The University of Alabama at Birmingham Center for Metabolic Bone Disease performed the histologic processing and sectioning of surrogates. Southern Research (Birmingham, AL) provided support for the manufacture of the bioreactor system. Funding was provided by the United States Department of Defense Breast Cancer Research Program (BC121367).

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</ol>

The authors declare that they have no competing financial interests.

这里，已经描述了三维培养物的方法，该方法结合了组织微环境的组件，包括细胞外基质（ECM）和人类间质成纤维细胞，在一个体积更接近的模型人乳腺癌，以允许一个概括性三维形态的发展。所描述的3D培养方法是更具有代表性的人类疾病比在多种细胞类型的传统的二维细胞培养物的掺入ECM的3D体积。人们已经注意到，这些参数（ 即，多种类型的细胞，细胞外基质，和3D体积）提供更拨上下文来研究生物过程，因为组织结构，正在考虑到1,8-从微环境，和维数的信号。三维培养的其它方法先前已被描述，包括，细胞上的3D矩阵29的顶部的培养，球状体30的产生，悬滴cutlures 31，次利用微流体装置32。虽然这些系统的具有独特的优势，整体他们不包括任一基质成分，基质细胞群，或代表维数。三维培养的方法，这里的描述包括每个参数的，并且可以使用的生物反应器系统，以允许对难以利用固体3D培养以实现更长的发育阶段来建立。此外，替代物的灌注提供固体代理人相比增长更大程度。 这种方法用于体外培养的另一个特征是在可以使用代用品"组织"要执行的分析类型的灵活性。各种端点可以间接培养过程中通过评估的条件培养基，或灌洗液，可溶性产品（被监控例如，作为LDH作为indicat细胞死亡，具体的分泌蛋白或代谢的指标或者或适当的表型功能）利用荧光或luminescently标记的细胞的非侵入性成像来评估生长和代理体系结构加时赛。以下的生长中，ECM可使用的ECM蛋白酶消化和细胞可被分离并用于进一步的分析或进一步的实验中，如流式细胞量化33。从ECM除去细胞的裂解物也可以基因组评估，在mRNA转录物的水平，或用于蛋白质表达。可替代地，端点可以直接从固定和处理的替代"组织"的实验已经结束后测定的。这些包括各种关于使用免疫组织学切片上进行H＆E染色的组织切片和其他分子分析（形态参数（ 例如，核/细胞形态和细胞聚集程度）的例如，作为小区的指示符Ki-67的表达增殖）或在原位杂交（ 例如 ，RNA的特异性基因产物的表达）。另外，也可以执行其他分子分析，例如实时，定量PCR或下一代测序，这些福尔马林固定，石蜡包埋的替代物;然而，由于通过固定和处理诱导的分子交联，速冻代用品优选​​用于这些类型的分析。这样的分子和形态学评估可以提供关于细胞生长，死亡，或响应于以下培养在整个生长的药物治疗以及有价值的信息。 使用CellProfiler程序中存在的代理人的组织学切片核细胞的密度的半自动化定量的主要优点是节省时间，它提供。