Name: preparation and metabolic assay of 3-dimensional spheroid co-cultures of pancreatic cancer cells and fibroblasts
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Description: Watch this Scientific Journal Video about Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts at JoVE.com

我们要感谢 Samuel Zirbel 协助优化培养条件和球体生长监测。我们非常感谢安捷伦科技提供的海马 XFe96 分析仪、 检测试剂和技术的见解。我们还感谢博士赫曼特科克 Barts 癌症研究所在联合王国提供 PS1 细胞。这项工作支持部分由沧桑 Magowitz 基金会胰腺肿瘤研究和站达癌症癌症研究英国乐园基金会胰癌梦团队研究的资助 (授权编号: SU2C-美国癌症研究学会-DT-20-16)。站起来对癌症是一个娱乐产业基金会的计划。研究经费由美国协会管理用于癌症研究，SU2C 的科学伙伴。

1.胰腺癌文化 3D 球体使用磁性生物 <ol><li>使用标准的无菌组培技术、 培养细胞的兴趣到 70-80%在适当增长媒体融合 T75 烧瓶。 
	注意: 通常情况下，从 70-80%汇合 T75 瓶 5 7 × 10 6 细胞收获。在这项研究-Patu8902 采用两种不同的细胞类型 （胰腺肿瘤细胞） 和 PS1 细胞 (胰腺星状细胞激活的 21)。这两种细胞的培养基中罗斯威尔公园纪念研究所媒体 1640 (RPMI 1640) 加 10%(v/v) 培养液，1 × 青霉素-链霉素 (p/s)。</li>
	<li>洗细胞一次 5 毫升的杜尔贝科 &#39; s 磷酸盐缓冲 saline(DPBS).</li>
	<li>塑料分离细胞表面由 trypsinizing 与 2 毫升的 0.05%胰蛋白酶-EDTA 为 5-10 分钟 37 ° C.检查在显微镜下观察细胞脱离。</li>
	<li>停用胰蛋白酶的加法到 8 毫升在生长培养基中分离细胞。避免过度胰酶消化，因为这可能会影响细胞的健康存活率。</li>
	<li>吸管向上和向下，生成单个细胞悬浮液和计数细胞密度使用自动的细胞计数器或一个血球计数板。</li>
	<li>在此期间，平衡 NS 对环境温度有一小瓶。</li>
	<li>计算播种所需的数目的球体 （井） 和一种新的 15 毫升锥形管分装所需的细胞数量。例如，对整个 96 好板与 5,000 细胞/井，分装至少 5.5 × 10 5 细胞的种子。</li>
	<li>仔细在步行 3 分钟 500 x g 离心收集细胞吸出或倒上清液和液重悬细胞在 1.0 x 10 6 细胞/毫升生长介质中的浓度。移液器轻轻地使用宽无聊的吸管尖避免剪切。例如，重悬 5.5 × 10 5 细胞在生长培养基中 550 微升。</li>
	<li>磁化的细胞使用平衡的 NS （步骤 1.6） 所需的量。
	<ol><li>直接向细胞悬液在生长培养基中添加 NS 和轻轻地搅动。为高效磁标记细胞，每 100 微升细胞 （再悬浮在 1 × 10 6 细胞/毫升） 使用 10 微升 NS。 
		注: 为典型的实验中，标签 5.5 × 10 5 Patu8902 细胞在 550 微升 55 µ L NS 和 1.1 × 10 6 PS1 在 1100年微升 （分别） 与 110 微升 NS。</li>
		<li>轻轻仰拱管几次以确保细胞悬浮液。暂时，转入单井的 24 孔板细胞 NS 混合。做这分别为每个单元格类型被磁化。盖板和孵育细胞在室温下 2 h 允许 NS 绑定到细胞表面下轻轻摇动。 
		注: 文化及测定球形开始无论从 Patu8902 或 PS1 单独的细胞，除了共培养球体开始与两个单元格类型预混在印刷磁驱动器上的进行了成功使用这种方法在独立之前实验。从 Patu8902 和 PS2 细胞产生共培养微丸的方法在随后的步骤如下所述。</li>
	</ol></li> <li>向上和向下的磁化的单元格的吸管轻轻拌匀，准备主混合包含所需单元格数目。对于播种球体，使用 15,000 细胞 （5000 Patu8902 + 10,000 PS1） 共并调整到每口井的 96 孔板 150 µ L 总体积。 
	注意: 最后一卷配发到每个井必须相同，使用的单元格数目无关。</li>
	<li>细胞驱蚊 96 孔板顶 96 井磁球体驱动器和免除 150 微升细胞组合成每口井。观察细胞慢慢收集到的远远高于磁铁中心。覆盖和孵育板一夜之间在一个 5%的 CO 2 孵化器与磁球体驱动器仍然连接设置在 37 ° C。删除磁力驱动和孵育进一步增长达 7 天。 
	注: 至关重要的球体印刷使用磁球体驱动器使用单元格拒生长板。确保使用更薄更小的磁体的球体驱动器，不控股驱动器。</li>
	<li>监视增长球体每一天。补充的新鲜培养基每三天通过放置生长板安装在磁驱动器举行角度和使用多通道的吸管抽出乏媒体。 
	注: Patu8902 和 PS1 细胞共同在 15,000 细胞/井将会长成 3D 球体直径为 400-600 µ m 5-7 天内。在这一点上，球体准备代谢表征、 药物评价和其他下游应用。</li>
</ol> 2。3D 胰腺肿瘤球体胞外的通量仪的生物能量学途径的代谢测定 注: 在本部分中，代谢功能的球体使用胞外的通量分析描述分析仪测定单枚专为 3D 球体。
<ol><li>洗涤和转让的球体从生长板到球体测定微孔反应板。 
	注: 可能为代谢检测使用球体当他们到达 ~ 500 微米直径。
	<ol><li>进行化验分析时前, 一天水合物检测 calibrant （200 微升/井） 在提供的实用板探测器或传感器墨盒和孵育过夜 (4-18 h) 在湿的孵化器 (非 CO 2) 设置在 37 ° C.</li>
