Work partially funded by Prostate Cancer Canada (grant # D2014-4 to SG and CJD) and the Canadian Institutes of Health Research (grant # MOP-111069 to SG). We would like to thank Dr. Richard Poulin for editorial assistance and Mrs. Chanel Dupont for technical assistance.

注:没有道德的考虑，因为动物和人类癌细胞购买或慷慨地提供给我们。 1.使胶原蛋白插头（伪原发灶） <ol><li>制备胶原分散液。 I型从鼠尾腱（RTT）既可以提取胶原消毒如先前报道17，或购买。分散用混合机冻干RTT胶原（3.25-3.50毫克/毫升0.02N的乙酸）;对于均匀的混合（高速设置5 2分钟运行）。 </li><li>收获（胰蛋白酶-EDTA，通常情况下），并使用台盼蓝排除了使用血球计数活细胞。调整到所需的细胞密度（每插头5×10 4细胞）。 </li><li>制备在无菌条件下的所有解决方案（氢氧化钠，胎牛血清，DMEM 5倍， 碳酸氢钠 ）分别（ 表1），并保持在冰上冷却。注:加入各种解决方案的顺序是重要的，以防止渗透或酸性震荡细胞。 </li><li>进行细胞分散液（1.25×10 6个细胞）到最终的胶原溶液（5毫升）尽快。拌匀（通过上下吹打），同时避免气泡，然后在96孔板的每个孔中迅速分布200微升准备使用的溶液。轻轻敲击细胞培养罩的工作区表面上的多孔板，以去除气泡，并均匀地散布在孔内的溶液。 </li><li>填充所有孔之后（此步骤需要每96孔板约15-20分钟），将其存储到孵化器。 </li><li>从2小时到过夜孵育，在37℃下的板。胶原凝胶化（ 即 ，原纤维）在30分钟内发生。培养基（100μl/孔）添加到培养以进行过夜孵育。 </li></ol> 2.纤维蛋白胶的第一层<ol><li>纤维蛋白原溶液制备方法。 注:同一批次的纤维蛋白胶的理想情况下应该使用更重现resulTS，如血纤维蛋白凝胶形成可以商业冻干纤维蛋白原的不同批次之间有所不同。 <ol><li>始终使用新鲜配制的纤维蛋白原溶液。在打开的小瓶，以避免水合物晶体形成之前使冻干的纤维蛋白原至室温。 </li><li>逐渐溶解在预热（37℃）的Hank氏平衡盐溶液（HBSS）以钙 /镁离子以3毫克/毫升的工作浓度（考虑制备15％过量的最小最终体积的所需的纤维蛋白原: 例如 ，17.25毫克5.75毫升为5ml的溶液）。 <ol><li>添加预热的HBSS滴加起初以溶解纤维蛋白原片段。分解较大的片段与在烧杯刮刀。搅动烧杯不时以促进混合。在手术过程中不要使用搅拌器板。通过上下吹打悬浮液溶解剩余的粉末。 </li></ol></li><li>保持纤维蛋白原溶液不冷不热，而免缝使其通过一个0.22微米的过滤器LIZING溶液。注意:如果HBSS是不够暖或血纤维蛋白原不完全溶解，该溶液可能堵塞过滤器。如果适用的话，更换过滤器一次或两次，这可以降低纤维蛋白原浓度，从而纤维蛋白凝块的刚度。 </li><li>暂停的利益（ 例如 ，内皮细胞）的细胞分化为准备使用的纤维蛋白原溶液中，同时调整其最终体积，作为替代程序。 </li></ol></li><li>准备凝血酶解决方案。 <ol><li>准备在DDH 2 O（50 NIH单位/ ml）的储备液，然后用0.22微米的过滤消毒的。 </li><li>使用的纤维蛋白原/凝血酶比率≥1:0.0075（体积/体积），以生成血纤维蛋白凝胶。 </li></ol></li><li>生成纤维蛋白胶。 <ol><li>保持无菌纤维蛋白原和在所有的后续步骤凝血酶在冰上原液。血纤维蛋白凝胶允许形成在24孔板中。 </li><li>及时覆盖日每个电子表面很好地与纤维蛋白原溶液（200μl/孔），同时避免气泡形成。一次处理6个孔。 </li><li>一旦纤维蛋白原溶液完全覆盖孔的表面，倾斜板以45°角和通过丢弃凝血酶入井的中心添加1.5微升的凝血酶溶液到第一井，然后轻轻水平摇动板1-2秒。 <ol><li>离开板在层流罩（5-10分钟），直至凝胶化/凝固过程已经完成下一个稳定的位置（注:在聚合过程中必须不被干扰， 例如 ，通过输送板的孵化器）。 </li></ol></li><li>一旦第一个六口井已经聚合，重复相同的序列（ 即 3之前的步骤），在接下来的6口井，直到所有的水井都被处理。 </li></ol></li></ol> 3.纤维蛋白胶并夹塞胶原第二层<ol><li>选项答:（使用胶原蛋白立即插件）。 <ol><li>确保纤维蛋白胶的第一层已在所有孔中通过微妙地倾斜板聚合。放置装有由侧的胶原凝胶插头侧与24孔板（包含纤维蛋白凝胶）的96孔板来缓解胶原插头传送。 </li><li>添加HBSS一滴到含有胶原塞板的各孔中。 </li><li>除去从井各胶原插头与安装在一注射器（用作把手）细针或使用微勺（见视频）。每个胶原插头传送到使用一个或两个微匙第一纤维蛋白凝胶层，同时确保胶原塞孔中心到井和无菌状态保持良好。 </li><li>用的纤维蛋白原溶液的第二层覆盖在预先形成的纤维蛋白胶（300微升/孔），并在2.3中描述介绍凝血酶，保持一个最小的1:在一个时间0.0075比率和六个孔中的序列&lt;/ LI&gt; </ol></li><li>选项​​B（涂敷胶原插头与生长因子减少基底膜（GFRBM）的薄层）。 <ol><li>酷派所有准备的解决方案和手段事先并自GFRBM冷冻等分处理过程中让他们在4℃或冰上（ 例如 ，移液器，吸头，试管）的解冻过程中过度加热速度非常敏感（按照制造商的说明） 。 </li><li>从板孔中以下的去除，浸泡各胶原插头2分钟在1.5毫升离心管中含有100μl的纯GRFBM溶液的冰上。 </li><li>每个涂覆插头转印到第一纤维层，同时确保它是良好居中，如前面所述。在37°C孵育含插件板5分钟，以允许GRFBM以形成凝胶。添加第二个纤维层在步骤3.1.4。 </li></ol></li></ol> 4.细胞培养基条件<ol><li>用培养基填充每口井（400微升）。培养基和补充剂将基于所述细胞系和实验条件中选择。 </li><li>在100激肽释放酶抑制剂单位（KIU）/ ml的终浓度添加抑肽酶，一个抗纤维蛋白溶解剂，培养基中。 注:在用于测试的细胞系的条件下的细胞培养孵化器存放板。 </li><li>补充用新鲜培养基培养隔日或根据实验时间表，并添加抑肽酶。加入新鲜培养基之前，稍有倾斜板（以30-35°角）和倾斜吸管针对井的侧，同时小心抽吸下恒定观测条件培养基。 </li></ol>