虽然时间每替代所需的总量是可变的，在很大程度上依赖于替代​​物的大小和图像的获得的数，它是ESTI这交配到达从代理图像的细胞密度需要4-5个小时的动手时，每使用CellProfiler程序时手动计数与代理每半小时的时间替代。大多数使用动手时间CellProfiler分析归因于ImageJ的代孕面积的测量。该CellProfiler程序确实需要时间来自动处理的图像，但不要求动手从比导入图像其他研究者时间。 有很多的方法和这里所描述的3D替代培养物的限制。如在图12中所示，使用灌注生物反应器系统的提供增速比固体培养物的一个更大的程度，其中细胞的积累和增生的速度相当慢。虽然这里使用的灌注生物反应器系统提供生长优势，灌注系统也可以有各自的局限性。例如，PDMS是常在生物反应器的制造中使用，因为它易于使用，惰性属性和重复性。然而，众所周知这种材料吸收来自细胞培养基疏水分子，并可能需要长达亲脂性药物34-36。尽管其它材料可以用来代替PDMS如果这些限制是令人关注的，可用的其它材料也带有自己的缺点，即必须考虑的。因为CellProfiler分析是依赖于输入的直径和核的圆的参数，这些输入为每个代理/细胞类型优化可能是必要的。也请注意，这里提出的细胞密度测量不细胞生存力的直接测量的。以前已经指出，细胞生存力的直接测量是在三维培养系统37挑战性。然而，核退化是凋亡和坏死的一部分，两个占优势的细胞死亡的形式。在细胞凋亡，细胞核片段，一个所谓的核碎裂，继染色质凝聚，或固缩38,39的过程。这不同于坏死，其中引起细胞膜的垂死细胞膨胀破裂并释放其细胞质的内容。组织学上，坏死的特征在于核溶解（ 即，核的溶解）中，随后固缩和碎裂40。已经经历了这些变化的核上可以看到H＆E染色组织切片，但不会被识别为CellProfiler程序完整的细胞核。出现这些变化的核然而在细胞死亡过程相对较晚。因此，量化每区有核细胞的数量可以包括在细胞死亡的早期阶段的细胞，但仍然会提供关于在替代定影时细胞生存力的有用信息。可能被用于指示活力或生长的其它方法包括灌流液的分析（ 例如，在Alamar蓝色或乳酸脱氢酶测定法）○在代孕"组织"增殖中的R评价（ 例如，通过Ki-67标记采用免疫组化）。 在协议为替代设置关键步骤包括ECM聚合，其中温度，pH和时间都必须进行监测，以确保以沐浴在介质中的替代完全聚合之前。此外，电镀前保持在冰上的细胞中+ ECM混合物中的8孔腔室载玻片或注射到生物反应器系统是为了防止过早聚合的关键。也为了防止替代的设置，用于增加pH值至7，应加入到最后的混合物在碳酸氢钠中聚合。最后，必须考虑在37℃下孵育足够的时间，以确保完全的ECM聚合; 45分钟已被证明是足够的。在替代制备的另一个重要因素是气泡形成避免。在固体和灌注系统两者，最好是避免bubbl的形成在细胞中+ ECM混合物作为下载到不创建营养素的培养过程中的流动的块。同样重要的是在灌注系统，其中的气泡可能作为空气栓塞作用，阻断介质流动介质导入期间避免气泡。 适当固定是下游分析很重要;因此，关注的标准化和一致的固定（包括固定的类型和时间中的固定剂的量）和加工是必要的，以获得复制的结果。持续时间和固定型较少此处描述的H＆E染色的问题，但这些参数可能会影响其他类型代理人的分子分析。这是因为福尔马林固定，特别是，使分子的交联，包括蛋白质，DNA，和RNA在有些时间依赖性和特异性分子的方式41,42。这是在免疫组化分析，特别重要的意义时，抗体识别可欣通过交联dered。

