这些研究提供资金，部分通过从替代研究与发展基金会（107588），美国国立卫生研究院（1U54CA136465-04S1），和加州乳腺癌研究发展计划（201B-0141）资助。我们感谢博士。安德鲁·威尔伯和R.斯科特·麦基弗的慷慨捐赠自己<pkt2 lubig="">转座子和PK / hUbiC-SB11转座质粒。我们非常感谢约翰Tamaresis博士对统计方法的建议，蒂莫西·布朗的表演术流，乔其纱亨里奇上迁移试验的意见，和南希Bellagamba为促进THR标本采集。 </pkt2>

股骨头从择期THR手术矫形外科系在斯坦福大学医学院的患者收集。所有的组织收集的去鉴定标本按照斯坦福大学的研究合规办公室法规。 1。选择乳腺癌细胞系（S） <ol><li>获得或转染所需的乳腺癌细胞系（S）与萤光素酶-GFP报道构建。下面实验使用的MDA-MB-231和MCF-7乳腺癌细胞系工程改造为萤火虫荧光素酶的稳定表达（fLuc）和增强型绿色荧光蛋白（EGFP）的睡美人转座子的质粒PKT2 / LuBiG进行转染和转座质粒PK / hUbiC-SB11 45,46。 注:将转染的细胞系被称为MDA-MB-231-fLuc-EGFP和MCF-7-fLuc-EGFP。 </li></ol> 2.隔离组织˚F从股骨头ragments <ol><li>以下THR手术，收集废弃股骨头在无菌容器中填充有生理盐水。样品转移到实验室，并在1-3小时的手术处理。注意:如果在处理的延迟，将样品在4℃下。 </li><li>无菌手套，手术咬骨钳，镊子，烧杯，用70％异丙醇冲洗仪器，以及组织培养容器的规定如下为每个类型的实验:通过将下列项目上的吸收垫在层流组织培养罩准备的工作区域。 </li><li>穿着无菌手术手套以保持圆形股骨头在一方面，使用咬骨钳在另一方面，提取从股骨的暴露上部轴所需的尺寸（〜2-5毫米）的小梁骨碎片。转移碎骨入培养容器（ 例如，6-，12-，或24孔组织培养板或迁移室），这取决于扩散的类型，山口onization或迁移实验中，如在步骤3，4或5所述。 注:在完成对每个具体的增殖，定植或迁移实验的最初步骤后上述骨组织碎片应该有所收获。碎片将尺寸范围， 如图2。片段可以之前或实验为标准化的目的之后被测量。 </li></ol> 3.联合培养乳腺癌细胞毗邻碎骨来衡量乳房细胞增殖使用BLI <ol><li>在实验前，制备含2×10 5个乳腺癌细胞/ 50微升的悬浮液，并准备骨蜡的插头用于固定骨头碎片中培养。切P200枪头到3件，并使用最大的一块的窄端的"俗套"的方式来切小（〜35毫克）的骨片蜡。使用最小片的宽端以弹出骨蜡塞到培养皿进行存储。 </li><li>将一块骨蜡在每口井的12点钟位置，轻轻按下，用无菌手套的食指。 </li><li>吸移管将50μl含1×10 5个乳腺癌细胞在DMEM-10％FBS +青霉素-链霉素的6孔板的每个孔的中心的细胞悬浮液。 （可替换地，种子细胞，如果需要的分散图案。）将板在37℃组织培养孵化器45分钟，以促进细胞附着。 </li><li>放置1骨块到1片骨蜡的每个实验井并应用温和的压力与咬骨钳将其固定。一式三份，从一个给定的THR检体，使得3碎骨放入三甲孔进行实验，而在底部的三个孔中含有骨蜡仅作为对照。 </li><li>使用5毫升吸管，轻缓地交付5毫升的DMEM-10％FBS的每个的侧面，以避免撞出乳房细胞或骨碎片。