		<li>准备适当测试培养基所需的代谢测定通过补充 （见材料表） 基地媒体与任一 2 毫米 L-谷氨酰胺对糖酵解压力测试 (GST) 测定或 1 毫米丙酮酸、 L-谷氨酰胺 2 毫米和 10 毫米为葡萄糖线粒体的压力测试 (MST) 测定. 
		注: 胞外通量分析仪用于测量线粒体呼吸 (OCR) 和糖酵解 （易凯） 活细胞在 96 孔板格式。这些费率根据调查样本细胞代谢功能的概述。请参阅在检测试剂盒的详细说明手册。</li>
		<li>添加测定具体的补充，（请参阅步骤 2.1.1） 后，暖补充的检测介质在 37 ° C 水浴和调节 pH 至 7.4 ± 0.1 用 0.1 n 氢氧化钠。直到使用保暖介质。</li>
		<li>流程进行再造测定试剂盒试剂推荐股票浓度的校准的检测中。 
		注: 这些都是在 10 × 井所需的最终浓度。葡萄糖、 寡霉素和 2-脱氧葡萄糖 (2-DG) 股票浓度分别为 100 毫米，100 微米和 500 毫米，。</li>
		<li>生长板在灯光下观察球体显微镜检查球体形态和整体均匀性; 形状和结构e 球体。</li>
		<li>转移到磁保持一个驱动器上的生长板和小心地吸取 ~ 120 µ L 生长介质。轻轻地洗 ~ 120 µ L 的温暖和预校准检测媒体的椭球、 抽吸和重复洗两次。检查在显微镜下再次确保球体不冲走球体。</li>
		<li>备球体测定微孔板将 180 µ L 温暖测定介质添加到每个井。</li>
		<li>设置为 10 微升 P20 微管吸吮和适合宽无聊的秘诀。如果宽无聊的技巧不是可用的切断提示经常吸液管中，用干净的剪刀或扩大孔的手术刀。 
		注: 这应该减少剪切和转移检测板的同时保留整体形态的球体。</li>
		<li>使用温暖 (37 ° C) 表面进行转移的球体。使用与白光灯温暖 x 射线胶片查看器表面，对比度增强和援助的球体在传输过程中的可视化。</li>
		<li>仔细抽吸单预洗的球体从使用 P20 微管吸吮细胞驱蚊生长板装有全孔尖，轻轻地转移球直接进入每个井进行代谢的球体检测板的中心测定。允许每个球体，轻柔地落进入中央微室的每口井的纯净的重力来填充测定板。 
		注: 通常需要为每个球体落入井的中心由重力 5 8 s。不拔出吸管直到球体在微室已安定下来了。做 4 角井的测定板 （A1、 A12、 H1、 H12） 因为这些存款球体不会用作背景井。</li>
		<li>监测的形态及位置的每个球体以确保每个球体是在每口井的中心。如有必要，重新定位到由吸出球体使用全井中心孔吸管尖和随后沉积由重力。</li>
		<li>一旦所有球体已被都转移，一个小时之前，测定放置在 37 ° C 加湿的空气孵化器 (非 CO 2) 测定板。</li>
	</ol></li> <li>加载传感器墨盒进行化验与化合物 <ol><li>准备 10 × 集中检测特定试剂在测定特定媒体第 2.1.2 步所述。例如，开展商品及服务税，重组葡萄糖、 寡霉素，2-脱氧试验测定手册所述的推荐量。GST 和 MST 检测进行了使用 Patu8902 PS1 球体。</li>
		<li>妥善安置传感器墨盒与行标记 A H 在左手边和放置传感器滤盒顶部加载指南。通过定向加载指南，以便对应的端口要加载的这封信是在左上角，确保所需的端口正确加载。</li>
		<li>使用多通道移液器，免除试剂直接进入注射端口。举行在用手指尖在整个过程的位置加载指南。 
		注: 每个试剂将注入在试验测定手册所述建议的端口。避免产生气泡，但也不会拍任何部分的墨盒，排除气泡。</li>
		<li>删除加载指南和一级眼时用墨盒位置直观地考察注射端口用于即使加载。</li>
		<li>创建使用仪器控制器浪潮软件 （从今往后，分析软件） 2.3 节中所述的实验测定设计的手段协议。</li>
	</ol></li> <li>测定设计和执行使用分析软件 <ol><li>创建实验测定模板 <ol><li>打开分析软件并单击 &#34; 模板 &#34; 或选择从现有的测定模板。双击 &#34; 空白模板 &#34;.</li>
			<li>定义实验组和条件。
			<ol><li>定义的喷油策略端口 A、 B 和 C.离开港口 D 空。 
				注: GST 测定，只有这些端口将加载与葡萄糖、 寡霉素和 2 DG。在 MST 检测的情况下将加载寡霉素、 FCCP 和鱼藤酮。</li>
				<li>定义预治疗如果微丸治疗与一个或多个测试化合物和对照组。定义检测媒体，包括源、 补充剂和其他信息。</li>
				<li>定义一个或多个单元格类型 (例如 Patu8902，PS1) 看到的密度和通道的数字信息。</li>
			</ol></li> <li>单击 &#34; 生成组 &#34;.</li>
			<li>单击 &#34; 板地图 &#34; 选项卡和组分配给对应的实验设计测定板。</li>
			<li>作为背景井选择的四个角井。确保这些水井有没有球体。</li>
		</ol></li></ol></li> <li>运行检测分析仪 <ol><li>仪器协议选项卡上的单击保持默认协议命令通过检查 &#34; 校准 &#34;，&#34; 平衡 &#34; 和 &#34; 基线测量周期 &#34;.</li>
		单击 <li>&#34; 注射 &#34;，并定义复合为每个端口。编辑测量周期到 6 个周期从默认的 3 个周期为球形。保持默认 3 分钟混合、 0 分钟的等待和 3 分钟测量时间. 
		注: 默认混合等措施时间分别为 3 分、 0 分、 3 分。三基础代谢率测量通常都在第一次测定复方注射之前。这些测量可以校准和调整根据实验设计.</li>
		<li>审查议定书和组摘要。</li>
		<li>检测设计模板保存。</li>
		<li>进行预检测校准一旦注射端口已加载测定底物。将墨盒与公用事业板块转移到胞外的通量分析仪. 
		注意: 这通常在 15-20 分钟内完成，并校准传感器进行 OCR 和易凯测量。</li>
		<li>后的墨盒校准，实用板替换包含 3D 球体预热的测定板和启动检测。测量完成后，将数据导出到电子表格或图形和统计软件来分析数据。</li>
	</ol>
	</li>
</ol>