三维能够更准确地模仿肿瘤体系结构和微环境在体内 （3D）培养模型对于旨在解剖细胞及其微环境之间复杂的相互作用，并以测试候选疗法的功效研究很重要。肿瘤维影响氧和养分梯度，药物暴露，间质性压力/血流量，以及3D结构1-4的均匀性。适当的基质微环境的存在有助于肿瘤维和影响细胞-ECM信令和基质细胞和恶性上皮细胞之间的旁分泌信号传导。肿瘤维和细胞功能的微环境的影响被很好地建立，以这两个因素改变药物反应1,3,5-8。此外，细胞的生长动力学，代谢率，以及细胞信号传导在三维二维（2D）培养和培养之间不同，这些因素AFFE电视机细胞反应1,3,8-10。 在体外 ，肿瘤替代微环境可以通过包括代表性的ECM成分和基质细胞群进行调制。恶性上皮细胞被ECM和癌症相关的基质细胞或以协同/保护方式，以促进肿瘤进展或以抑制的方式影响以抑制进一步肿瘤传播5,6,10。在任一情况下，基质可以影响通过旁分泌信号和/或通过在导致肿瘤增加间质的压力降低药物递送1,6-治疗反应和药物递送。因此，在加入ECM和基质细胞的成临床前模型有助于概括不能在二维培养以及建模的肿瘤的各个方面。 在此一方法建立乳腺癌代理人，纳入一个概括性的微环境，包括细胞外基质成分和stromal细胞，在3D体积中描述。在乳腺癌中，基质细胞群主要是由癌相关成纤维细胞（CAF）和基质细胞外基质的主要是由I型胶原的与基质组分的比例较小了在基底膜找到，包括层粘连蛋白和IV型胶原1,4,11-13。因此，乳腺癌微环境的这些组件（ 例如，CAF，I型胶原和基底膜）已经被纳入代理人。该方法可用于产生固体，未灌注3D代用品（ 图1A）或可适于通过经由生物反应器系统（ 图1B）的替代，以包括介质的灌注。这两种方法如下所述。这种方法也可修改为包括其它基质元素，如肿瘤相关巨噬细胞，或通过调节细胞和细胞外基质成分，根据与其他实体瘤模型。 <p这里描述类="jove_content"&gt;对于乳腺癌替代，我们已利用了MDA-MB-231（231），乳腺癌细胞系，CAF先前从人乳腺癌14分离，和一个ECM组成90％的胶原蛋白I的（ 6毫克/毫升）和10％的生长因子减少基底膜材料（BM）。代孕是在8孔玻片（固体代理）或者种植或生物反应器系统是利用提供持续的营养灌注（灌注替代）。任何灌注生物反应器系统，该系统可容纳含有ECM的细胞的体积可以使用15。作为一个例子，我们描述了我们的生物反应器系统的组织替代物的准备。这个系统是在内部开发并且不是可商购的。因为我们的重点是在这里对​​3D组织替代物的制备和分析，我们还没有进入对我们的生物反应器系统的制造和装配的具体细节丰富。然而，详细描述此系统及其发展已经出版16。在该生物反应器系统中，聚二甲基硅氧烷（PDMS）流动通道用于容纳所述替代，这是由一个PDMS泡沫支持的（使用类似于由Calcagnile 等 17中所述的方法形成的）。此体积是由4微通道（每个400微米的直径），其被连续地由介质经由microphysiologic泵灌注供应氧气和营养物的替代穿透。 的替代物的合适的分析是至关重要获得关于对治疗的反应或其他操作的细胞功能相关的信息。代理人可通过各种方法，包括完整的替代物的直接成像使用共聚焦显微镜或非侵入性成像的其他方法，通过测定条件培养基，或灌洗液间接细胞分析进行分析，对于分泌的产品，或固定和加工后的组织切片的分析至石蜡。可以在组织学切片进行评估一个这样的参数是细胞密度。我们提出了以测量细胞密度的一种方法（ 即，每截面积的有核细胞的数目），使用施加到用苏木精和曙红（H＆E）染色的替代组织切片的显微照片半自动化图像处理技术。细胞密度可以用作细胞数随时间的相对变化的一个指标，或从不同的生长条件和处理结果。 <img alt="图1" src="/files/ftp_upload/54004/54004fig1.jpg" /> 图1. 3D体积和生物反应器系统。 A）的过程示意图产生固体3D代理人。上图:含ECM（粉红色），上皮细胞（黄色）和CAF（橙色）固体体积的3D卡通;下图:含8孔腔滑代理人的俯视图B）原理的过程来产生3D灌注代理人。上图:CA具有通道3D体积的rtoon以允许介质灌注和含有ECM（粉），上皮癌细胞（黄色），和CAF（橙色）;中间:含PDMS的泡沫（黑色箭头）的PDMS流动通道的图像与细胞中+ ECM被注入并通过聚合物涂布的不锈钢丝（粉红色箭头）直径为400微米的侵入;下:含有替代，并连接到生物反应器系统，以允许连续介质灌注的PDMS流动通道的图像（蠕动泵和介质贮存器未示出）℃）培养后固体和灌注代理人的处理步骤的图像。左图:包含样品处理凝胶和替代的cryomold的形象;中东:含固定和加工替代石蜡块的图像;右:用替代的H＆E染色组织切片载玻片上的图像<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54004/54004fig1large.jpg" target="_blank">，请点击这里查看</a>更大的版本这个数字。