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The authors have no disclosure.

作为一个重要的技术脚注，至关重要的是，没有间隙存在于中央和周边的凝胶之间的接口。否则，可能会降低细胞迁移/侵袭纤维蛋白凝胶的能力。如果凝血酶未被适当地稀释的胶原蛋白和纤维蛋白凝胶之间的空间可以培养的第24小时的过程中可形成。它也可能是测试可能导致的胶原蛋白凝胶的细胞系培养过程中收缩，从而导致相当大的空间都凝胶之间形成。当基质细胞中的胶原凝胶与癌细胞由于肌纤维母细胞样活性18沿混合，这是尤其期望，为了避免这种伪像，胶原插头应该在37℃孵育一段较长的时间（≥48小时）来诱导之前组装的血纤维蛋白的​​三明治凝胶收缩。 还与测定所遇到的一个问题是存在的可辨别纤维蛋白纤维或结块。这赋予浑浊，而不是半透明方面的纤维蛋白胶，这是由于癌细胞迁徙途径的扰动数据解释复杂化。这样的问题可能在在培养板中的聚合方法，从血纤维蛋白原溶液的摇晃不充分或过度产生。它也可能是由于加入了不当凝血酶成纤维蛋白原溶液（步骤2.3）。一个正确或阴天纤维蛋白胶不能固定一次凝固。胶原或纤维蛋白胶可以含有，在大多数情况下，将被自发后在孵化几天释放出气泡;尽管如此，适当的移液最大限度地减少其发生。建议在细胞培养周期的开始引入的抗纤维蛋白溶解剂。细胞培养物的经过长时间，可能会发生由于细胞活性和的C内的蛋白水解酶（ 即 ，纤维蛋白溶酶原活化剂）活化纤溶ollagen插头和卫星的肿瘤，这取决于癌症的细胞类型，并从降级的血纤维蛋白凝胶和细胞附着在板的底部的胶原插头的其攻击性19 .The释放是过度纤溶两种后果（ 见表故障诊断部2）。本技术的一个限制是该纤维蛋白胶，它可以使得难以通过整个凝胶观察细胞的厚度。然而，使用一个显微镜具有长工作距离聚光有助于避开这一限制，并监测沿整个Z轴的细胞。共焦或活体显微镜也可以提供替代可视化的细胞中的纤维蛋白凝胶。 之一的发展此三维细胞培养物模型中的主要目标是提供一种细胞外基质隔室通过该癌细胞能够自由迁移和发展。纤维蛋白被选择，因为它的肿瘤中存在，这归功于增加由炎症，坏死和改变的血管生成诱导的血管渗透性，并由于纤维蛋白也是已知的促进肿瘤细胞侵入20,21。在两个隔室之间的界面呈现很大的兴趣，因为它模仿生产新的支持性基质，如纤维蛋白，在肿瘤。此外，氧气和养分扩散，并在较长的运行，pH稳定性，都可能在胶原隔室失调相比周边血纤维蛋白隔室，其中扩散变得容易。由于大量癌细胞最初聚集在中央胶原塞，坏死/凋亡的迹象是在原发肿瘤内培养期间观察到的，模拟的原发肿瘤的患者22的膨胀过程中观察到的现象。此外，卫星的肿瘤的形成显着地相关的癌细胞的攻击性以及它们的转移行为。取决于细胞系和O型˚F实验，观察相关转移性过程可能长达30天所需的潜伏期。这通常比其他三维测定较长，但增加的延迟带来学习的模型更具有代表性的原位肿瘤的优势。 它众所周知，在3D培养模型中生长的细胞显示在细胞信号传导和基因表达的差异相比单层细胞培养物5,23。在三维生长的癌细胞也影响其对化疗药物的敏感性24-26。此三维细胞培养模型的一个重要的和有吸引力的特征在于修改的ECM浓度，因而凝胶的刚性，这可能是感兴趣的那些对细胞 - 细胞和细胞-ECM的生物力学特性工作的可能性的相互作用，以及作为基质性质对细胞侵袭的影响/迁移处理27。这里提出的三维细胞培养物测定法可以适应版，以适应不同的设置和/或实验条件。例如，该测定可以扩大以容纳需要大量的细胞的研究;通过加倍材料的量和细胞数，胶原塞可以在一个48孔板来制备，并在6孔板层叠在纤维蛋白凝胶。另一方面，可能难以小型化的高通量研究并且由于血纤维蛋白凝胶的不一致均匀性测量的培养体系。 在两种凝胶隔室之一引入非癌细胞的可能修改癌症细胞的行为。例如，当前列腺癌细胞系PC3与纤维母细胞和内皮细胞14混合卫星肿瘤的形成而加剧。备选地，荧光标记的细胞系可以在凝胶隔间被引入到调查细胞 - 细胞相互作用和/或培养期间跟踪细胞。此外，喜stological部分可以凝胶固定后来制备然而，免疫组化结果的质量可能抗体之间，因为可能会发生纤维蛋白交叉反应有所不同。如用苏木精和曙红（H＆E）组织学切片的常规染色可以揭示凝胶基质（ 图3D）内细胞组织。 这是比较容易从纤维蛋白胶除去胶原插头（ 例如 ，通过用玻璃吸管拔出插头）。因此，细胞和存在于纤维蛋白胶卫星肿瘤可以分别从那些在胶原插头发现生长。来自卫星的菌落细胞可通过与外科剪刀或手术刀提取菌落并且在不脱离纤维蛋白凝胶抑肽酶到游离细胞在培养基中他们成长收获。从复合凝胶的不同隔室收集单元可以允许各种进一步的研究，包括基因和蛋白质表达分析。它可以也可以用来分离与侵袭性表型的细胞群为进一步在体外或体内研究。例如，这种三维培养模型用于鉴定参与黑素瘤转移16个基因。所有这些代表性的应用清楚地表明通过在其细胞共培养可以进行的双隔室的3D细胞培养系统所允许的灵活性。 总之，我们的三维培养系统的设计非常灵活，可以提供新的途径，调查在肿瘤细胞凋亡，迁移和转移，以及抗癌药物评价和生物分子事件。