将板在3776;Ç组织培养孵化20-24小时。 </li><li>执行生物发光成像（BLI）从培养箱取出板并添加荧光素底物（以达到300微克/ ml的浓度）至各孔中。图像使用下列参数立即将板的IVIS成像系统平台上:光圈1，小分级，1秒的曝光时间，水平D.测量每个的信号强度以及平均辐射率（光子/秒/平方厘米/球面度）。 </li><li>使用图像处理软件来定义感​​兴趣区域（ROI）的地区，以定量的平均四射的所有实验和对照孔。出口平均辐射值到Excel表进行统计并生成图表。 </li></ol>直接种植于碎骨4.联合培养乳腺癌细胞研究殖民化和细胞数量<ol><li>使用镊子直接将骨碎片在24孔组织培养板的空孔中（1片段/孔）。 </li><li>果仁ETTE的DMEM-10％FBS的含有直接在各个骨片段的顶部1×10月3日至6日的乳 ​​腺癌细胞的50微升细胞悬浮液液滴。将板在37℃组织培养孵化器45分钟，以促进细胞附着。轻轻加1-2毫升的DMEM-10％FBS的成的各孔，例如在一侧的片段是完全浸没在介质。孵育所述板在37℃组织培养箱为20-24小时。 </li><li>温育后，用钳子向每个骨组织片段转移到新鲜的24孔板中含有1ml的DMEM-10％FBS的每个孔中。要执行BLI，按照上面的步骤3.6-3.7。此外，片段可使用荧光显微镜进行观察，或冲刷如在步骤6中所述，得到的骨髓人口为乳腺癌细胞数量和性质进行分析。 </li><li>通过荧光显微镜下BLI，吸管5微升荧光标记的双膦酸盐溶液（预做准备完整的片段进行定性评估根据制造商的说明缩减）到每个孔来标记碎骨的矿化骨针。孵育板在组织培养培养箱中另外24小时。 </li><li>以下培养中的荧光标记的双膦酸盐溶液中的24小时后，用钳子向片段转移到含有酚红的培养基，并查看使用配置以图像的GFP（485纳米）和二膦酸标签（680 nm）的荧光显微镜组织培养板中。 </li></ol> 5.测量乳腺癌细胞移植到骨组织碎片和养殖骨头碎片上清<ol><li>迁移朝向骨组织培养上清液。 注:以一式三份进行实验，从一个给定的样品THR使得3个片段被用来产生3上清。对于每个试验，迁移横跨含有骨上清液与仅含对照培养基三口井三口井相比。 <ol><li>生成骨炎通过将骨碎片成12孔板每孔含有2.2毫升的DMEM-10％FBS中的孔中的上清液告。文化在37℃组织培养孵化24小时。退出介质在24小时，更换2.2毫升新鲜的DMEM-10％FBS。治疗对照孔含有DMEM-10％FBS的方式相同。 注:该介质是在24小时变更为除去分泌响应于组织创伤手术产生的因素。因为在生成骨组织上清FBS含含含FBS中只包括以控制FBS的乳腺癌细胞迁移的贡献中，水井。 </li><li>在48小时后，收集0.9毫升每种上清液/控制介质和吸管的一部接收机板的孔中。刀片将在后面被放入这些孔中。孵育接收板在组织培养孵化器同时播种的刀片。注意:剩余的上清液（〜蒸发后的1毫升）中，可收集的分泌组实际分析RS如果需要44。 </li><li>种子的Transwell小室，将它们放入一个标准的，空的24孔培养板。吸移管350微升细胞悬浮液含1×10 5个乳腺癌细胞在DMEM-10％FBS的到各刀片的上部，内膜表面， 如图1A所示 。放置含种子插入到37℃组织培养箱中在24孔板45分钟，以促进附着。 </li><li>一旦细胞已附连到插入件中，使用镊子根据制造商的说明将插入传送到接收器板。