新的肿瘤药物。

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

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

Tumor spheroids have been developed as a three-dimensional (3D) cell culture model in cancer research and anti-cancer drug discovery. However, currently, high-throughput imaging modalities utilizing bright field or fluorescence detection, are unable to resolve the overall 3D structure of the tumor spheroid due to limited light penetration, diffusion of fluorescent dyes and depth-resolvability. Recently, our lab demonstrated the use of optical coherence tomography (OCT), a label-free and non-destructive 3D imaging modality, to perform longitudinal characterization of multicellular tumor spheroids in a 96-well plate. OCT was capable of obtaining 3D morphological and physiological information of tumor spheroids growing up to about 600 &#181;m in height. In this article, we demonstrate a high-throughput OCT (HT-OCT) imaging system that scans the whole multi-well plate and obtains 3D OCT data of tumor spheroids automatically. We describe the details of the HT-OCT system and construction guidelines in the protocol. From the 3D OCT data, one can visualize the overall structure of the spheroid with 3D rendered and orthogonal slices, characterize the longitudinal growth curve of the tumor spheroid based on the morphological information of size and volume, and monitor the growth of the dead-cell regions in the tumor spheroid based on optical intrinsic attenuation contrast. We show that HT-OCT can be used as a high-throughput imaging modality for drug screening as well as characterizing biofabricated samples.

Optical coherence tomography (OCT), a three-dimensional imaging technology, was used to monitor and characterize the growth kinetics of multicellular tumor spheroids. Precise volumetric quantification of tumor spheroids using a voxel counting approach, and label-free dead tissue detection in the spheroids based on intrinsic optical attenuation contrast, were demonstrated.

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, ...

科研

癌症研究

Production of Large Numbers of Size-controlled Tumor Spheroids Using Microwell Plates

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically relevant than conventional adherent-sheet cultures, they more accurately recapitulate the complexity and interactions present in real tumors. In order to harness this model to better assess tumor biology, or the efficacy of novel therapeutic agents, it is necessary to be able to generate spheroids reproducibly, in a controlled manner and in significant numbers.
The AggreWell system consists of a high-density array of pyramid-shaped microwells, into which a suspension of single cells is centrifuged. The numbers of cells clustering at the bottom of each microwell, and the number and ratio of distinct cell types involved depend only on the properties of the suspension introduced by the experimenter. Thus, we are able to generate tumor spheroids of arbitrary size and composition without needing to modify the underlying platform technology. The hundreds of microwells per square centimeter of culture surface area in turn ensure that extremely high production levels may be attained via a straightforward, nonlabor-intensive process. We therefore expect that this protocol will be broadly useful to researchers in the tumor spheroid field.

We introduce a protocol for the generation of large numbers (thousands to hundreds of thousands) of uniform size- and composition-controlled tumor spheroids, using commercially available microwell plates.

生产尺寸控制肿瘤细胞球体采用微孔板大数

Tumor spheroids are increasingly recognized as an important in vitro model for the behavior of tumor cells in three dimensions. More physiologically ...

生物工程

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

In this protocol, we outline the procedure for generation of tumor spheroids within 384-well hanging droplets to allow for high-throughput screening of anti-cancer therapeutics in a physiologically representative microenvironment. We outline the formation of patient derived cancer stem cell spheroids, as well as, the manipulation of these spheroids for thorough analysis following drug treatment. Specifically, we describe collection of spheroid morphology, proliferation, viability, drug toxicity, cell phenotype and cell localization data. This protocol focuses heavily on analysis techniques that are easily implemented using the 384-well hanging drop platform, making it ideal for high throughput drug screening. While we emphasize the importance of this model in ovarian cancer studies and cancer stem cell research, the 384-well platform is amenable to research of other cancer types and disease models, extending the utility of the platform to many fields. By improving the speed of personalized drug screening and the quality of screening results through easily implemented physiologically representative 3D cultures, this platform is predicted to aid in the development of new therapeutics and patient-specific treatment strategies, and thus have wide-reaching clinical impact.

This protocol describes generation of patient-derived spheroids, and downstream analysis including quantification of proliferation, cytotoxicity testing, flow cytometry, immunofluorescence staining and confocal imaging, in order to assess drug candidates&#8217; potential as anti-neoplastic therapeutics. This protocol supports precision medicine with identification of specific drugs for each patient and stage of disease.

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 ...

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.

A protocol for robotic printing of cancer cell spheroids in a high throughput 96-well plate format using an aqueous two-phase system is presented.