调查癌细胞侵袭/迁移和随后的转移机构的基本和生物医学特征是强烈的研究1,2的主题。转移是癌症的最终阶段，临床管理仍然遥遥无期。在细胞和分子水平上更好地了解转移将使更有效的疗法3的发展。 转移细胞的几个属性可以在体外 4包括它们的干性和潜在的获得的过渡状态（ 例如 ，上皮-间质转化）迁移和内部以及从原发肿瘤5侵入进行探讨。然而，侵袭/转移过程的体外评估一直是一个挑战，因为它几乎排除了血液/淋巴循环的贡献。在胶原凝胶中嵌入肿瘤碎片器官文化有页上一页狡猾被用于监测癌症的侵袭性。虽然肿瘤的复杂性被保留（ 例如 ，非癌细胞的存在），肿瘤片段暴露于有限介质扩散，以采样变化，并且对基质细胞6的过度生长。另一种方法包括在细胞外基质（ECM），它模仿了三维（3D）细胞环境的组分中生长的癌细胞。的乳腺癌细胞系中胶原凝胶和/或基底膜衍生基质的扩散是之中三维细胞培养物的最佳表征的例子。通过使用特定的三维细胞培养物的环境中，在标准条件下生长的乳腺癌细胞中观察到的紊乱组件可被反向以自发形成乳腺腺泡和管状结构7-10的。此外，来自腺癌细胞的多细胞肿瘤球状体的形成使用不同的技术（聚集例如，悬滴，浮球体，琼脂嵌入）现在构成了最常用的三维细胞培养物测定法11-13。然而，该测定是由限制集合的癌细胞系可以形成球状体，并通过提供研究细胞在这些条件下，短周期的限制。 在这种可视化技术，我们这里所介绍其中感兴趣癌细胞嵌入在胶原凝胶，以允许可替代地涂覆有基底膜衍生矩阵的伪原发肿瘤的体外形成复杂的三维细胞培养物测定法。一旦形成，所述伪原发性肿瘤，然后夹在无细胞基质（在本例中纤维蛋白胶），它允许癌细胞跨越两个矩阵隔间之间的界面（参见图1）。有趣的是，从伪原发性肿瘤始发侵袭性癌细胞沿继发性肿瘤样结构出现在纤维蛋白胶。这样的三维培养系统提供调查所需的灵活性，例如，抗癌药物，基因表达和细胞-细胞和/或细胞-ECM相互作用14-16。 <img alt="图1" src="/files/ftp_upload/54322/54322fig1.jpg" /> 图 1: 该方法的概述 ，生成三维细胞培养系统作为癌症研究的模型方法的原理总结<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54322/54322fig1large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a>

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Freeze-dried collagen</td>
			<td>Sigma-Aldrich</td>
			<td>C7661</td>
			<td>from rat tail tendon (soluble dispersion) or home-made (see Rajan et al., ref.#14)</td>
		</tr>
		<tr>
			<td>Fibrinogen (freeze-dried)</td>
			<td>Sigma-Aldrich&#160;</td>
			<td>F8630</td>
			<td>Type I-S, 65-85% protein with &#8805;75% of protein is clottable</td>
		</tr>
		<tr>
			<td>Thrombin</td>
			<td>EMD Chemicals Inc.</td>
			<td>605157</td>
			<td>&#160;Gibbstown, NJ; NIH units/mg dry weight&#160;</td>
		</tr>
		<tr>
			<td>Growth factor-reduced Matrigel&#160;</td>
			<td>Corning</td>
			<td>356234</td>
			<td>Previously from BD Biosciences</td>
		</tr>
		<tr>
			<td>Aprotinin</td>
			<td>Sigma-Aldrich</td>
			<td>A6279&#160;</td>
			<td>&#160;solution at 5-10TIU/ml (Trypsin Inhibitor Unit)&#160;</td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;Micro-spoons</td>
			<td>Fisher Scientific</td>
			<td>2140115</td>
			<td>Fisherbrand Handi-Hold Microspatula</td>
		</tr>
		<tr>
			<td>96 well plate, round base</td>
			<td>Sarstedt</td>
			<td>3925500</td>
			<td></td>
		</tr>
		<tr>
			<td>24 well plate</td>
			<td>Sarstedt</td>
			<td>3922</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s Medium</td>
			<td>Sigma Chemical, Co.</td>
			<td>D5546</td>
			<td>DMEM</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>VWR</td>
			<td>CAA15-701</td>
			<td>FBS, Canadian origin.</td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Sigma Chemical, Co.</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>Hank&#8217;s Balanced Salt Solution&#160;</td>
			<td>Sigma Chemical, Co.</td>
			<td>H8264</td>
			<td>HBSS</td>
		</tr>
	</tbody>
</table>

three-dimensional culture assay to explore cancer cell invasiveness and satellite tumor formation