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作者乔治 · w · 罗杰斯和安德鲁 · 尼尔森是生产试剂和仪器在这篇文章中使用的安捷伦科技的员工/股东。所有其他作者宣称他们有没有经济利益的竞争。

使用胰腺癌, 3rd导致的癌症相关死亡的美国24, 作为一个例子和模型, 建立了快速和一致的方法生长和测定3D 椭球利用胰腺癌细胞培养和活化的胰腺成纤维细胞。利用磁性打印, 从直径400-600 µm 的尺寸中获得了椭球范围。这些被接受了新陈代谢的试验成功地测试了培养的3D 椭球的功能。这种方法简单, 一致, 可以很容易地适应其他细胞类型。
虽然这一方法将加速和增加的转化值的测试执行与3D 椭球, 它可以改进的技术, 允许复用的球体操作和处理。这将进一步减少进行化验所需的总时间、成本和劳力。此外, 还可以使用球体卷来规范 OCR 信号, 如其他人所报告的那样6。按单元格的平均体积归一化的球体体积可用于确定每个单元格的 ocr, 但由于椭球的增长不会完全相同, 因此计算每个单元格的绝对 ocr 可能会有挑战性。目前, 这种方法是有限的, 缺乏一个有效的工具, 多复用球体处理和转移从生长板到化验环境。
所描述的方法是修改和改编自磁性打印技术, 并进一步验证, 以显示功能相关性通过应用培养椭球的下游代谢试验。该方法提供了一个重要的工具, 以产生强大的, 可行的和功能椭球, 其中包含两个主要的细胞类型发现在大多数肿瘤组织, 癌细胞和肿瘤相关的成纤维细胞, 可用于其他研究化验,例如药物屏幕。与其他方法25相比, NS 方法的相对易用性和效率是一个主要的改进, 如挂起方法2、26的椭球生成。
强健的椭球产生的, 因此提供了一个体外肿瘤模型, 其中包括基质细胞, 这往往是从许多3D 文化研究缺少。应用3D 椭球模型的可能性是巨大的。例如, 这种模型可以用来研究细胞间的相互作用, 生物转移和测试的遗传易感性和依赖性的各种治疗方案。3D 椭球, 重述的在体内肿瘤微环境是非常相关和有用的工具的药物筛选研究和那些调查的作用机制的电流和新疗法。代谢分析探索使用椭球培养的突变细胞的能量通路可能有助于揭示这些基因和相关通路在病理的相关3D 癌症模型中的作用, 如本研究中所描述的椭球。
这种方法的成功应用首先依赖于磁性印刷所需的细胞的健康, 这就是为什么在对数相中生长的细胞应该被捕获和磁化。关键是使用磁性球体驱动器打印磁化细胞, 而不是持有驱动器, 这应该只在球体洗涤和媒体补充步骤的描述方法。另一个最关键的方面为成功的读出新陈代谢的分析是保持形态学的椭球在转移过程中从生长板到化验板。
总的来说, 这项协议已经建立了3D 椭球, 既包含癌症和基质细胞, 继随后的代谢检测椭球使用细胞外通量分析仪。该方法的主要特点是: 快速、易适应、前后一致、与体内肿瘤微环境相关并模拟, 比较便宜, 可作为研究肿瘤生物学和发展抗癌的新工具。代理.