癌症细胞球体机器人生产与药物测试的双水相系统

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing ...

A Three-Dimensional Spheroid Model to Investigate the Tumor-Stromal Interaction in Hepatocellular Carcinoma

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver cancer, rationalize its rank as the second most common cause of cancer-related death. Preclinical models need to be adapted to recapitulate the human conditions to select the best therapeutic candidates for clinical development and aid the delivery of personalized medicine. Three-dimensional (3D) cellular spheroid models show promise as an emerging in vitro alternative to two-dimensional (2D) monolayer cultures. Here, we describe a 3D tumor spheroid&#160;model which exploits the ability of individual cells to aggregate when maintained in hanging droplets, and is more representative of an in vivo environment than standard monolayers. Furthermore, 3D spheroids can be produced by combining homotypic or heterotypic cells, more reflective of the cellular heterogeneity in vivo, potentially enabling the study of environmental interactions that can influence progression and treatment responses. The current research optimized the cell density to form 3D homotypic and heterotypic tumor spheroids by immobilizing cell suspensions on the lids of standard 10 cm3 Petri dishes. Longitudinal analysis was performed to generate growth curves for homotypic versus heterotypic tumor/fibroblasts spheroids. Finally, the proliferative impact of fibroblasts (COS7 cells) and liver myofibroblasts (LX2) on homotypic tumor (Hep3B) spheroids was investigated. A seeding density of 3,000 cells (in 20 &#181;L media) successfully yielded Huh7/COS7 heterotypic spheroids, which displayed a steady increase in size up to culture day 8, followed by growth retardation. This finding was corroborated using Hep3B homotypic spheroids cultured in LX2 (human hepatic stellate cell line) conditioned medium (CM). LX2 CM triggered the proliferation of Hep3B spheroids compared to control tumor spheroids. In conclusion, this protocol has shown that 3D tumor spheroids can be used as a simple, economical, and prescreen in vitro tool to study tumor-stromal interactions more comprehensively.

Comprehensive in vitro models that faithfully recapitulate the relevant human disease are lacking. The current study presents three-dimensional (3D) tumor spheroid creation and culture, a reliable in vitro tool to study the tumor-stromal interaction in human hepatocellular carcinoma.

一种三维球体模型研究肝细胞癌肿瘤-基质相互作用

The aggressiveness and lack of well-tolerated and widely effective treatments for advanced hepatocellular carcinoma (HCC), the predominant form of liver ...

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

Despite remarkable advances in understanding tumor biology, the vast majority of oncology drug candidates entering clinical trials fail, often due to a lack of clinical efficacy. This high failure rate illuminates the inability of the current preclinical models to predict clinical efficacy, mainly due to their inadequacy in reflecting tumor heterogeneity and the tumor microenvironment. These limitations can be addressed with 3-dimensional (3D) culture models (spheroids) established from human tumor samples derived from individual patients. These 3D cultures represent real-world biology better than established cell lines that do not reflect tumor heterogeneity. Furthermore, 3D cultures are better than 2-dimensional (2D) culture models (monolayer structures) since they replicate elements of the tumor environment, such as hypoxia, necrosis, and cell adhesion, and preserve the natural cell shape and growth. In the present study, a method was developed for preparing primary cultures of cancer cells from individual patients that are 3D and grow in multicellular spheroids. The cells can be derived directly from patient tumors or patient-derived xenografts. The method is widely applicable to solid tumors (e.g., colon, breast, and lung) and is also cost-effective, as it can be performed in its entirety in a typical cancer research/cell biology lab without relying on specialized equipment. Herein, a protocol is presented for generating 3D tumor culture models (multicellular spheroids) from primary cancer cells and evaluating their sensitivity to drugs using two complementary approaches: a cell-viability assay (MTT) and microscopic examinations. These multicellular spheroids can be used to assess potential drug candidates, identify potential biomarkers or therapeutic targets, and investigate the mechanisms of response and resistance.

The present protocol describes generating 3D tumor culture models from primary cancer cells and evaluating their sensitivity to drugs using cell-viability assays and microscopic examinations.