在单层哺乳动物细胞培养被广泛用于研究各种生理和分子过程。然而，这种方法来研究生长的细胞往往会产生不希望的痕迹。因此，在一个三维（3D）环境的细胞培养，通常使用细胞外基质组分，成为一个有趣的选择，因为它接近相似的体内组织或器官中的原生。我们开发了使用两个室，即（i）一个三维细胞培养系统包含嵌入在胶原凝胶充当伪主macrospherical肿瘤和（ii）的周边无细胞隔室制成的纤维蛋白凝胶的癌细胞的中心隔室， 即细胞外基质组分从在中心使用的不同，其中，癌症细胞可以迁移（侵入前）和/或形成代表仲或卫星肿瘤微球肿瘤。卫星肿瘤的外围隔室的形成是显着相关与天然肿瘤细胞的已知攻击性或转移性原点，这使得这种三维培养系统是唯一的。这种细胞培养物的方法可以考虑以评估癌细胞侵袭和运动性，细胞 - 细胞外基质相互作用并作为评价抗癌药物性能的方法。

如前面提到的，这个三维细胞培养物测定法的一个有趣的特征是肿瘤细胞不仅可以从胶原插头向相邻的血纤维蛋白凝胶迁移，而且还建立继发性肿瘤（ 例如 ，卫星瘤样结构）。这可以在用凝胶厚度低和高放大倍数的倒置相差显微镜直接观察到，尤其是具有长工作距离聚光镜（ 图2）。采用这种三维细胞培养的方法，公知的转移性细胞的行为可以容易地相比，在非转移性细胞中发现，与不同的实验装置15反复证明。例如，许多卫星肿瘤发现随机分散在与转移性细胞系B16F10纤维蛋白凝胶，而只有很少的微小卫星肿瘤可以因此与它的非转移性对应，B16F0细胞系（ 图2来检测）。 <img alt="图2" src="/files/ftp_upload/54322/54322fig2.jpg" /> 图 2: 在3D细胞培养系统小鼠黑素瘤B16F10（A）和B16F0（B）的细胞的行为的B16F10和B16F0细胞系已知是转移性和弱转移性，分别。该复合凝胶的上部视图是在培养15天示于24孔板中。胶原插头（*）相邻，其中的细胞已迁移（箭头）以及形成卫星的肿瘤如在较高的放大倍数观察的血纤维蛋白凝胶层。比较方便，这些细胞系表达高水平的黑色素，可直接可视化（ 即不染色）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54322/54322fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 迁移前和卫星肿瘤的形状可以作为FUNC变化的细胞系的特性化。例如，鼠乳腺癌4T07细胞，这是不好的转移性，但局部非常侵入性的，可以形成一个迁移前径向延伸入纤维蛋白胶（ 图3A）。在培养14天后，这些细胞从迁徙前拉远，形成恒星的形状的结构非常类似乳腺结构（ 图 3B-D）。 <img alt="图3" src="/files/ftp_upload/54322/54322fig3.jpg" /> 图3: 小鼠乳腺癌4T07细胞 。这些细胞表现出在纤维蛋白胶（A）迁移能力强。虚线描绘的胶原塞和箭头表示的迁移前的方向。细胞均匀地侵入纤维蛋白基质（A），并精心组织，可以在三维观察恒星形管状结构（B的维C）。根据相衬显微镜的看法。（A - C）和伊红（D）染色的组织切片显示<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54322/54322fig3large.jpg" target="_blank">，请点击这里查看该图的放大版本。</a> 与此三维培养系统，特别是在卫星肿瘤，所观察到的细胞结构可通过平面投影如先前16所示染色用亚甲蓝溶液凝胶后进行量化。在细胞培养的各个阶段染色用Hoechst 33258凝胶样品使得能够可视化下萤光倒置显微镜（图4）的细胞的细胞核。 <img alt="图4" src="/files/ftp_upload/54322/54322fig4.jpg" /> 图4: 激素引起的乳腺癌细胞。乳腺癌细胞MCF-7细胞暴露于他莫昔芬的不同浓度。用相差显微镜观察在纤维蛋白凝胶（ 上排）卫星的肿瘤。从胶原凝胶（指示细胞在伪原发性肿瘤的存在下）（下排）的DNA，被用Hoechst 33258染色并使用荧光显微镜观察。正如预期的那样，他莫昔芬诱导的DNA片段化细胞凋亡的结果。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54322/54322fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 卷 </td></tr><tr><td> DMEM 5X（无碳酸氢钠）脱粉准备</td><td> 1毫升</td></tr><tr><td> FBS </td><td> 0.5毫升</td></tr><tr><td> 0.26μm的碳酸氢钠 </td><td> 0.5毫升</td></tr><tr><td> 1N的NaOH </td><td> 20微升</td></tr><tr><td>蒸水</td><td> 100微升</td></tr><tr><td colspan="2">保持冰这个解决方案</td></tr><tr><td>细胞悬液</td><td> 880微升</td></tr><tr><td colspan="2">拌匀冰，最后迅速补充: </td></tr><tr><td> RTT胶原（3.5毫克/毫升） </td><td> 2毫升</td></tr><tr><td>总成交量</td><td> 5毫升</td></tr></tbody></table> 表 1: 胶原蛋白插头所需的试剂和准备18-19插头胶原溶液中。 