在过去的十年中, 大量的体外3D 模型已经开发, 以重述和调查的体内肿瘤生物学, 微环境和生长条件的癌细胞1,2。二维 (2D) 单层细胞培养系统, 具有均匀暴露于生物化学因素和研究化合物, 不能复制本机3D 的肿瘤-基质相互作用暴露在一个梯度的化合物扩散通过细胞外基质蛋白 (ECM)3,4。因此, 与2D 组织培养模型相比, 3D 癌症模型已经显现出更好的模拟肿瘤微环境的潜能, 并成为更好地了解体内肿瘤特征的重要工具, 如缺氧、纤维组织, 休眠, 药物显, 毒性和治疗抵抗5,6。为此, 3D 模型有潜力通过模仿体内肿瘤的特点来弥合2D 细胞培养和整个动物模型之间的差距, 同时相对便宜和优化的快速生成和一致性。这些优势正在被利用, 以加快在许多领域的转化研究, 包括癌症生物学, 形态发生和组织工程7,8。
在激增的3D 组织培养方法, 磁悬浮技术最近开发和描述的增长, 检测和成像的椭球衍生的各种细胞类型9,10,11,12. 磁性3D 打印利用磁力来设计组织, 用具有生物相容性的纳米粒子磁化细胞, 并以井的格式印刷它们。这使得快速生产一致, 接近相同的3D 椭球, 这可以被利用和使用的大量下游应用生物化学和生物物理调查10。在这里, 我们已经适应了磁性打印技术使用一种生物相容性的材料称为 Nanoshuttle (NS) 组成的氧化铁, 聚 l-赖氨酸和金纳米粒子标记胰腺癌细胞和成纤维细胞。NS 附加静电到等离子膜, 不知道绑定到任何特定的受体, 并释放出细胞表面在一周内。它需要非常低的磁力 (30pN), 足以聚集但不伤害细胞, 不影响细胞活力, 新陈代谢或增殖, 使其极为生物相容的3D 培养10,13,14 ,15。
本研究以胰腺癌为例和模型, 描述了3D 癌细胞-成纤维细胞椭球的生成和代谢测定。从2D 血管培养的细胞开始, 我们用磁性打印说明了胰腺肿瘤-成纤维细胞共培养椭球的生长条件。培养的椭球, 然后使用细胞外通量分析仪的功能代谢分析, 技术表明, 同时测量两个主要的能量产生途径, 糖酵解和线粒体呼吸, 在各种生活细胞和组织16,17,18,19,20。糖酵解被测量作为细胞外酸化率 (ECAR) 的变化, 而线粒体呼吸或氧化磷酸化被测量为耗氧率 (OCR)。我们建议, 该方法开发的胰腺肿瘤椭球可作为一个骨干的优化和翻译3D 肿瘤球体的生成和分析其他细胞/组织类型。

<table><tbody><tr><td>0.05% Trypsin EDTA</td><td>Sigma</td><td>25300-054</td><td></td></tr><tr><td>96 well cell repellant plate</td><td>USA Scientific/ Griener Bio-One</td><td>655970</td><td>spheroid growth plate</td></tr><tr><td>Cellometry Cell counting slides</td><td>Nexcelom&amp;nbsp;</td><td>CHT4-SD100-002</td><td></td></tr><tr><td>D-Glucose</td><td>Sigma</td><td>D9434</td><td></td></tr><tr><td>DPBS</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Glycolysis Stress Test Kit</td><td>Agilent Technologies</td><td>103020-100</td><td></td></tr><tr><td>L-Glutamine</td><td>Sigma</td><td>59202C</td><td></td></tr><tr><td>&lt;span class=&quot;font5&quot;&gt;Mitochondrial&lt;/span&gt;&lt;span class=&quot;font5&quot;&gt; Stress Test Kit&lt;/span&gt;</td><td>Agilent Technologies</td><td>103015-100</td><td></td></tr><tr><td>n3D Magnetic Spheroid drive</td><td>Nano3D Bioscience</td><td>655841</td><td>Part of &lt;span class=&quot;font5&quot;&gt;Spheroid Bioprinting &lt;/span&gt;&lt;span class=&quot;font5&quot;&gt;Kit&lt;/span&gt;</td></tr><tr><td>n3D Magnetic Spheroid holding drive</td><td>Nano3D Bioscience</td><td>655841</td><td>Part of &lt;span class=&quot;font5&quot;&gt;Spheroid Bioprinting &lt;/span&gt;&lt;span class=&quot;font5&quot;&gt;Kit&lt;/span&gt;</td></tr><tr><td>n3D NanoShuttle-PL&amp;nbsp;</td><td>Nano3D Bioscience</td><td>655841</td><td>Part of Spheroid Bioprinting &lt;span class=&quot;font5&quot;&gt;Kit&lt;/span&gt;&lt;span class=&quot;font5&quot;&gt;:&amp;nbsp; store at 4 &amp;deg;C&lt;/span&gt;</td></tr><tr><td>Patu8902 cells</td><td>Laboratory Stock</td><td></td><td>pancreatic ductal adenocarcinoma cells</td></tr><tr><td>PS1 cells</td><td>Laboratory Stock</td><td></td><td>activated pancreatic stellate cells</td></tr><tr><td>RPMI1640</td><td>Gibco</td><td>11875&lt;span class=&quot;font5&quot;&gt;-093&lt;/span&gt;</td><td>growth media for cells, spheroids</td></tr><tr><td>SeaHorse XF&lt;span class=&quot;font12&quot;&gt;&lt;sup&gt;e&lt;/sup&gt;&lt;/span&gt;&lt;span class=&quot;font5&quot;&gt;96 extracellular flux analyzer&lt;/span&gt;</td><td>Agilent Technologies</td><td></td><td>extracellular flux analyzer</td></tr><tr><td>Sodium Pyruvate</td><td>Sigma</td><td>S8636</td><td></td></tr><tr><td>XF Base media</td><td>Agilent Technologies</td><td>102365-100</td><td>base media for metabolic assays on XF&lt;span class=&quot;font12&quot;&gt;&lt;sup&gt;e&lt;/sup&gt;&lt;/span&gt;&lt;span class=&quot;font5&quot;&gt;96&lt;/span&gt;</td></tr></tbody></table>

preparation and metabolic assay of 3-dimensional spheroid co-cultures of pancreatic cancer cells and fibroblasts