角度放置到接收器以及防止插入膜和接收机以及上清液之间形成气泡时每次插入。孵育接收机板与刀片20小时在37℃组织培养孵化器。 注意:检查接收板的底侧为插入膜和接收机以及supernatan之间的气泡吨。如果气泡是可见的，删除特定的插件，并再次将它们插入接收机很好，直到无气泡存在。 </li><li>经过20小时的使用镊子从接收机板取出刀片。在步骤使用程序3.6-3.7执行BLI图像已经迁移入并连接到接收器板孔底部的细胞。 </li></ol></li><li>移民对骨组织碎片。 注:以一式三份进行实验，从一个给定的样品THR使得3个片段被放入3个独立的刀片，和图3的玻璃珠放入3个独立的刀片作为对照。 <ol><li>吹打0.9毫升的DMEM-10％FBS的入每个孔制备接收机板。将其放入组织培养的孵化器而准备的刀片。 </li><li>种子的插入，颠倒它们的塑料离心管中，有一个盖的表面上。吸取的DMEM-10％FBS的含1×10 5个乳腺癌细胞D的50微升细胞悬浮液的液滴irectly到每个插入膜外，底表面（其朝上的反相插入）。覆盖离心管箱与盖裂了开，并将其传输到37℃组织培养箱中45分钟，以促进细胞附着。 </li><li>转移离心管方框，组织培养罩，并使用镊子打开种子插入右侧向上，并将它们传送到接收器板的孔中。吸取0.450毫升DMEM-10％FBS为每个插入杯中。使用镊子，传输一个骨组织片段（〜3毫米），为每个准备插入杯中。或者，用玻璃珠作为阴性对照额外的井。将板在组织培养培养箱中20小时。 </li><li>为了测量迁移，制备含有每孔1ml的DMEM-10％FBS的标准的24孔组织培养板。从孵化器中取出接收器板，然后使用镊子，从每个插入转移骨头碎片和珠子到标准板的不同孔。添加荧光素衬底（达到300微克/ ml的终浓度）至每个孔，并利用在步骤3.6-3.7中的过程执行BLI。 </li></ol></li></ol> 6.附加前和后的实验分析<ol><li>分析冲洗骨髓。 <ol><li>传送一个骨片段插入一个70微米的细胞滤网放置在50ml锥形管的顶部。使用10毫升注射器与地下25针大力冲洗片段用10ml的PBS中，以获得在锥形管骨髓细胞的悬浮液。离心细胞悬浮液3分钟，在300×g离心并21℃下，吸出上清，重悬沉淀的细胞在PBS或流式细胞其它所需的解决方案。 注:再悬浮量将取决于细胞沉淀后离心分离，和应用的大小而变化。 </li><li>对于存活测定的PBS〜75-300微升体积是合适的，并且这些混悬剂可以分析使用市售fluorescenc根据制造商的说明ë基于活力测定法，吸移到常规血球，并且使用倒置荧光显微镜计数。 </li><li>对于流式细胞仪分析，或基于检测GFP阳性乳腺癌细胞的分选，重悬含有2％FBS，2mM的EDTA的细胞沉淀在1ml PBS中，和10mM的HEPES，使得浓度为1×10 6个细胞/ ml。 </li></ol></li><li> H＆E染色及免疫组化。 <ol><li>处理用于苏木精 - 伊红（H＆E）染色和/或免疫组织化学的片段，放置在塑料盒标有一个号码2铅笔的片段，并淹没在容器中填充有10％的福尔马林缓冲液24小时。 </li><li>过程中组织进行石蜡包埋，切片和使用，其中包括一个脱钙一步占每个片段中的骨针矿化例行协议染色。 </li></ol></li></ol>

