We thank Marleen De Meulemeester for demonstrating the assay technique in the video film. B. I. R. is a Postdoctoral Research Fellow of the Research Foundation &#8211; Flanders (FWO &#8211; Vlaanderen). L.M.M. is a recipient of an Emmanuel van der Schueren grant from the Flemish League against Cancer (Vlaamse Liga tegen Kanker).

<img alt="图1" src="/files/ftp_upload/52792/52792fig1.jpg" /> 图的不同检测步骤1.示意图。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 1.准备预培养心的碎片（的PHF） <ol><li>孵育受精鸡卵在37℃9天。通过允许在4天后续准备的PHF（ 例如，在星期四）日期和最后的对抗肿瘤细胞聚集完成这个孵化（ 例如，在鸟巢星期一）。 </li><li>消毒壳与在水中的70％（体积/体积）乙醇。进行使用的无菌溶液和材料所有进一步操作在组织培养橱。打开外壳使用时钝钳胚胎极。 </li><li>拉出胚胎由HOL丁俊晖与眼球摘除勺子（ 图2）颈部。放置在玻璃培养皿的胚胎用直径为5厘米含有5毫升林格氏盐溶液。 <ol><li>打开腹胸部皮肤被撕开使用锋利的镊子双手肌肤，由同一类型的操纵去除胸骨，并用显微虹膜切除术剪刀将其切断大血管解剖出来的心脏。执行与下一个校准目镜网格macroscope所有进一步的操作。 </li></ol></li></ol><img alt="图2" src="/files/ftp_upload/52792/52792fig2.jpg" /> 图2.抬起出胚胎用勺子去核卵细胞中。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="4"><li>的心脏转移到一个玻璃培养皿一个直径为5厘米含有用5％胎牛血清5毫升MEM-安瑞嘉3培养基。通过采用切除显微剪刀虹膜切除术心脏的上部颅第三卸下心房和相关的船只。从解剖心室心包有一对锋利的钳子。 </li><li>请使用显微剪刀虹膜切除术心室矢状半切，并清除血液通过用锋利的钳轻轻晃动。 </li><li>转移脑室到另一个玻璃培养皿的直径为5厘米含有用5％胎牛血清加入5ml新鲜MEM-安瑞嘉3培养基中。用显微虹膜切除术剪刀剪心室成约为0.4毫米直径件。一个心脏可以产生约100片段。 注:一个心脏可以产生约100片段</li><li>旋转培养皿轻轻地来驱动所有心室碎片朝菜的中心。删除所有语料库槲用不锈钢针用Sep从心室片段arating它们和驱动它们朝向培养皿的周边。 </li><li>的心脏的片段，用玻璃巴斯德吸管转移到含有培养基（MEM-安瑞嘉3加10％体积/体积的胎牛血清）的2-至3ml 50毫升锥形瓶中。 注意:确切培养基体积取决于个体烧瓶的可变底部凸部:其底部的中心应涵盖的仅液体的薄膜。 </li><li>气的烧瓶（多个），用5％的CO 2的空气中经由塞子的混 ​​合物，并温育于旋转摇床烧瓶（多个），在37℃下以70转/分钟（rpm）离心24小时。此悬浮培养步骤的理由是为了获得适合于与肿瘤细胞聚集随后对抗球形心脏组织碎片。 </li><li>的心脏的片段和它们的培养基转移至培养皿。丢弃语料库槲，坏死（深）的片段，并用针心脏碎片集团。 </li><li>如在步骤1.9中所述另一个60小时孵育在含有6毫升培养基的另外50毫升的锥形烧瓶中剩余的心脏片段。 注:在此潜伏期，碎片将成为球形。它们包括成肌细胞的芯和一薄层成纤维细胞在外围的。 </li><li>选择球状片段表现出成纤维细胞的薄，均匀的层（形成半透明胶囊）中，并由macroscope和针的装置的直径为0.4毫米。一鸡心脏将产生约20合适的PHF。转移这些的PHF，其中许多将收缩节奏在37℃下，于含有新鲜培养基另一个培养皿。这些都是的PHF准备与测试细胞对抗。 </li></ol> 2.编写和测试球状细胞聚集对抗<ol><li>制备半固体琼脂培养基:通过煮沸三次溶解在15毫升林格氏盐溶液100mg的琼脂。