很多种癌症，包括胰腺癌，有致密的纤维基质中发挥重要的作用，在肿瘤的生长和侵袭。活化的癌相关成纤维细胞是肿瘤基质与肿瘤细胞相互作用和支持他们的成长和生存的关键组成部分。复述的癌症细胞相互作用并激活成纤维细胞的模型是为基质的生物学研究和开发的抗肿瘤药物的重要工具。 在这里，一种方法快速生成的鲁棒的三维 (3D) 球体共同文化的胰腺癌细胞和活化胰腺的成纤维细胞，可以用于随后的生物研究。此外，描述 3D 球体进行功能代谢的检测方法，探讨细胞生物能量通路使用胞外通量分析仪的使用与配对球体微孔板。胰腺癌细胞 (Patu8902) 和激活胰腺成纤维细胞 (PS1) 共同培养和磁化使用生物相容性纳米粒子组装。磁化的细胞迅速被罗使用 96 井格式，在生长介质生成球体直径为 5-7 天的文化内 400-600 微米之间的磁驱动器。使用 Patu8902 PS1 球体的功能代谢检测然后进行了利用胞外通技术探讨细胞能量的途径。此处是简单允许一致产生的癌症细胞成纤维细胞球体共同文化、 方法可以潜在地适应当前的描述方法优化后其他癌症细胞类型。

一般工作流程磁性生物和 3D 球体胞外通量分析 
图 1描述了本议定书所述的整个过程的概述。胰腺癌细胞 (Patu8902) 和星状细胞激活 (PS1) 条件下培养，组织培养瓶含有适当增长媒体到合流的 70-80%。细胞分离从 2D 培养器皿，并洗，细胞密度测定和细胞最后再悬浮生长培养基中于 1 × 106细胞/毫升。NS、 黄金、 铁的氧化物和聚-L-赖氨酸组成的纳米粒子组装用于磁化细胞。然后混合和印在 96 孔板细胞驱蚊生长板装入磁球体驱动器上单独磁化的 Patu8902 和 PS1 细胞。我们已经优化 15,000 要接种单井 （混有一万个 PS1 细胞生成单个球体的 5,000 Patu8902 细胞） 细胞总数。后 5-7 天的适当的组织培养条件下的孵化，3 维球体得到了，在这段时间这些球体生长监测和记录。洗完后用测定媒体，球体被身体转嫁球体微孔板来进行代谢实验使用胞外通量分析仪同时测量的糖酵解和线粒体的呼吸。
文化和生长的胰腺肿瘤成纤维细胞球体 若要获取功能和鲁棒性的球体上, 皮癌细胞线 Patu8902 和活化星状细胞 PS1 的培养基中辅以胎牛血清的 rpmi1640。Pat8902 和磁化与 NS 的 PS1 细胞被然后混合在一起的比为 1:2，以种子每井在生长板的 15,000 细胞总数。24h 内印刷磁驱动器上的细胞，细胞聚焦到井的中心正好在每个井下每个磁针。Patu8902 PS1 球体增长由 2、 4、 7 和 9 天后种子细胞成像进行了监测。图 2显示了上述各时间点的量化的代表性球体的平均直径。球体的直径增加从 414 µ m 日到 7 日 596 µ m 2 后印刷。那里是增长对天 7 和 9，无显著差异，因此所有进一步实验是限于 7 天的球体生长。我们注意到球体获得更清晰的形态，特别是周围的外边缘和 NS 逐步更加均匀地扩散到整个球体的体积与文化中更多天。我们也估计球形 10 微丸，基于其主要和次要的轴的长度。平均的球形是 0.92 ± 0.04 Patu8902 独自 （肿瘤）、 0.95 ± 0.04 为 PS1 （基质） 单独和 0.94 ± 0.02 的共同文化球体。
使用的胰腺 3D 球体的功能代谢测定胞外通量分析仪 若要测试微丸的功能，我们采用代谢和生物能学分析，以探讨在球体能量通道。我们开展了对微丸体外培养如上文所述的糖酵解压力测试。为此，从 Patu8902 或 PS1 除了所产生的两种细胞共培养的细胞生成的球体遭到含量测定。有趣的是，所有三种类型的球体，是否来自不同或混合细胞，展出 GST 检测生物功能响应。经注射葡萄糖饱和浓度，球体类型测试显示增加在糖酵解途径易凯 (图 3) 增加可见一斑。当线粒体 ATP 生产被抑制使用寡霉素时，能源生产转移到糖酵解和细胞推向最大的糖酵解能力，视为易凯进一步增加。使用 2-DG，竞相绑定葡萄糖激酶，易凯，确认易凯事先增加由于糖酵解活动减少导致的葡萄糖模拟的糖酵解的抑制作用。我们注意到在本研究中测试的球体回应以这种方式，虽然 PS1 独自球体 (平均直径 250 µ m) 有一个相对较低的信号，可能是由于其体积小、 糖酵解能力较低，相比 Patu8902 癌症(平均直径 600 µ m) 的单元格。一般情况下，更高的易凯信号从肿瘤细胞相比，这些成纤维细胞 PS1 相对较小的球体派生球体，可能可以归因于癌症细胞的固有代谢倾向于糖酵解22， 23。所有三种类型的球体，源于播种相同数目的单元格，虽然我们注意到每种类型，与 PS1 独自球体被最小的球体大小变异紧接着共同文化的扁球体，和 Patu8902 单独球体被最大。
要测试中 3D 球体的线粒体功能，我们将共同文化球体遭受 MST 测定。MST 措施通过直接测量细胞 （图 4A和4B） OCR 的线粒体功能的关键参数。化合物 （寡霉素、 FCCP 和各种鱼藤酮和霉素 A） 串行注射用于测量参数线粒体 ATP 生产，最大的呼吸和非线粒体呼吸等。这些参数和基底的呼吸率进一步用于计算质子漏和备用呼吸能力 (图 4)。寡霉素回应跌幅 OCR ATP 合酶抑制剂 (复杂 V) 关联到线粒体的呼吸与细胞 ATP 生产关联。球体孵育与寡霉素更长的时间，以允许最大渗透和高效的响应时间。第二次注射用 FCCP 解偶联剂刺激最大耗氧量和 OCR 相应增加。FCCP 刺激 OCR 用于计算备用呼吸能力，测定细胞对增加的能源需求作出反应的能力。第三次注射;鱼藤酮，混合 (一种复杂我抑制剂)，和霉素 A （一种复合体 III 抑制剂） 阻止线粒体呼吸，视为 OCR (图 4B大幅下降ong >)。
总体来看，我们的观察确认培养球体使用胞外通量分析仪作为衡量其代谢足迹/活动两方面的糖酵解和线粒体潜力，如上文所述的功能。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56081/56081fig1.jpg"> 
图 1.文化和胰腺癌细胞/成纤维细胞微丸的含量测定方法的示意图。Patu8902 和 PS1cells 是培养、 胰酶、 洗净和计数。播种每个球体，Patu8902 （5,000 细胞/井） 和 PS1 成纤维细胞 （10,000 细胞/井） 被磁化使用 NS 和印在单元格拒生长板在第 1 天。后播种，生长板被安装在一个磁球体的驱动器 （96 井格式生长板每个井下的一个磁铁） 和允许为 2-7 天时间来获得 3D 球体的文化。由此产生的球体被洗和转移到球体测定微孔反应板进行代谢检测细胞外流量仪表。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56081/56081fig1large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56081/56081fig2.jpg"> 
图 2.生长的胰腺肿瘤成纤维细胞球体。（Patu8902 PS1 共培养球体对应于文化中的一天 A) 代表图像显示在和中的球体 (µ m) 直径的大小量化下面。椭球大小和 NS 粒子 （黑点） 在其体积扩散会逐步增加至 7 天内 2 显而易见。球体在文化中存放额外 2 天之后，没有导致球体直径显著增加。（B） 代表图片的球形肿瘤细胞 (Patu8902)，基质细胞 (PS1) 和共培养的细胞 （Patu8902 + PS1） 是显示。（C） 球形数据被描绘从 10 个人的球体，从每个组。误差线代表标准偏差 (SD)。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56081/56081fig2large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56081/56081fig3.jpg"> 
图 3.糖酵解压力测试 (GST) 与胰腺球体。（A） 与各种典型的糖酵解作用测定示意图测定参数描述。（B） 以 GST 试验的实例对胰腺的球体培养使用描述的方法进行。葡萄糖注射液加大糖酵解视为在易凯，随着增加进一步增加另一方面寡霉素关闭线粒体呼吸。易凯瀑布 2-脱氧葡萄糖，竞争模拟响应通过阻断糖酵解。所有的球体被指出展示 GST 检测功能响应。（C) 输出的 GST 检测使用的制造商报告生成器描绘 GST 检测三个主要参数。误差线代表均值 (SEM) 的标准误差。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56081/56081fig3large.jpg" target="_blank">请点击这里查看此图的大版本。</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56081/56081fig4.jpg"> 
图 4。线粒体压力测试 (MST) 与 Patu8902 PS1 球体。（A） 与各种检测参数的典型的线粒体功能测定的示意图被描绘。（显示了 B） 对胰腺肿瘤成纤维细胞球体的 MST 的示例。（C） 共培养 3D 球体陈列 MST 检测功能响应。不出所料，球体与寡霉素，ATP 合酶抑制剂的挑战时注意到线粒体功能测量作为 OCR 的跌幅。使用 FCCP 电子流加大导致最大的 OCR，立即崩溃之时抑制线粒体呼吸使用-线粒体复合抑制剂的鸡尾酒。误差线代表均值 (SEM) 的标准误差。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56081/56081fig4large.jpg" style="font-size: 14px;" target="_blank">请点击这里查看此图的大版本。</a>