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梅拉等人以前描述了死后收集和骨标本分析转移性前列腺癌患者收获在尸检时，露出高质量组织和验证的使用人骨样品来研究转移过程47。在这里，我们已经描述了协议，用于收集，处理和使用从下面的THR手术在短期共培养系统来研究乳腺癌细胞的迁移，定居和增殖丢弃股骨头获得的人骨组织碎片。大约300,000髋关节置换手术是在美国（矫形外科医生的美国学院; Orthoinfo.aaos.org）每年...

肿瘤微环境是目前公认的癌细胞行为乳腺癌进展和转移过程中的关键决定因素传播1-3。这里提出的方法的目标是促进人体骨组织，乳腺癌转移4-6的最频繁的部位的微环境中的乳腺癌细胞的研究。骨是由矿化和骨髓室7-9，这两个怀有细胞和分泌的因子牵连转移进展10,11。尽管广泛使用在体内小鼠模型促进转移过程12-15系统性研究，骨架内细胞的相互作用是不进行观察和直接扰动容易获得。模型系统研究和培养小鼠骨髓组织和小鼠骨髓细胞提供更好的访问，并已经取得了对串扰许多见解和机制基本乳腺癌细胞米etastasis小鼠骨骼16-22。然而，研究表明，有可能是osteotropism的物种特异性的模式为骨组织23,24。这些模式可以反映固有物种特异性在骨组织性质的差异，从改变的骨髓群体中的免疫缺陷小鼠11产生的差异，和/或小鼠的相对年轻的年龄在实验中使用的，所有这些都在骨的可能的决定因素微环境。 体外方法用于人骨共培养研究乳腺癌细胞已经典型地集中于特定骨的细胞类型，如骨髓来源的成骨细胞或基质细胞作为单层培养的或工程化的三维模型系统25-35内。虽然2维细胞培养模型已形成的在体外的支柱接近为癌症研究，人们早就认识到，细胞的行为是从根本上改变在米onolayer培养系统18,36,37。这导致了设计的微环境，模仿的3-维活组织的复杂性，包括矩阵可与天然材料，如胶原蛋白构成的支架的基于模型的开发，或合成的聚合物接种有特定的细胞类型，以创建组织样的微环境36-41。设计方案还包括使用生物反应器平台的控制和研究微18,19,41-43体内的荷尔蒙环境。虽然仿生模型和生物反应器提供了一个复杂的组织微环境中的受控环境的许多元素，并且已成功地应用到推进乳腺癌转移到骨18,19,35,42,43的研究中，工程化的模型系统一般不掺入天然骨环境中存在的细胞类型和细胞外基质组分的全谱。 直到最近，乳腺癌细胞尚未研究天然的，完整的，三维人体骨组织的微环境中。我们最近报道使用人体骨组织碎片从全髋关节置换分离（THR）的手术标本44的共培养模型的发展。这些标本既怀有必要研究潜在的微转移在短期内培养机制的矿化和骨髓车厢。在以前的工作中，我们建立了验证性原则，使用生物发光成像（BLI）监测荧光素酶表达乳腺癌细胞（MDA-MB-231-fLuc）共培养骨碎片44的扩散。在这里，我们提出详细的实验方案为研究使用人骨组织的外植体的天然微环境的上下文中乳腺细胞癌的增殖，定植，和迁移。 首先，我们提出了一个协议相邻骨骼碎片来衡量乳房共培养乳腺癌细胞使用BLI细胞增殖。在这一节中，乳腺癌细胞悬液接种作为细胞的斑点在邻近骨头碎片，这是由骨蜡件固定6孔板中。对照孔含有骨蜡，但是没有骨片段。一旦细胞斑附着，培养基，并将该板是24小时，在这之后的生物发光信号的强度（与细胞数目相关联）的测量是用BLI培养。在接下来的步骤中，我们提出直接接种到骨碎片研究定居和增殖共培养的乳腺癌细胞的方法。这里的乳房细胞悬浮液被直接吸移到所述骨组织碎片，其通过BLI和荧光显微镜在培养监测随着时间的推移追踪定植和细胞数。在该方法中，骨髓隔室可以从骨碎片被刷新为在定植乳腺癌细胞通过流式细胞分析的任何时间点术，或骨髓存活测定。 此外，我们取消划线两种不同的方法，用于测量乳腺癌细胞迁移。在第一种方法的步骤进行了概述，用于测量朝向骨组织培养上清液迁移。在这个协议中的乳腺癌细胞接种到Transwell小室的内，上表面插入膜具有8微米的孔径（ 如图1A）。插入件，然后放入一个接收机板用含有骨组织上清液或对照培养基的孔中。超过20小时的温育期的过程中，少量的乳腺癌细胞通过插入膜迁移入下面的组织培养孔中，其中它们附着于检测通过BLI。在第二迁移方法的乳腺癌细胞接种到Transwell小室的插入件的膜的下部，外表面。骨组织碎片放入插入杯子和乳腺癌细胞向上迁移通过膜拓殖骨碎片（ 见图1B），其然后用成像BLI。