好立即混合加入胶原后（通过反转容器上下），同时避免气泡，然后迅速分发200μl到每个孔中。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 脚步 </td><td><strong&gt;问题</td><td> 可能的原因 </td><td> 解决方案 </td></tr><tr><td rowspan="4"> 步骤1。 胶原塞 </td><td rowspan="2">气泡</td><td rowspan="2">不适当的移液* </td><td>拌匀，同时避免过多的泡沫。 </td></tr><tr><td>绘制每200微升加样器活塞一路下来，仅释放200微升（反向移液）。* </td></tr><tr><td>胶原蛋白收缩插头</td><td>保温过夜当胶原插头与某些细胞系收缩，特别是。 </td><td>检查细胞开始实验前诱导胶原蛋白的收缩。它建议以监测收缩超过几天。否则，插头可在夹在凝胶收缩。 </td></tr><tr><td>凝胶化，不会发生</td><td>检查新鲜度，摩尔浓度，pH值等）的试剂;确保无试剂已经不见了踪影。 </td><td>从BEG再次启动一局。 </td></tr><tr><td rowspan="5"> 第2步。 纤维蛋白胶的第一层 </td><td>气泡</td><td>同样的意见。*;需要实践。 </td><td>相同的溶液。*气泡可保留在纤维蛋白凝胶，但它们通常在37℃下随着时间推移消失。 </td></tr><tr><td rowspan="2">凝胶化，不会发生</td><td rowspan="2">用纤维蛋白原的错误数量;检查凝血酶浓度或降解。 </td><td>从重新开始。 </td></tr><tr><td>假定凝血酶溶液不足;略微增加的浓度和/或凝血酶的量（ 例如 ，两次）。 </td></tr><tr><td>凝胶化后残留的液相</td><td>纤维蛋白原溶液和凝血酶不能很好混合。 </td><td>从重新开始。 </td></tr><tr><td>纤维蛋白是不均质的（ 即原纤维结构，团块） </td><td>凝血酶已充分扩散到凝胶;恩cessive或不充分搅拌</td><td>需要实践;从重新开始。 </td></tr><tr><td rowspan="4"> 步骤3。 三明治/ 第二层纤维蛋白 </td><td colspan="3">相同的注释中的步骤2。 </td></tr><tr><td>胶原蛋白插头是迅速（几个小时）包围在纤维空区域</td><td>纤维蛋白并没有与胶原凝胶接触​​充分聚合</td><td>从重新开始</td></tr><tr><td rowspan="2">胶原蛋白插头可能会逐步（天周）由纤维蛋白空白区域包围</td><td>当地裂解; </td><td rowspan="2">如果被限制到约塞了几个地方，这个实验可以继续。否则，考虑到从从头开始。可能是由于分泌纤溶酶原激活物过量。 </td></tr><tr><td>不要忘了抗纤维蛋白溶解剂！ </td></tr><tr><td rowspan="2"> 步骤4和5。 切尔升培养和后续</td><td>纤溶</td><td>问题的抗纤维蛋白溶解剂（降解，次优剂量等 ）。 </td><td>如果卫星的肿瘤或侵入的广泛量，增加抗纤溶剂的剂量。 </td></tr><tr><td>酸化</td><td>过度的细胞生长。 </td><td>要经常更换细胞培养基。 </td></tr></tbody></table> 表2:排除可能存在的问题，并提出了解决方案。

Watch this Scientific Journal Video about Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation at JoVE.com

Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells ...

three-dimensional-culture-assay-to-explore-cancer-cell-invasiveness

Universidad San Sebastian

Research

JoVE Journal

Medicine

10.3K 次观看.  CHU de Québec Research Centre. Cancer cells are embedded in a collagen gel and then sandwiched in an acellular fibrin gel to generate a 3D culture system in which the invasiveness and formation of satellite tumors may be monitored.