Watch this Scientific Journal Video about Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts at JoVE.com

Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

本文介绍了一种制备3维 (3D) 椭球共培养胰腺癌细胞和成纤维细胞的方法, 其次是利用细胞外流量分析仪测量代谢功能。

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

Many cancer types, including pancreatic cancer, have a dense fibrotic stroma that plays an important role in tumor progression and invasion. Activated ...

preparation-metabolic-assay-3-dimensional-spheroid-co-cultures

Nanyang Technological University

Research

JoVE Journal

Cancer Research

12.1K 次观看.  Translational Genomics Research Institute. 本文介绍了一种制备3维 (3D) 椭球共培养胰腺癌细胞和成纤维细胞的方法, 其次是利用细胞外流量分析仪测量代谢功能。

视频: Preparation and Metabolic Assay of 3-dimensional Spheroid Co-cultures of Pancreatic Cancer Cells and Fibroblasts

Assessing Cell Viability and Death in 3D Spheroid Cultures of Cancer Cells

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. The power of this preparation lies in its ability to mimic many aspects of the in vivo conditions of tumors while being fast, cheap, and versatile enough to allow relatively high-throughput screening. The spheroid culture conditions can recapitulate the physico-chemical gradients in a tumor, including the increasing extracellular acidity, increased lactate, and decreasing glucose and oxygen availability, from the spheroid periphery to its core. Also, the mechanical properties and cell-cell interactions of in vivo tumors are in part mimicked by this model. The specific properties and consequently the optimal growth conditions, of 3D spheroids, differ widely between different types of cancer cells. Furthermore, the assessment of cell viability and death in 3D spheroids requires methods that differ in part from those employed for 2D cultures. Here we describe several protocols for preparing 3D spheroids of cancer cells, and for using such cultures to assess cell viability and death in the context of evaluating the efficacy of anticancer drugs.

Here, we present several simple methods for evaluating viability and death in 3D cancer cell spheroids, which mimic the physico-chemical gradients of in vivo tumors much better than the 2D culture. The spheroid model, therefore, allows evaluation of the cancer drug efficacy with improved translation to in vivo conditions.