<table><tbody><tr><td>DMEM (1X) + GlutaMAX-l</td><td>Life Technologies</td><td>10569-010</td><td>Supplement all DMEM with 10% fetal bovine serum and 1% Pen/Strep</td></tr><tr><td>McCoy&#039;s 5A Medium (1X)</td><td>Life Technologies</td><td>16600-082</td><td>Supplement all McCoy&#039;s&amp;nbsp; with 10% fetal bovine serum and 1% Pen/Strep</td></tr><tr><td>Fetal bovine serum</td><td>Life Technologies</td><td>16000-044</td><td></td></tr><tr><td>Penicillen Streptomycin</td><td>Life Technologies</td><td>15140-122</td><td></td></tr><tr><td>Blastocidin (10 mgl/ml)</td><td>InvivoGen&amp;nbsp;</td><td>ant-bl</td><td>Supplement media used for transfected cell lines.</td></tr><tr><td>Falcon&amp;reg; Cell Culture Inserts for 24 Well Plate with Transparent PET Membrane (8.0 &amp;mu;m pore size)</td><td>Corning Incorporated</td><td>353097</td><td></td></tr><tr><td>Falcon&amp;reg; 24 Well TC-Treated Polystyrene Cell Culture Insert Companion Plate</td><td>Corning Incorporated</td><td>353504</td><td></td></tr><tr><td>LIVE/DEAD Viability/Cytotoxicity Kit *for mammalian cells*</td><td>Invitrogen Detection Technologies</td><td>L-3224</td><td></td></tr><tr><td>FluoroBrite DMEM</td><td>Life Technologies</td><td>A18967-01</td><td></td></tr><tr><td>D-luciferin firefly, potassium salt 5 g (30 mg/ml)</td><td>Life Technologies</td><td>L-8220</td><td></td></tr><tr><td>Sterile Cell Strainer 70 &amp;mu;m</td><td>Fisher Scientific</td><td>22363548</td><td></td></tr><tr><td>6 Well TC-Treated Cell Culture Cluster</td><td>Corning Incorporated</td><td>3516</td><td></td></tr><tr><td>12 Well TC-Treated Cell Culture Cluster</td><td>Corning Incorporated</td><td>3513</td><td></td></tr><tr><td>24 Well TC-Treated Cell Culture Cluster</td><td>Corning Incorporated</td><td>3526</td><td></td></tr><tr><td>Friedman-Pearson Rongeur</td><td>Fine Science Tools</td><td>16021-14</td><td></td></tr><tr><td>Bone wax</td><td>Surgical Specialties Corporation</td><td>903</td><td></td></tr><tr><td>IVIS 50 Imaging Platform</td><td>Caliper Life Sciences/PerkinElmer</td><td></td><td></td></tr><tr><td>Living Image Software Program&amp;nbsp;</td><td>Caliper Life Sciences/PerkinElmer</td><td>133026</td><td></td></tr><tr><td>EVOS FL Imaging System</td><td>Life Technologies</td><td>Version 4.2</td><td></td></tr><tr><td>OsteoSense 680 EX</td><td>PerkinElmer</td><td>NEV10020EX</td><td></td></tr><tr><td>MCF-7 breast cancer cells</td><td>ATCC</td><td>HTB-22</td><td></td></tr><tr><td>MDA-MB-231 breast cancer cells</td><td>ATCC</td><td>HTB-26</td><td></td></tr><tr><td>Monoclonal mouse anti-human cytokeratin antibody</td><td>DAKO Cytomation</td><td>Clone (AE1/AE3)</td><td></td></tr></tbody></table>

methods for culturing human femur tissue explants to study breast cancer cell colonization of the metastatic niche

骨是乳腺癌转移的最常见的部位。虽然它已被广泛接受的微环境影响癌细胞行为，知之甚少人体骨组织的天然微环境中的乳腺癌细胞的特性和行为。我们已开发的方法来跟踪，量化和的微环境中调节人乳腺癌细胞培养的人骨组织碎片从以下全髋关节置换手术丢弃股骨头隔离。使用设计用于萤光素酶和增强型绿色荧光蛋白（EGFP）表达的乳腺癌细胞中，我们能够后可重复地定量迁移和增殖型态使用生物发光成像（BLI），轨道细胞相互作用使用荧光显微镜的骨碎片内，并评估乳腺细胞定植流式细胞仪。这种模式的主要优势包括:1）一个人，建筑完好无损组织microenvironment包括有关人体的细胞类型，以及2）直接进入微环境，这有利于快速定量和定性监测和乳腺癌和骨细胞的特性，行为和交互作用扰动。一个主要的限制，目前，是组织片段，该组合限制住研究短期培养的窗口的有限的可行性。该模型系统的用途包括研究乳腺癌和其他骨寻求恶性肿瘤的基本生物学的转移性利基内，并且开发治疗策略，以有效地针对乳腺癌细胞在骨组织中。