<table><tbody><tr><td>Dulbecco&#039;s Modified Eagel Medium 1x (DMEM)</td><td>Corning CellGro</td><td>10-014-CV</td><td></td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Atlanta Biologicals</td><td>S11150</td><td></td></tr><tr><td>0.25% Trypsin + 2.21&amp;nbsp;mM EDTA 1x</td><td>Corning</td><td>25-053-CI</td><td></td></tr><tr><td>Tissue Culture plates, 100&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229620</td><td>Sterile</td></tr><tr><td>Tissue Culture plates, 35&amp;nbsp;mm</td><td>CellTreat Scientific Products</td><td>229638</td><td>For PDMS foam formation&amp;nbsp;</td></tr><tr><td>9&quot; Glass pipette</td><td>Fisher&amp;nbsp;</td><td>13-678-20D</td><td>Sterile</td></tr><tr><td>10 ml pipette</td><td>CellTreat Scientific Products</td><td>229210B</td><td>Sterile</td></tr><tr><td>1,000 &amp;micro;l piptette tips</td><td>FisherBrand</td><td>02-717-166</td><td>Sterile Filtered</td></tr><tr><td>200 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-141</td><td>Sterile Filtered</td></tr><tr><td>10 &amp;micro;l pipette tips</td><td>FisherBrand</td><td>02-717-158</td><td>Sterile Filtered</td></tr><tr><td>15 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229410</td><td>Sterile</td></tr><tr><td>50 ml conical tubes</td><td>CellTreat Scientific Products</td><td>229422</td><td>Sterile</td></tr><tr><td>1.5 ml microcentrifuge tubes</td><td>FisherBrand</td><td>05-408-129</td><td>Sterile</td></tr><tr><td>Trypan blue</td><td>Corning Cellgro</td><td>25-900-CI</td><td>Sterile</td></tr><tr><td>Sylgard 184</td><td>Electron Microscopy Sciences</td><td>24236-10</td><td>PDMS elastomer and curing agent. Used for our in-house bioreactor.</td></tr><tr><td>PDMS Foam</td><td></td><td></td><td>Made in-house for use in our in-house bioreactor.</td></tr><tr><td>High Concentration Bovine Collagen Type I</td><td>Advanced Biomatrix</td><td>5133-A</td><td>FibriCol ~10 mg/ml</td></tr><tr><td>Growth Factor Reduced Matrigel (Basement Membrane)</td><td>Corning</td><td>354230</td><td>Basement membrane material</td></tr><tr><td>Sodium Bicarbonate</td><td>Sigma</td><td>S8761</td><td></td></tr><tr><td>Molecular Biology Grade Water</td><td>Fisher</td><td>BP2819-1</td><td></td></tr><tr><td>DMEM 10x&amp;nbsp;</td><td>Sigma-Aldrich</td><td>D2429</td><td></td></tr><tr><td>Nunc Lab-Tek Chamber Slide System&amp;nbsp;</td><td>Thermo Scientific</td><td>177402</td><td>8-well</td></tr><tr><td>Bioreactor</td><td></td><td></td><td>Made in-house.</td></tr><tr><td>Spring-Back 304 Stainless Steel&amp;mdash;Coated with PTFE polymer</td><td>McMaster-Carr</td><td>1749T19</td><td>Stainless steel wires to generate microchannels in our in-house&amp;nbsp; bioreactor system. 0.016&quot; Diameter</td></tr><tr><td>BioPharm Plus platinum-cured silicone pump tubing, L/S 14</td><td>Masterflex</td><td>EW-96440-14</td><td>For use in our in-house bioreactor system. Tubing ID: 1.6 mm, Hose barb size: 1/16 in.</td></tr><tr><td>2-Stop Tubing Sets, non-flared PVC, 1.52&amp;nbsp;mm ID</td><td>Cole-Parmer&amp;nbsp;</td><td>EW-74906-36</td><td>For use in our in-house bioreactor system (with microperistalitic pump).&amp;nbsp;</td></tr><tr><td>Six Channel precision micro peristaltic pump</td><td>Cole-Parmer</td><td>EW-74906-04</td><td>For use with our in-house bioreactor system</td></tr><tr><td>Tuberculin Syringes</td><td>BD Medical</td><td>309625</td><td>26 gauge 3/8 in. needle; Sterile</td></tr><tr><td>Dissecting Tissue Forceps</td><td>FisherBrand</td><td>13-812-36</td><td>5.5 inch</td></tr><tr><td>Mini Tube Rotator</td><td>Boekel Scientific</td><td>260750</td><td>Equipment option for surrogate rotation. Used with carousel for 50 ml tubes (model number 260753)</td></tr><tr><td>50 ml tube carousel</td><td>Boekel Scientific</td><td>260753</td><td>Used with mini tube rotator</td></tr><tr><td>Bambino&amp;nbsp; Hybridization Oven</td><td>Boekel Scientific</td><td>230301</td><td>Equipment option for surrogate rotation</td></tr><tr><td>HistoGel Specimen Processing Gel</td><td>Thermo Scientific</td><td>HG-4000-012</td><td>Specimen Processing Gel described in Step 5.2</td></tr><tr><td>Cryomold</td><td>Andwin Scientific</td><td>4566</td><td>15 mm x 15 mm x 5 mm</td></tr><tr><td>Tissue Marking Dye</td><td>Cancer Diagnostics, inc.&amp;nbsp;</td><td>03000P</td><td>Can be used to mark surrogates,&amp;nbsp; allowing multiple samples to be included in one tissue cassette&amp;nbsp;</td></tr><tr><td>Hinged tissue cassettes&amp;nbsp;</td><td>FisherBrand</td><td>22-272-416</td><td></td></tr><tr><td>Formalin</td><td>Fisher</td><td>23-245-685</td><td></td></tr><tr><td>GoldSeal Plain Glass Slides</td><td>Thermo Scientific</td><td>3048-002</td><td></td></tr><tr><td>Xylene</td><td>Fisher</td><td>X3P-1GAL</td><td></td></tr><tr><td>Ethanol, 200 proof (100%), USP</td><td>Decon Laboratories, Inc.</td><td>2805M</td><td></td></tr><tr><td>Hematoxylin</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7211</td><td></td></tr><tr><td>Clarifier</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7401</td><td></td></tr><tr><td>Bluing Solution</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7301</td><td></td></tr><tr><td>Eosin Y</td><td>Thermo Scientific Richard-Allan Scientific</td><td>7111</td><td></td></tr><tr><td>Cytoseal XYL mounting media</td><td>Thermo Scientific Richard-Allan Scientific</td><td>83124</td><td></td></tr><tr><td>Coverslips</td><td>Fisher Scientific</td><td>12-548-5G</td><td></td></tr></tbody></table>