评估癌细胞3D球形培养中的细胞活力和死亡

Three-dimensional spheroids of cancer cells are important tools for both cancer drug screens and for gaining mechanistic insight into cancer cell biology. ...

科研

癌症研究

Monitoring Cancer Cell Invasion and T-Cell Cytotoxicity in 3D Culture

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited number of assays allow for direct monitoring and mechanistic insights into the interactions between tumor and immune cells, amongst which, T-cells play a significant role in executing the cytotoxic response of the adaptive immune system to cancer cells. Most assays are based on two-dimensional (2D) co-culture of cells due to the relative ease of use but with limited representation of the invasive growth phenotype, one of the hallmarks of cancer cells. Current three-dimensional (3D) co-culture systems either require special equipment or separate monitoring for invasion of co-cultured cancer cells and interacting T-cells.
Here we describe an approach to simultaneously monitor the invasive behavior in 3D of cancer cell spheroids and T-cell cytotoxicity in co-culture. Spheroid formation is driven by enhanced cell-cell interactions in scaffold-free agarose microwell casts with U-shaped bottoms. Both T-cell co-culture and cancer cell invasion into type I collagen matrix are performed within the microwells of the agarose casts without the need to transfer the cells, thus maintaining an intact 3D co-culture system throughout the assay. The collagen matrix can be separated from the agarose cast, allowing for immunofluorescence (IF) staining and for confocal imaging of cells. Also, cells can be isolated for further growth or subjected to analyses such as for gene expression or fluorescence activated cell sorting (FACS). Finally, the 3D co-culture can be analyzed by immunohistochemistry (IHC) after embedding and sectioning. Possible modifications of the assay include altered compositions of the extracellular matrix (ECM) as well as the inclusion of different stromal or immune cells with the cancer cells.

The presented approach simultaneously evaluates cancer cell invasion in 3D spheroid assays and T-cell cytotoxicity. Spheroids are generated in a scaffold-free agarose multi-microwell cast. Co-culture and embedding in type I collagen matrix are performed within the same device which allows to monitor cancer cell invasion and T-cell mediated cytotoxicity.

监测三D培养中的癌细胞入侵和T细胞毒性

Significant progress has been made in treating cancer with immunotherapy, although a large number of cancers remain resistant to treatment. A limited ...

Assessment of Mitochondrial Health in Cancer-Associated Fibroblasts Isolated from 3D Multicellular Lung Tumor Spheroids

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and progression. Complexity within the tumor microenvironment, including tumor secretome, low-grade inflammation, hypoxia, and redox imbalance, fosters heterotypic interaction and allows the transformation of inactive resident fibroblasts to become active CAFs. CAFs are metabolically distinguished from normal fibroblasts (NFs) as they are more glycolytically active, produce higher levels of reactive oxygen species (ROS), and overexpress lactate exporter MCT-4, leading to the opening of the mitochondrial permeability transition pore (MPTP). Here a method has been described to analyze the mitochondrial health of activated CAFs isolated from the multicellular 3D tumor spheroids comprising of human lung adenocarcinoma cells (A549), human monocytes (THP-1), and human lung fibroblast cells (MRC5). Tumor spheroids were disintegrated at different time intervals and through magnetic-activated cell sorting, CAFs were isolated. The mitochondrial membrane potential of CAFs was assessed using JC-1 dye, ROS production by 2',7'-dichlorodihydrofluorescein diacetate (DCFDA) staining, and enzyme activity in the isolated CAFs. Analyzing the mitochondrial health of isolated CAFs provides a better understanding of the reverse Warburg effect and can also be applied to study the consequences of CAF mitochondrial changes, such as metabolic fluxes and the corresponding regulatory mechanisms on lung cancer heterogeneity. Thus, the present study advocates an understanding of tumor-stroma interactions on mitochondrial health. It would provide a platform to check mitochondrial-specific drug candidates for their efficacies against CAFs as potential therapeutics in the tumor microenvironment, thereby preventing CAF involvement in lung cancer progression.

Multicellular 3D tumor spheroids were prepared with lung adenocarcinoma cells, fibroblasts, and monocytes, followed by the isolation of cancer-associated fibroblasts (CAFs) from these spheroids. Isolated CAFs were compared with normal fibroblasts to assess mitochondrial health by studying the mitochondrial transmembrane potential, reactive oxygen species, and enzymatic activities.

评估从3D多细胞肺肿瘤球体中分离的癌症相关成纤维细胞的线粒体健康

Cancer-associated fibroblasts (CAFs) are among the most abundant stromal cells present in the tumor microenvironment, facilitating tumor growth and ...

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 ...

Three-dimensional Co-culture Model for Tumor-stromal Interaction

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal cells. The tumor microenvironment is comprised of a variety of cell types, such as fibroblasts, immune cells, vascular endothelial cells, pericytes and bone-marrow-derived cells, embedded in the extracellular matrix (ECM). Cancer-associated fibroblasts (CAFs) have a pro-tumorigenic role through the secretion of soluble factors, angiogenesis and ECM remodeling. The experimental models for cancer cell survival, proliferation, migration, and invasion have mostly relied on two-dimensional monocellular and monolayer tissue cultures or Boyden chamber assays. However, these experiments do not precisely reflect the physiological or pathological conditions in a diseased organ. To gain a better understanding of tumor stromal or tumor matrix interactions, multicellular and three-dimensional cultures provide more powerful tools for investigating intercellular communication and ECM-dependent modulation of cancer cell behavior. As a platform for this type of study, we present an experimental model in which cancer cells are cultured on collagen gels embedded with primary cultures of CAFs.

Here we present a protocol to co-culture in three-dimensions, which is useful for investigating multicellular interactions and extracellular matrix-dependent modulation of cancer cell behavior. In this experimental model, cancer cells are cultured on collagen gels embedded with human cancer-associated fibroblasts.