从股骨头分离组织碎片股骨头从男性和女性患者（44-90岁）在接受矫形外科系选修全髋关节置换手术在斯坦福大学医学院的数量相等收集。每个丢弃股骨头含有股骨上端，其中窝藏小梁骨组织的矿化骨针和骨髓组成的一小部分。此组织是可透过轴（图2A）的使用外科咬骨钳提取小片段的外科手术暴露的横截面。所提取的片的组织学示于图2B中 ，揭示了?...

Watch this Scientific Journal Video about Methods for Culturing Human Femur Tissue Explants to Study Breast Cancer Cell Colonization of the Metastatic Niche at JoVE.com

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

协议描述为研究乳腺癌细胞的迁移，增殖和定植于人体骨组织外植体模型系统。

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 ...

methods-for-culturing-human-femur-tissue-explants-to-study-breast

Research

JoVE Journal

Medicine

12.4K 次观看.  Stanford University School of Medicine. 协议描述为研究乳腺癌细胞的迁移，增殖和定植于人体骨组织外植体模型系统。 

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

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 ...

科研

癌症研究

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 ...

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 ...

医学

Preparation and Analysis of In Vitro Three Dimensional Breast Carcinoma Surrogates

Three dimensional (3D) culture is a more physiologically relevant method to model cell behavior in vitro than two dimensional culture. Carcinomas, including breast carcinomas, are complex 3D tissues composed of cancer epithelial cells and stromal components, including fibroblasts and extracellular matrix (ECM). Yet most in vitro models of breast carcinoma consist only of cancer epithelial cells, omitting the stroma and, therefore, the 3D architecture of a tumor in vivo. Appropriate 3D modeling of carcinoma is important for accurate understanding of tumor biology, behavior, and response to therapy. However, the duration of culture and volume of 3D models is limited by the availability of oxygen and nutrients within the culture. Herein, we demonstrate a method in which breast carcinoma epithelial cells and stromal fibroblasts are incorporated into ECM to generate a 3D breast cancer surrogate that includes stroma and can be cultured as a solid 3D structure or by using a perfusion bioreactor system to deliver oxygen and nutrients. Following setup and an initial growth period, surrogates can be used for preclinical drug testing. Alternatively, the cellular and matrix components of the surrogate can be modified to address a variety of biological questions. After culture, surrogates are fixed and processed to paraffin, in a manner similar to the handling of clinical breast carcinoma specimens, for evaluation of parameters of interest. The evaluation of one such parameter, the density of cells present, is explained, where ImageJ and CellProfiler image analysis software systems are applied to photomicrographs of histologic sections of surrogates to quantify the number of nucleated cells per area. This can be used as an indicator of the change in cell number over time or the change in cell number resulting from varying growth conditions and treatments.

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, ...

生物工程

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 ...

Heteromulticellular Stromal Cells in Scaffold-free 3D Cultures of Epithelial Cancer Cells to Drive Invasion

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully recapitulate aspects of cancer progression such as proliferation and invasion. Although patient-derived organoids and patient-derived xenograft organoids are pathophysiologically relevant, they are costly to propagate, difficult to manipulate, and comprised primarily of the most proliferative cell types within the tumor microenvironment (TME). These limitations prevent their use for elucidating cellular mechanisms of disease progression that depend upon tumor-associated stromal cells which are found within the TME and known to contribute to metastasis and therapy resistance. 
Here, we report on methods for cultivating epithelial-stromal multicellular 3D cultures. The advantages of these methods include a cost-effective system for rapidly generating organoid-like 3D cultures within scaffold-free environments that can be used to track invasion at single-cell resolution within hydrogel scaffolds. Specifically, we demonstrate how to generate these heteromulticellular 3D cultures using BT-474 breast cancer cells in combination with fibroblasts (BJ-5ta), monocyte-like cells (THP-1), and/or endothelial cells (EA.hy926). Additionally, differential fluorescent labeling of cell populations enables time-lapse microscopy to define 3D culture assembly and invasion dynamics. 
Notably, the addition of any two stromal cell combinations to 3D cultures of BT-474 cells significantly reduces circularity of the 3D cultures, consistent with the presence of organoid-like or secondary spheroid structures. In tracker dye experiments, fibroblasts and endothelial cells co-localize in the peripheral organoid-like protrusions and are spatially segregated from the primary BT-474 spheroid. Finally, heteromulticellular 3D cultures of BT-474 cells have increased hydrogel invasion capacity. Since we observed these protrusive structures in heteromulticellular 3D cultures of both non-tumorigenic and tumorigenic breast epithelial cells, this work provides an efficient and reproducible method for generating organoid-like 3D cultures in a scaffold-free environment for subsequent analyses of phenotypes associated with solid tumor progression.