视频: Three-Dimensional Culture Assay to Explore Cancer Cell Invasiveness and Satellite Tumor Formation

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

人类神经内分泌肿瘤细胞株为研究人类神经内分泌肿瘤治疗的三维模型

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

科研

医学

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

来自患者的细胞培养和分离的CD133<sup&gt; +</sup&gt;假定的肿瘤干细胞的恶性黑色素瘤

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

The Use of Reverse Phase Protein Arrays (RPPA) to Explore Protein Expression Variation within Individual Renal Cell Cancers

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment resistance is thought to be the molecular complexity of the disease 1. Targeted therapy such as the tyrosine kinase inhibitor (TKI)-sunitinib have been utilized, but only 40% of patients will respond, with the overwhelming majority of these patients relapsing within 1 year 2. As such the question of intrinsic and acquired resistance in renal cell cancer patients is highly relevant 3.
In order to study resistance to TKIs, with the ultimate goal of developing effective, personalized treatments, sequential tissue after a specific period of targeted therapy is required, an approach which had proved successful in chronic myeloid leukaemia 4. However the application of such a strategy in renal cell carcinoma is complicated by the high level of both inter- and intratumoral heterogeneity, which is a feature of renal cell carcinoma5,6 as well as other solid tumors 7. Intertumoral heterogeneity due to transcriptomic and genetic differences is well established even in patients with similar presentation, stage and grade of tumor. In addition it is clear that there is great morphological (intratumoral) heterogeneity in RCC, which is likely to represent even greater molecular heterogeneity. Detailed mapping and categorization of RCC tumors by combined morphological analysis and Fuhrman grading allows the selection of representative areas for proteomic analysis.
Protein based analysis of RCC8 is attractive due to its widespread availability in pathology laboratories; however, its application can be problematic due to the limited availability of specific antibodies 9. Due to the dot blot nature of the Reverse Phase Protein Arrays (RPPA), antibody specificity must be pre-validated; as such strict quality control of antibodies used is of paramount importance. Despite this limitation the dot blot format does allow assay miniaturization, allowing for the printing of hundreds of samples onto a single nitrocellulose slide. Printed slides can then be analyzed in a similar fashion to Western analysis with the use of target specific primary antibodies and fluorescently labelled secondary antibodies, allowing for multiplexing. Differential protein expression across all the samples on a slide can then be analyzed simultaneously by comparing the relative level of fluorescence in a more cost-effective and high-throughput manner.

The Use of Reverse Phase Protein Arrays RPPA to Explore Protein Expression Variation within Individual Renal Cell Cancers

RPPA enables the protein expression of hundreds of samples, printed on nitrocellulose slides to be interrogated simultaneously, using fluorescently labelled antibodies. This technique has been applied to study the effect of drug treatment heterogeneity within clear cell renal carcinoma.

使用反时相蛋白的阵列（RPPA）探索在个人的肾细胞癌蛋白表达的变化

Currently there is no curative treatment for metastatic clear cell renal cell cancer, the commonest variant of the disease. A key factor in this treatment ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

A Mouse Tumor Model of Surgical Stress to Explore the Mechanisms of Postoperative Immunosuppression and Evaluate Novel Perioperative Immunotherapies

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to the development of metastatic disease in animal models and in cancer patients. Preclinical work from our group and others has demonstrated a profound suppression of innate immune function, specifically NK cells in the postoperative period and this plays a major role in the enhanced development of metastases following surgery. Relatively few animal studies and clinical trials have focused on characterizing and reversing the detrimental effects of cancer surgery. Using a rigorous animal model of spontaneously metastasizing tumors and surgical stress, the enhancement of cancer surgery on the development of lung metastases was demonstrated. In this model, 4T1 breast cancer cells are implanted in the mouse mammary fat pad. At day 14 post tumor implantation, a complete resection of the primary mammary tumor is performed in all animals. A subset of animals receives additional surgical stress in the form of an abdominal nephrectomy. At day 28, lung tumor nodules are quantified. When immunotherapy was given immediately preoperatively, a profound activation of immune cells which prevented the development of metastases following surgery was detected. While the 4T1 breast tumor surgery model allows for the simulation of the effects of abdominal surgical stress on tumor metastases, its applicability to other tumor types needs to be tested. The current challenge is to identify safe and promising immunotherapies in preclinical mouse models and to translate them into viable perioperative therapies to be given to cancer surgery patients to prevent the recurrence of metastatic disease.