preparation and analysis of in vitro three dimensional breast carcinoma surrogates

三维（3D）培养是一个更生理学相关方法细胞行为体外比二维培养建模。癌，包括乳房癌，是癌上皮细胞和间质组分，包括成纤维细胞和胞外基质（ECM）组成的复杂的三维的组织。然而，大多数体外乳腺癌模型由唯一的癌症上皮细胞，省略了基质和，因此， 在活体内肿瘤的三维结构。癌适当的3D建模是肿瘤生物学，行为和对治疗的反应准确的了解非常重要。然而，培养和3D模型的体积的持续时间是由培养中的氧和营养物质的可用性限制。在此，我们展示了一个在其中乳腺癌上皮细胞和基质成纤维细胞掺入的ECM来产生三维乳腺癌的替代，其包括基质和可作为中培养方法固体三维结构或通过使用灌注生物反应器系统以提供氧和营养物质。以下设置和初始生长期，代理人可用于临床前药物测试。可替代地，替代的细胞和基质成分可以被修饰以解决各种生物的问题。培养后，代理人被固定并加工以石蜡，以类似于临床乳腺癌标本的处理的方式，为的感兴趣参数的评价。哪里的ImageJ和CellProfiler图像分析软件系统被施加到代理人的组织切片的显微照片，进行了说明，这样的一个参数，本细胞的密度的评价来量化每区有核细胞的数量。这可以作为在一段时间或在细胞数的变化，从不同的生长条件和治疗导致细胞数的变化的指标。

两者都如上述和生长7天制备固体和灌注三维乳腺癌代理人。接着，替代物是固定的，加工以石蜡，切片，并用苏木精和曙红染色，如上所述。测定单位面积的有核细胞（包括231细胞和CAF）各代理的数量。如在图12中可以看出，H＆E染色的切片的代表性显微照片表明存在于比固体代理人（ 图12B）的灌注代理人（ 图12A）的细胞的浓度较高时，即使细胞的密度起初掺入的替代物在固体和灌注条件相同。细胞生长的这种可视表示被每面积（密度）细胞的较高的平均数目在灌注代理人支持（N = 3）相比，固体代理人（N = 3），如用t确定他CellProfiler分析（ 图12C）。以得到在图12C中的数据，每个代理的完整组织学横切片成像时，需要为每个代理多个图像，并且如上所述进行分析的图像。然后，核为每个代理的总数是由代理的总面积除以（表示为核/ 1×10 6个像素2的数量），从而为每个代理的细胞密度。来验证核细胞定量使用CellProfiler，采用CellProfiler程序的结果与通过简单地手动计数在每个6代理人（ 表3）的本单位面积的细胞数得到的那些进行比较。如上述计算出的各替代的细胞密度。无显著差异，在通过手动计数和CellProfiler分析或灌注代用品（p值= 0.855），或固体替代物获得的细胞密度实测值（<html> P = 0.553）。为了支持固态和灌注代理人之间的细胞密度的差异，在细胞生长的差异相关，每个代理的细胞增殖通过Ki-67标记（ 图12D）27的免疫组化分析评估。同样的趋势被发现，与在灌注的代理人增殖细胞的显著更高的百分比（n = 3时）相比，固体代理人（N = 3），这表明在灌注代理人要更高的生长速率，并与增加的细胞密度相关这些代理人。而细胞类型（ 即 ，乳腺癌上皮或CAF）没有在这些测量中进行评价，免疫染色细胞类型特异性标记物可以被用于更好地了解一个特定的细胞群体的生长或分布。协议详细介绍这个过程已经被先前公布的25,28。 <html> LES / ftp_upload / 54004 / 54004fig12.jpg"/&gt; 图12.代表性的成果。 A）从灌注替代品。B）从固体替代生长7天介质的H＆E染色部分代表图像的H＆E染色部分生长7天（400X原放大倍数）的形象代表，每2天更换（400X原始放大倍数）。13 C）单位面积，或细胞密度有核细胞的平均数的比较，随后的7天的生长后灌注（没有的媒体变化）和固体代理人的（介质每2天更换）。D）Ki-的比较67标记指数（ 即 ，细胞的一个群体中表达Ki-67的比例），细胞增殖的量度，以下灌流和固体代理人7天的生长。数据表示平均值±SEM，n = 3的每个条件，**表示P≤0.01和***表示P≤0.001（学生t检验）。tp_upload / 54004 / 54004fig12highres.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 实验 </td><td> CellProfiler:单位面积有核细胞 </td><td> 手册:单位面积有核细胞 </td><td> 平均CellProfiler </td><td> 平均手册 </td><td> 的P值（配对t检验） </td></tr><tr><td>灌注替代1 </td><td> 88.178 </td><td> 75.532 </td><td> 97.225 </td><td> 99.901 </td><td> 0.855 </td></tr><tr><td>灌注替代2 </td><td> 107.528 </td><td> 117.812 </td><td></td><td></td><td></td></tr><tr><td>灌注替代3 </td><td> 95.967 </td><td> 106.359 </td><td></td><td></td><td></td></tr><tr><td>小号奥利德替代1 </td><td> 19.797 </td><td> 17.480 </td><td> 16.491 </td><td> 12.991 </td><td> 0.553 </td></tr><tr><td>固体替代2 </td><td> 8.612 </td><td> 5.650 </td><td></td><td></td><td></td></tr><tr><td>固体替代3 </td><td> 21.065 </td><td> 15.844 </td><td></td><td></td><td></td></tr></tbody></table> 通过手动或CellProfiler分析3固体和3灌注代理人得到表3的细胞密度。