There is a critical need for 3D cancer models that capture heterocellular crosstalk to study cancer metastasis. Our study presents the generation of heteromulticellular stromal-epithelial in a scaffold and scaffold-free environment that can be used to study invasion and cellular spatial distributions.

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

Breast cancer is the second leading cause of cancer-related death among women in the U.S. Organoid models of solid tumors have been shown to faithfully ...

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later distally, is a major factor in patient prognosis. Experimental systems of breast cancer rely on cell lines usually derived from primary tumours or pleural effusions. Two major obstacles hinder this research: (i) some known sub-types of breast cancers (notably poor prognosis luminal B tumours) are not represented within current line collections; (ii) the influence of the tumour microenvironment is not usually taken into account.
We demonstrate a technique to culture primary breast cancer specimens of all sub-types. This is achieved by using three-dimensional (3D) culture system in which small pieces of tumour are embedded in soft rat collagen I cushions. Within 2-3 weeks, the tumour cells spread into the collagen and form various structures similar to those observed in human tumours1. Viable adipocytes, epithelial cells and fibroblasts within the original core were evident on histology. Malignant epithelial cells with squamoid morphology were demonstrated invading into the surrounding collagen. Nuclear pleomorphism was evident within these cells, along with mitotic figures and apoptotic bodies.
We have employed Optical Projection Tomography (OPT), a 3D imaging technology, in order to quantify the extent of tumour spread in culture. We have used OPT to measure the bulk volume of the tumour culture, a parameter routinely measured during the neo-adjuvant treatment of breast cancer patients to assess response to drug therapy. 
Here, we present an opportunity to culture human breast tumours without sub-type bias and quantify the spread of those ex vivo. This method could be used in the future to quantify drug sensitivity in original tumour. This may provide a more predictive model than currently used cell lines.

We have developed a collagen-based in vitro assay which promotes proliferation and invasion from samples of all breast cancer subtypes. Optical Projection Tomography, a three dimensional microscopy technique was utilised to visualise and quantify tumour expansion. This assay may be used to quantify drug response of individual tumour samples.

长期培养的人类乳腺癌标本，并利用光学投影层析成像分析

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

生物学

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 ...

Isolation of Mammary Epithelial Cells from Three-dimensional Mixed-cell Spheroid Co-culture