A mouse tumor model of surgical stress is used to explore how postoperative immune suppression promotes metastatic disease and to evaluate immunostimulatory perioperative therapies.

手术应激探索术后免疫抑制的机制和评估新围手术期免疫疗法的小鼠肿瘤模型

Surgical resection is an essential treatment for most cancer patients, but surgery induces dysfunction in the immune system and this has been linked to ...

Three Dimensional Cultures: A Tool To Study Normal Acinar Architecture vs. Malignant Transformation Of Breast Cells

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The interplay of signals between cancer cells and their microenvironment exerts a powerful influence on breast cancer growth and biological behavior1. However, most of these signals from the extracellular matrix are lost or their relevance is understudied when cells are grown in two dimensional culture (2D) as a monolayer. In recent years, three dimensional (3D) culture on a reconstituted basement membrane has emerged as a method of choice to recapitulate the tissue architecture of benign and malignant breast cells. Cells grown in 3D retain the important cues from the extracellular matrix and provide a physiologically relevant ex&#160;vivo system2,3. Of note, there is growing evidence suggesting that cells behave differently when grown in 3D as compared to 2D4. 3D culture can be effectively used as a means to differentiate the malignant phenotype from the benign breast phenotype and for underpinning the cellular and molecular signaling involved3. One of the distinguishing characteristics of benign epithelial cells is that they are polarized so that the apical cytoplasm is towards the lumen and the basal cytoplasm rests on the basement membrane. This apico-basal polarity is lost in invasive breast carcinomas, which are characterized by cellular disorganization and formation of anastomosing and branching tubules that haphazardly infiltrates the surrounding stroma. These histopathological differences between benign gland and invasive carcinoma can be reproduced in 3D6,7. Using the appropriate read-outs like the quantitation of single round acinar structures, or differential expression of validated molecular markers for cell proliferation, polarity and apoptosis in combination with other molecular and cell biology techniques, 3D culture can provide an important tool to better understand the cellular changes during malignant transformation and for delineating the responsible signaling.

Three dimensional culture of mammary epithelial cells on a reconstituted basement membrane is a useful method to recapitulate the in vivo architecture of the benign breast, and to differentiate the malignant phenotype from the benign breast phenotype. Importantly, this system can be applied to study invasive carcinomas in other tissues.

三维文化:一种工具来研究正常腺泡结构与恶性转化的乳腺细胞

Invasive breast carcinomas are a group of malignant epithelial tumors characterized by the invasion of adjacent tissues and propensity to metastasize. The ...

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional (3D) Model

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the extracellular matrix (ECM) has been demonstrated to be a critical regulator of cell behavior in culture and homeostasis in vivo. The current approach of culturing cells on two-dimensional (2D), plastic surfaces results in the disturbance and loss of complex interactions between cells and their microenvironment. Through the use of three-dimensional (3D) culture assays, the conditions for cell-microenvironment interaction are established resembling the in vivo microenvironment. This article provides a detailed methodology to grow breast cancer cells in a 3D basement membrane protein matrix, exemplifying the potential of 3D culture in the assessment of cell invasion into the surrounding environment. In addition, we discuss how these 3D assays have the potential to examine the loss of signaling molecules that regulate epithelial morphology by immunostaining procedures. These studies aid to identify important mechanistic details into the processes regulating invasion, required for the spread of breast cancer.

Quantification of Breast Cancer Cell Invasiveness Using a Three-dimensional 3D Model

This article provides detailed methodologies for the use of three-dimensional (3D) assays to quantify breast cancer cell invasion. Specifically, we discuss the procedures required to set up such assays, quantification, and data analysis, as well as methods to examine the loss of membrane integrity that occurs when cells invade.

乳腺癌细胞侵袭定量使用三维（3D）模型

It is now well known that the cellular and tissue microenvironment are critical regulators influencing tumor initiation and progression. Moreover, the ...

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 ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

从人乳腺癌组织及细胞系微球体形成分析

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...

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 ...