Watch this Scientific Journal Video about Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates at JoVE.com

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

制备及分析<em&gt;体外</em&gt;三维乳腺癌代理项

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, ...

preparation-analysis-vitro-three-dimensional-breast-carcinoma

Nanyang Technological University

Research

JoVE Journal

Bioengineering

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

10.6K 次观看.  University of Alabama at Birmingham. We demonstrate a method to generate 3D breast cancer surrogates, which can be cultured using a perfusion bioreactor system to deliver oxygen and nutrients. Following growth, surrogates are fixed and processed to paraffin for evaluation of parameters of interest. The evaluation of one such parameter, cell density, is explained.

视频: Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

研究使用正交乳腺癌模型的三阴性乳腺癌

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

科研

癌症研究

Establishment and Analysis of Three-Dimensional (3D) Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred to as patient-derived organoids (PDO), are an invaluable resource for studying the mechanism of tumorigenesis and metastasis of prostate cancer. Their main advantage is that they maintain the distinctive genomic and functional heterogeneity of the original tissue compared to conventional cell lines that do not. Furthermore, 3D cultures of PDO can be used to predict the effects of drug treatment on individual patients and are a step towards personalized medicine. Despite these advantages, few groups routinely use this method in part because of the extensive optimization of PDO culture conditions that may be required for different patient samples. We previously demonstrated that our prostate cancer bone metastasis PDX model, PCSD1, recapitulated the resistance of the donor patient&rsquo;s bone metastasis to anti-androgen therapy. We used PCSD1 3D organoids to characterize further the mechanisms of anti-androgen resistance. Following an overview of currently published studies of PDX and PDO models, we describe a step-by-step protocol for 3D culture of PDO using domed or floating basement membrane (e.g., Matrigel) spheres in optimized culture conditions. In vivo stitch imaging and cell processing for histology are also described. This protocol can be further optimized for other applications including western blot, co-culture, etc. and can be used to explore characteristics of 3D cultured PDO pertaining to drug resistance, tumorigenesis, metastasis and therapeutics.

Establishment and Analysis of Three-Dimensional 3D Organoids Derived from Patient Prostate Cancer Bone Metastasis Specimens and their Xenografts

Three-dimensional cultures of patient BMPC specimens and xenografts of bone metastatic prostate cancer maintain the functional heterogeneity of their original tumors resulting in cysts, spheroids and complex, tumor-like organoids. This manuscript provides an optimization strategy and protocol for 3D culture of heterogeneous patient derived samples and their analysis using IFC.

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

Three-dimensional (3D) culture of organoids from tumor specimens of human patients and patient-derived xenograft (PDX) models of prostate cancer, referred ...

Isolation and Functional Assessment of Human Breast Cancer Stem Cells from Cell and Tissue Samples

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major contributors to breast cancer initiation, relapse, metastasis and therapy resistance. It is imperative to understand the biology of breast cancer stem cells in order to identify novel therapeutic targets to treat breast cancer. Breast cancer stem cells are isolated and characterized based on expression of unique cell surface markers such as CD44, CD24 and enzymatic activity of aldehyde dehydrogenase (ALDH). These ALDHhighCD44+CD24- cells constitute the BCSC population and can be isolated by fluorescence-activated cell sorting (FACS) for downstream functional studies. Depending on the scientific question, different in vitro and in vivo methods can be used to assess the functional characteristics of BCSCs. Here, we provide a detailed experimental protocol for isolation of human BCSCs from both heterogenous populations of breast cancer cells as well as primary tumor tissue obtained from breast cancer patients. In addition, we highlight downstream in vitro and in vivo functional assays including colony forming assays, mammosphere assays, 3D culture models and tumor xenograft assays that can be used to assess BCSC function.