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work has been done using classical two-dimensional cell culture models, which allow for easy cell manipulation. It has become clear that intracellular signaling pathways are affected by extracellular forces, including dimensionality and cell surface tension3-4. Multiple approaches have been taken to develop three-dimensional models that more accurately represent biologic tissue architecture3. While these models incorporate multi-dimensionality and architectural stresses, study of the consequent effects on cells is less facile than in two-dimensional tissue culture due to the limitations of the models and the difficulty in extracting cells for subsequent analysis.
The important role of the microenvironment around tumors in tumorigenesis and tumor behavior is becoming increasingly recognized4. Tumor stroma is composed of multiple cell types and extracellular molecules. During tumor development there are bidirectional signals between tumor cells and stromal cells5. Although some factors participating in tumor-stroma co-evolution have been identified, there is still a need to develop simple techniques to systematically identify and study the full array of these signals6. Fibroblasts are the most abundant cell type in normal or tumor-associated stromal tissues, and contribute to deposition and maintenance of basement membrane and paracrine growth factors7.
Many groups have used three dimensional culture systems to study the role of fibroblasts on various cellular functions, including tumor response to therapies, recruitment of immune cells, signaling molecules, proliferation, apoptosis, angiogenesis, and invasion8-15. We have optimized a simple method for assessing the effects of mammary fibroblasts on mammary epithelial cells using a commercially available extracellular matrix model to create three-dimensional cultures of mixed cell populations (co-cultures)16-22. With continued co-culture the cells form spheroids with the fibroblasts clustering in the interior and the epithelial cells largely on the exterior of the spheroids and forming multi-cellular projections into the matrix. Manipulation of the fibroblasts that leads to altered epithelial cell invasiveness can be readily quantified by changes in numbers and length of epithelial projections23. Furthermore, we have devised a method for isolating epithelial cells out of three-dimensional co-culture that facilitates analysis of the effects of fibroblast exposure on epithelial behavior. We have found that the effects of co-culture persist for weeks after epithelial cell isolation, permitting ample time to perform multiple assays. This method is adaptable to cells of varying malignant potential and requires no specialized equipment. This technique allows for rapid evaluation of in vitro cell models under multiple conditions, and the corresponding results can be compared to in vivo animal tissue models as well as human tissue samples.

A simple method is described for analyzing effects of tissue fibroblasts on associated epithelial cells. The combination of this method and three-dimensional tissue culture can facilitate analysis of cells after isolation from 3D. The technique is applicable to cells of varying malignant potential, allowing systematic study of effects of tumor-associated stroma on tumor cells.

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

While enormous efforts have gone into identifying signaling pathways and molecules involved in normal and malignant cell behaviors1-2, much of this work ...

Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without breaching the collagenous basement membrane. While DCIS is a non-obligate precursor to invasive breast cancers, the molecular mechanisms and cell populations that permit progression to invasive cancer are not fully known. To determine if progenitor cells capable of invasion existed within the DCIS cell population, we developed a methodology for collecting and culturing sterile human breast tissue at the time of surgery, without enzymatic disruption of tissue.
Sterile breast tissue containing ductal segments is harvested from surgically excised breast tissue following routine pathological examination. Tissue containing DCIS is placed in nutrient rich, antibiotic-containing, serum free medium, and transported to the tissue culture laboratory. The breast tissue is further dissected to isolate the calcified areas. Multiple breast tissue pieces (organoids) are placed in a minimal volume of serum free medium in a flask with a removable lid and cultured in a humidified CO2 incubator. Epithelial and fibroblast cell populations emerge from the organoid after 10 - 14 days. Mammospheres spontaneously form on and around the epithelial cell monolayer. Specific cell populations can be harvested directly from the flask without disrupting neighboring cells. Our non-enzymatic tissue culture system reliably reveals cytogenetically abnormal, invasive progenitor cells from fresh human DCIS lesions.

Primary cell culture using intact tissue organoids provides a model system that mimics the multi-cellular in vivo microenvironment. We developed a serum-free primary breast epithelium tissue culture model that perpetuates mixed cell culture lineages and exhibits differentiated morphology, without enzymatic tissue disruption. Breast organoids remain viable for &#62;6 months.

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

Breast ductal carcinoma in situ (DCIS), by definition, is proliferation of neoplastic epithelial cells within the confines of the breast duct, without ...

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

摘要

探索更多视频

此视频中的章节

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

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

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

制备及分析

对于肿瘤基质相互作用的三维共培养模式

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

长期培养的人类乳腺癌标本，并利用光学投影层析成像分析

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

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

摘要

探索更多视频

此视频中的章节

从细胞和组织样本中分离和功能评估人类乳腺癌干细胞

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

三维组织培养模型研究人类原发性骨髓和恶性肿瘤

制备及分析

对于肿瘤基质相互作用的三维共培养模式

上皮癌细胞无支架 3D 培养物中的异多细胞基质细胞驱动侵袭

长期培养的人类乳腺癌标本，并利用光学投影层析成像分析

三维培养试验探索肿瘤细胞浸润和卫星肿瘤形成

从三维混合细胞球体共培养的乳腺上皮细胞的隔离

非酶中，预浸润性乳腺癌病变的无血清组织培养的微球体的自然发生

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