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 of 3D Tumor Spheroids for Drug Evaluation Studies

In recent decades, in addition to monolayer-cultured cells, three-dimensional tumor spheroids have been developed as a potentially powerful tool for the evaluation of anticancer drugs. However, the conventional culture methods lack the ability to manipulate the tumor spheroids in a homogeneous manner at the three-dimensional level. To address this limitation, in this paper, we present a convenient and effective method of constructing average-sized tumor spheroids. Additionally, we describe a method of image-based analysis using artificial intelligence-based analysis software that can scan the whole plate and obtain data on three-dimensional spheroids. Several parameters were studied. By using a standard method of tumor spheroid construction and a high-throughput imaging and analysis system, the effectiveness and accuracy of drug tests performed on three-dimensional spheroids can be dramatically increased.

This article demonstrates a standardized method for constructing three-dimensional tumor spheroids. A strategy for spheroid observation and image-based deep-learning analysis using an automated imaging system is also described.

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 ...

Quantifying Antibody-Dependent Cellular Cytotoxicity in a Tumor Spheroid Model: Application for Drug Discovery

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of action of the antibodies is antibody-dependent cellular cytotoxicity (ADCC), where the antibody binds to the cancer cells and engages the cellular component of the immune system, e.g., natural killer (NK) cells, to kill the tumor cells. The effectiveness of these therapies could be improved by identifying adjuvant compounds that increase the sensitivity of the cancer cells or the potency of the immune cells. In addition, undiscovered drug interactions in cancer patients co-medicated for previous conditions or cancer-associated symptoms may determine the success of the antibody therapy; therefore, such unwanted drug interactions need to be eliminated. With these goals in mind, we created a cancer ADCC model and describe here a simple protocol to find ADCC-modulating drugs. Since 3D models such as cancer cell spheroids are superior to 2D cultures in predicting in vivo responses of tumors to anticancer therapies, spheroid co-cultures of EGFP-expressing HER2+ JIMT-1 breast cancer cells and the NK92.CD16 cell lines were set up and induced with Trastuzumab, a monoclonal antibody clinically approved against HER2-positive breast cancer. JIMT-1 spheroids were allowed to form in cell-repellent U-bottom 96-well plates. On day 3, NK cells and Trastuzumab were added. The spheroids were then stained with Annexin V-Alexa 647 to measure apoptotic cell death, which was quantitated in the peripheral zone of the spheroids with an automated microscope. The applicability of our assay to identify ADCC-modulating molecules is demonstrated by showing that Sunitinib, a receptor tyrosine kinase inhibitor approved by the FDA against metastatic cancer, almost completely abolishes ADCC. The generation of the spheroids and image acquisition and analysis pipelines are compatible with high-throughput screening for ADCC-modulating compounds in cancer cell spheroids.

Here, we present a method to identify compounds that modulate the ADCC mechanism, an important cancer cell-killing mechanism of antitumor antibodies. The cytotoxic effect of NK cells is measured in breast cancer cell spheroids in the presence of Trastuzumab. Image analysis identifies live and dead killer and target cells in spheroids.

量化肿瘤球状体模型中抗体依赖性细胞毒性:药物发现应用

Monoclonal antibody-based immunotherapy targeting tumor antigens is now a mainstay of cancer treatment. One of the clinically relevant mechanisms of ...

Three-Dimensional (3D) Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in particular (e.g., brain tumors such as glioblastoma multiforme and squamous cell carcinoma of the head and neck &#8211; SCCHN) it is a cause of severe morbidity and can be life-threatening even in the absence of distant metastases. In addition, cancers which have relapsed following treatment unfortunately often present with a more aggressive phenotype. Therefore, there is an opportunity to target the process of invasion to provide novel therapies that could be complementary to standard anti-proliferative agents. Until now, this strategy has been hampered by the lack of robust, reproducible assays suitable for a detailed analysis of invasion and for drug screening. Here we provide a simple micro-plate method (based on uniform, self-assembling 3D tumor spheroids) which has great potential for such studies. We exemplify the assay platform using a human glioblastoma cell line and also an SCCHN model where the development of resistance against targeted epidermal growth factor receptor (EGFR) inhibitors is associated with enhanced matrix-invasive potential. We also provide two alternative methods of semi-automated quantification: one using an imaging cytometer and a second which simply requires standard microscopy and image capture with digital image analysis.

Three-Dimensional 3D Tumor Spheroid Invasion Assay

Invasion of surrounding normal tissues is a defining characteristic of malignant tumors. We provide here a simple, semi-automated micro-plate assay of invasion into a natural 3D biomatrix that has been exemplified with a number of models of advanced human cancers.

三维（3D）肿瘤球体侵袭试验

Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

医学

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Microtumors使用3D人体Biogel培养体系和病人源性胶质母细胞瘤的Kinomic剖析和药物反应测试的生成

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...