This experimental protocol describes the isolation of BCSCs from breast cancer cell and tissue samples as well as the in vitro and in vivo assays that can be used to assess BCSC phenotype and function.

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

Breast cancer stem cells (BCSCs) are cancer cells with inherited or acquired stem cell-like characteristics. Despite their low frequency, they are major ...

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a complex three-dimensional (3D) microenvironment involving layers of cancerous and non-cancerous cells surrounded by an extracellular matrix (ECM). The ECM provides physical and biological support and contributes to disease progression, patient prognosis, and therapeutic response.
This paper describes a protocol for assembling a 3D scaffold-based system to mimic the neuroblastoma microenvironment using neuroblastoma cell lines and collagen-based scaffolds. The scaffolds are supplemented with either nanohydroxyapatite (nHA) or glycosaminoglycans (GAGs), naturally found at high concentrations in the bone and bone marrow, the most common metastatic sites of neuroblastoma. The 3D porous structure of these scaffolds allows neuroblastoma cell attachment, proliferation and migration, and the formation of cell clusters. In this 3D matrix, the cell response to therapeutics is more reflective of the in vivo situation.
The scaffold-based culture system can maintain higher cell densities than conventional two-dimensional (2D) cell culture. Therefore, optimization protocols for initial seeding cell numbers are dependent on the desired experimental timeframes. The model is monitored by assessing cell growth via DNA quantification, cell viability via metabolic assays, and cell distribution within the scaffolds via histological staining.
This model&#39;s applications include the assessment of gene and protein expression profiles as well as cytotoxicity testing using conventional drugs and miRNAs. The 3D culture system allows for the precise manipulation of cell and ECM components, creating an environment more physiologically similar to native tumor tissue. Therefore, this 3D in vitro model will advance the understanding of the disease pathogenesis and improve the correlation between results obtained in vitro, in vivo in&#160;animal models, and human subjects.

Three-Dimensional In Vitro Biomimetic Model of Neuroblastoma Using Collagen-Based Scaffolds

This paper lists the steps required to seed neuroblastoma cell lines on previously described three-dimensional collagen-based scaffolds, maintain cell growth for a predetermined timeframe, and retrieve scaffolds for several cell growth and cell behavior analyses and downstream applications, adaptable to satisfy a range of experimental aims.

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

Neuroblastoma is the most common extracranial solid tumor in children, accounting for 15% of overall pediatric cancer deaths. The native tumor tissue is a ...

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its progression. Although the mechanisms involved in promoting breast cancer growth have been intensively studied using monocultures of breast cancer cells such as MCF-7 cells, the contribution of other cell types, such as vascular and lymphatic endothelial cells that are intimately involved in tumor growth, has not been investigated in depth. Cell-cell interaction plays a key role in tumor growth and progression. Neoangiogenesis, or the development of vessels, is essential for tumor growth, whereas the lymphatic system serves as a portal for cancer cell migration and subsequent metastasis. Recent studies provide evidence that vascular and lymphatic endothelial cells can significantly influence cancer cell growth. These observations imply a need for developing in vitro models that would more realistically reflect breast cancer growth processes in vivo. Moreover, restrictions in animal research require the development of ex vivo models to elucidate better the mechanisms involved.
This article describes the development of breast cancer spheroids composed of both breast cancer cells (estrogen receptor-positive MCF-7 cells) and vascular and/or lymphatic endothelial cells. The protocol describes a detailed step-by-step approach in creating dual-cell spheroids using two different approaches, hanging drop (gold standard and cheap) and 96-well U-bottom plates (expensive). In-depth instructions are provided for how to delicately pick up the formed spheroids to monitor growth by microscopic sizing and assessing viability using dead and live cell staining. Moreover, procedures to fix the spheroids for sectioning and staining with growth-specific antibodies to differentiate growth patterns in spheroids are delineated. Additionally, details for preparing spheroids with transfected cells and methods to extract RNA for molecular analysis are provided. In conclusion, this article provides in-depth instructions for preparing multi-cell spheroids for breast cancer research.

Mammary Epithelial and Endothelial Cell Spheroids as a Potential Functional In vitro Model for Breast Cancer Research

Crosstalk between mammary epithelial cells and endothelial cells importantly contributes to breast cancer progression, tumor growth, and metastasis. In this study, spheroids have been made from breast cancer cells together with vascular and/or lymphatic endothelial cells and demonstrate their applicability as an in vitro system for breast cancer research.

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

Breast cancer is the leading cause of mortality in women. The growth of breast cancer cells and their subsequent metastasis is a key factor for its ...

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 ...