对于肿瘤基质相互作用的三维共培养模式

Cancer progression (initiation, growth, invasion and metastasis) occurs through interactions between malignant cells and the surrounding tumor stromal ...

医学

<meta charset="utf8"></head><body>捕获结缔组织增生的 3D-PDAC 模型用于治疗评估

Growing Desmoplastic Three-Dimensional Pancreatic Cancer Spheroids from Co-Culture

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the dense desmoplastic extracellular matrix (ECM) that surrounds the tumor and reduces vascularization, generally termed desmoplasia. A variety of drug combinations and formulations have been tested to treat the cancer, and although many of them show success pre-clinically, they fail clinically. It, therefore, becomes important to have a clinically relevant model available that can predict the response of the tumor to therapy. This model has been previously validated against resected clinical tumors. Here a simple protocol to grow desmoplastic three-dimensional (3D)-coculture spheroids is described that can naturally generating a robust ECM and do not require any external matrix sources or scaffold to support their growth.
Briefly human pancreatic stellate cells (HPaSteC) and PANC-1 cells are used to prepare a suspension containing the cells in a 1:2 ratio, respectively. The cells are plated in a poly-HEMA coated, 96-well low attachment U-well plate. The plate is centrifuged to allow the cells to form an initial pellet. The plate is stored in the incubator at 37 &#176;C with 5% CO2, and media is replaced every 3 days. Plates can be imaged at designated intervals to measure spheroid volume. Following 14 days of culture, mature desmoplastic spheroids are formed (i.e. average volume of 0.048 + 0.012 mm3&#160;(451 &#181;m x 462.84 &#181;m)) and can be utilized for experimental therapy assessment. Mature ECM components include collagen-I, hyaluronic acid, fibronectin, and laminin.

<meta charset="utf8"></head><body>作者聚焦:使用结缔组织增生性球状体模型验证癌症治疗反应

Pancreatic cancer remains one of the toughest cancers to treat. Therefore, it is critical that pre-clinical models evaluating treatment efficacy are reproducible and clinically relevant. This protocol describes a simple co-culture procedure to generate reproducible, clinically relevant desmoplastic spheroids.

通过共培养的 Desmoplastic 三维胰腺癌球状体

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers with a 5-year survival rate of &#60;12%. The biggest barrier to therapy is the ...

Semi-Automated Phenotypic Analysis of Functional 3D Spheroid Cell Cultures

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell cultures. The clinostat mimics an environment where cells can assemble as highly reproducible spheroids with low shear forces and active nutrient diffusion. We demonstrate that both cancer and non-cancer hepatocytes (HepG2/C3A and THLE-3 cell lines) require 3 weeks of growth prior to achieving functionalities comparable to liver cells. This protocol highlights the convenience of utilizing incubators for 3D cells with cameras monitoring the cell growth, as snapshots can be taken to count and measure spheroids upon treatment. We describe the comparison of THLE-3 and HepG2/C3A cell lines, showing how non-cancerous cell lines can be grown as well as immortalized cancer cells. We demonstrate and illustrate how proteomics experiments can be conducted from a few spheroids, which can be collected without perturbing cell signaling, i.e., no trypsinization required. We show that proteomics analysis can be used to monitor the typical liver phenotype of respiratory chain metabolism and the production of proteins involved in metal detoxification and describe a semi-automated system to count and measure the spheroid's area. Altogether, the protocol presents a toolbox that comprises a phenotypic characterization via image capture and a proteomics pipeline to experiment on 3D cell culture models.

We present a protocol for growing high-reproducible spheroids and their phenotypic characterization using image capture and proteomics.

功能性 3D 球状体细胞培养物的半自动表型分析

We present a protocol that describes the properties and advantages of using a standalone clinostat incubator for growing, treating, and monitoring 3D cell ...

生物化学

A Cancer Cell Spheroid Assay to Assess Invasion in a 3D Setting

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential. Most traditional assays, however, do not examine these invasive features in a three-dimensional environment and the resulting data suffer from reduced biological applicability. Here an approach is presented to visualize the invasive ability of cell lines in a physiologically relevant setting. The cancer cell spheroid invasion assay first utilizes gravity to generate spheroids within drops of media that hang from the lid of a cell culture dish. Next, these spheroids are embedded in a 3D matrix consisting of a mixture of basement membrane materials and type I collagen. Cancer cell egression from the spheroids into the surrounding matrix is then monitored over time. The method described here can be modified to examine invasion after coculture of different cell types, inclusion of drugs/inhibitors, or alterations in extracellular matrix (ECM) constituents.

This method evaluates cancer cell invasion from spheroids into a surrounding 3D matrix. Spheroids are generated via the hanging drop culture method and then embedded in a matrix comprised of basement membrane materials and type I collagen. Invasion out of the spheroids is subsequently monitored.

癌细胞球体检测，以评估入侵的3D设置

The invasive nature of cancer cell lines is thought to correlate with their metastatic potential.  Most traditional assays, however, do not examine these ...

3维球形共培养胰腺癌细胞和成纤维细胞的制备及代谢试验

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评估癌细胞3D球形培养中的细胞活力和死亡

监测三D培养中的癌细胞入侵和T细胞毒性

评估从3D多细胞肺肿瘤球体中分离的癌症相关成纤维细胞的线粒体健康

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

对于肿瘤基质相互作用的三维共培养模式

通过共培养的 Desmoplastic 三维胰腺癌球状体

通过共培养的 Desmoplastic 三维胰腺癌球状体

通过共培养的 Desmoplastic 三维胰腺癌球状体

功能性 3D 球状体细胞培养物的半自动表型分析

癌细胞球体检测，以评估入侵的3D设置