医学

Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little is known about breast cancer cell properties and behaviors within the native microenvironment of human bone tissue.We have developed approaches to track, quantify and modulate human breast cancer cells within the microenvironment of cultured human bone tissue fragments isolated from discarded femoral heads following total hip replacement surgeries. Using breast cancer cells engineered for luciferase and enhanced green fluorescent protein (EGFP) expression, we are able to reproducibly quantitate migration and proliferation patterns using bioluminescence imaging (BLI), track cell interactions within the bone fragments using fluorescence microscopy, and evaluate breast cells after colonization with flow cytometry. The key advantages of this model include: 1) a native, architecturally intact tissue microenvironment that includes relevant human cell types, and 2) direct access to the microenvironment, which facilitates rapid quantitative and qualitative monitoring and perturbation of breast and bone cell properties, behaviors and interactions. A primary limitation, at present, is the finite viability of the tissue fragments, which confines the window of study to short-term culture. Applications of the model system include studying the basic biology of&#160;breast cancer and other bone-seeking malignancies within the metastatic niche, and developing therapeutic strategies to effectively target breast cancer cells in bone tissues.

Protocols are described for studying breast cancer cell migration, proliferation and colonization in a human bone tissue explant model system.

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

Bone is the most common site of breast cancer metastasis. Although it is widely accepted that the microenvironment influences cancer cell behavior, little ...

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 ...

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

Mammalian cell culture in monolayers is widely used to study various physiological and molecular processes. However, this approach to study growing cells often generates unwanted artifacts. Therefore, cell culture in a three-dimensional (3D) environment, often using extracellular matrix components, emerged as an interesting alternative due to its close similarity to the native in vivo tissue or organ. We developed a 3D cell culture system using two compartments, namely (i) a central compartment containing cancer cells embedded in a collagen gel acting as a pseudo-primary macrospherical tumor and (ii) a peripheral cell-free compartment made of a fibrin gel, i.e. an extracellular matrix component different from that used in the center, in which cancer cells can migrate (invasion front) and/or form microspherical tumors representing secondary or satellite tumors. The formation of satellite tumors in the peripheral compartment is remarkably correlated to the known aggressiveness or metastatic origin of the native tumor cells, which makes this 3D culture system unique. This cell culture approach might be considered to assess cancer cell invasiveness and motility, cell-extracellular matrix interactions and as a method to evaluate anti-cancer drug properties.

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 ...

Direct Bioprinting of 3D Multicellular Breast Spheroids onto Endothelial Networks

Bioprinting is emerging as a promising tool to fabricate 3D human cancer models that better recapitulate critical hallmarks of in vivo tissue architecture. In current layer-by-layer extrusion bioprinting, individual cells are extruded in a bioink together with complex spatial and temporal cues to promote hierarchical tissue self-assembly. However, this biofabrication technique relies on complex interactions among cells, bioinks and biochemical and biophysical cues. Thus, self-assembly may take days or even weeks, may require specific bioinks, and may not always occur when there is more than one cell type involved. We therefore developed a technique to directly bioprint pre-formed 3D breast epithelial spheroids in a variety of bioinks. Bioprinted pre-formed 3D breast epithelial spheroids sustained their viability and polarized architecture after printing. We additionally printed the 3D spheroids onto vascular endothelial cell networks to create a co-culture model. Thus, the novel bioprinting technique rapidly creates a more physiologically relevant 3D human breast model at lower cost and with higher flexibility than traditional bioprinting techniques. This versatile bioprinting technique can be extrapolated to create 3D models of other tissues in additional bioinks.

The goal of this protocol is to directly bioprint breast epithelial cells as multicellular spheroids onto pre-formed endothelial networks to rapidly create 3D breast-endothelial co-culture models which can be used for drug screening studies.

制备及分析

摘要

探索更多视频

研究使用正交乳腺癌模型的三阴性乳腺癌

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

使用胶原基支架的神经母细胞瘤三维体外仿生模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型

乳腺癌细胞侵袭定量使用三维（3D）模型

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

三维（3D）肿瘤球体侵袭试验

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

将3D多细胞乳房球体直接生物打印到内皮网络

制备及分析

摘要

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研究使用正交乳腺癌模型的三阴性乳腺癌

从患者前列腺癌骨转移标本及其异种移植物中提取的三维（3D）有机物的建立与分析

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

使用胶原基支架的神经母细胞瘤三维 体外 仿生模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

乳腺癌细胞侵袭定量使用三维（3D）模型

方法培养人股骨组织外植体，究其利基转移性乳腺癌细胞定居

三维（3D）肿瘤球体侵袭试验

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

将3D多细胞乳房球体直接生物打印到内皮网络

使用胶原基支架的神经母细胞瘤三维体外仿生模型

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能体外模型