冷静该悬浮液至40℃并加入7.5林格氏盐溶液毫升/蛋白（1:1）和7.5毫升胎牛血清。 </li><li>制备6-毫升含在50ml锥形瓶中1×10 5个测试细胞/在其适当的培养基ml的悬浮液。为此3天对抗文化开始计划之前。孵育在旋转摇床烧瓶在37℃在70rpm下培养3天。气的烧瓶用5％或10％（体积/体积） 的 CO 2在空气中的混合物中，这取决于所用的培养基的类型。 </li><li>查看骨料下配有校准的目镜网格macroscope。选择（用针）球状细胞聚集体的直径为0.2毫米。 </li><li>使用巴斯德吸移管向在培养基中的最小量的传送8所选的PHF（直径= 0.4毫米），含半固体琼脂培养基中13的胚胎手表玻璃。单独移动的PHF用针，以形成一个圆。吸过量的培养基用一小片滤纸（ 见图3）。 </li></ol><img alt="图3" src="/files/ftp_upload/52792/52792fig3.jpg" /> 图3.吸气各地的PHF多余的液体 。八的PHF被放置在半固体琼脂中的胚胎表玻璃，和流体过量时有小的三角片运滤纸除去。箭头指示的8个独立的PHF。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="5"><li>带上十大评选球状细胞聚集（直径= 0.2 MM）到使用巴斯德吸管圈。吸过量的培养基。移动一个骨料相向与针的PHF的，直到它们使彼此接触。用一小片滤纸抽吸过量的培养基。 </li><li>密封该手表玻璃用石蜡的盖，和INCU软化在37℃下进行4 - 24小时，根据测试细胞对的PHF的粘合性能。 </li><li>浸入面临对用预温（37℃）培养液中，并用巴氏吸管转移的每个单独的一对进含有1.5毫升培养培养基的5毫升锥形瓶中。 </li><li>孵育在旋转摇床，在37℃下调整至120转的烧瓶中。气用5％或10％（体积/体积）的CO 2的烧瓶中的空气，这取决于所用的试验电池（ 见图4）培养基的类型。 <ol><li>为了避免媒体的浓度，通过两个补充5毫升锥形瓶中填充用2ml林格氏盐溶液通过使它们润湿气体。刷新培养基每8天。 </li></ol></li></ol><img alt="图4" src="/files/ftp_upload/52792/52792fig4.jpg" /> 图4.小锥形瓶上gyrotory SHAK顶部。呃用1.5ml培养基的烧瓶被密封用装有气体中硅氧烷瓶塞-和出口针，并移动以120rpm在37℃。该气体通过入口管（1），以较大的Erlenmeyer烧瓶（2）充满无菌林格氏盐溶液主导，并进一步分散（3和4）含有与个别相对的对培养基小瓶（5和6）。最后，废气被收集在一个更大的烧瓶用无菌水器（7），从那里可以逸出到空气中（8）。该图显示了两套上自制的平台板顶部文化电池。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 3.组织学的面对文化<ol><li>准备布安 - 奥朗德的固定液。 <ol><li>溶解将2.5g乙酸铜（中性）的100ml单蒸馏水和慢慢加入4.0克苦味酸。 </li><li>过滤通过纸过滤器的溶液。加入10 mL福尔马林和1ml乙酸。拌9份的该溶液与1份的单蒸馏水饱和氯化汞溶液中。 注意:该解决方案包含多种有害物质。福尔马林是吸入有毒，与皮肤接触和吞食。乙酸具有腐蚀性的口腔和肠道消化后。苦味酸是过敏和爆炸时迅速加热或敲击。氯化汞是高度腐蚀性粘膜和nefrotoxic。穿戴防护服和手套准备布安 - 奥朗德的解决方案，它在处理火灾通风良好的地方偏僻。另一种固定方法是基于在磷酸缓冲盐水的4％甲醛。 </li></ol></li><li>经过数天或数周的潜伏期如下固定个体培养物。首先，将培养物转移到林格氏盐几秒钟溶液以除去血清蛋白。接着，沉浸在培养皿中，直径为3厘米的装有3毫升布安-奥朗德的固定溶液约2小时。 </li><li>孵育它们在水中2小时以除去尽可能多的固定溶液尽可能之前冲洗培养在3ml去矿物水洗涤三次。最后，转移到3毫升70％（体积/体积）乙醇在水中。保持在该溶液中O / N培养物，但是这可以延长天数。 </li><li>通过包含每个2小时100毫升的96％（体积/体积）乙醇在水中，100％乙醇或100％的异丙醇，和100％的二甲苯罐依次转印脱水。 <ol><li>各培养转移到一个独立的玻璃盖玻片（用二甲苯的最小量），放置在酒瓶胶囊石蜡包埋底部的盖玻片，并覆盖固定材料与液体石蜡在56℃。孵育在56℃下放置24小时，然后冷却该嵌入材料至RT。 注意:由于苯衍生物二甲苯可吸入后有毒，应通风良好的区域仅在处理。 </li></ol></li><li>取出酒瓶胶囊和盖玻片，并切出其中包含的固定文化蜡块。 </li><li>请用切片机14的整个相对的一对8微米厚的石蜡切片，并收集三个交替显微镜玻片已预先用组织粘合剂溶液作为粘着剂的所有部分。避免蛋清作为粘着剂，因为它可能会干扰免疫。 注意:在显微镜玻片的预处理是通过提供1小滴的组织粘合剂溶液和3小滴软化水用巴斯德移液管，并且将它们混合，以覆盖滑动完成。 </li><li>在装有100毫升100％二甲苯10分钟一罐浸泡两次取下第一张幻灯片的石蜡。 </li><li>再水化的载玻片浸入10秒中罐子含有100ml的下列溶液:二甲苯/乙醇（1:1），100％的乙醇，96％（体积/体积）的水乙醇，70％在水（v / v）的乙醇，最后软化水。 </li><li>溶解通过浸入氯化汞晶体中含有100ml的0.5％（重量/体积）中的碘96％（体积/体积）的水乙醇进行2分钟的罐子。 </li><li>清除在含有100毫升5％（重量/体积）硫代硫酸钠在水中10秒的罐部分。然后在蒸馏水中彻底冲洗的幻灯片。 </li><li>孵育在含有100ml哈里斯&#39;苏木2分钟一个罐子的幻灯片，在0.1M的HCl简要淹没，然后洗在流动的自来水10分钟。 </li><li>孵育在含有100ml曙红0.1％（重量/体积）的水进行1分钟的罐子。 </li><li>通过短暂浸没在含有100ml以下一系列溶液的罐脱水:软化水，70％（体积/体积）乙醇，96％（体积/体积）乙醇，100％乙醇洗涤两次，二甲苯 - 乙醇（1:1） ，最后在100％二甲苯。 </li><li>安装幻灯片通过提供2个独立的介质液滴在载玻片安装介质，让这些扩展通过将一个盖玻片在它们上面，并让它在室温下硬化24小时。 </li></ol> 4.免疫组化<ol><li>制备的Tris缓冲盐水（TBS）。溶解将6.0g三（羟甲基）氨基甲烷（Tris碱）45.0克氯化钠在4.5升的去离子水中。带来至pH 7.6，用1M的HCl（约42毫升）。添加蒸馏水至5 L. </li><li>制备的Tris-HCl缓冲液中。溶解60克Tris碱的在800毫升软化水中。带来至pH 7.6用6M盐酸（约65毫升）。加蒸馏水至1升稀释10倍的蒸馏水后使用。 </li><li>准备的Tris-BSA 0.1％的缓冲。溶解12.1克Tris碱的45.0克NaCl的4.5升的去离子水中。带来至pH 8.2，用1M的HCl（约42毫升）。加将5.0g牛血清白蛋白（BSA）和6.5克的NaN 3。添加软化水，使5 L. 注意:叠氮化钠是一种高度有毒产品。与酸接触释放剧毒气体。戴上手套，避免摄入一切手段。叠氮化钠与形式铜，铅和其他金属非常敏感的爆炸性化合物。冲洗水槽用大量的水冲洗。另一种防腐剂是0.01％硫柳汞。 </li><li>按照项目3.2至3.10。 </li><li>浸在TBS或三-0.1％BSA的缓冲幻灯片，擦去周围用纸巾部分多余的液体。 </li><li>在TBS或在5％（重量/体积）牛血清白蛋白的Tris-0.1％应用150微升正常山羊血清以1:20稀释（重量/体积）BSA缓冲液在高湿度室中30分钟（一个封闭的盒子用于共-incubation用开水冲收件人确保内高湿度）的幻灯片。然后去除多余的液体用纸巾。 </li><li>应用150微升初级抗血清稀释在Tris-0.1％（重量/体积）牛血清白蛋白在高湿度室中补充有1％（体积/体积）正常山羊血清，至少2小时。确定初级抗血清的最佳稀释度empiri地作为备用。 注意:免疫组织化学可用于检测鸡心脏抗原但是，使用特异性抗体，鸡心脏抗原所描述的技术可以应用到其他抗原（如绿色荧光蛋白）15为好。 </li><li>在Tris-0.1％洗两次幻灯片w / v的BSA在摇表5-10分钟。 </li><li>用纸巾为至少1小时在高湿度室中除去多余的流体，并应用150微升的山羊抗兔抗血清在过量（ 例如 ，在TBS中1:20）。 </li><li>用TBS在摇表5-10分钟洗两次。 </li><li>用纸巾去除多余的液体。所适用的过氧化物酶 - 抗过氧化物酶复合物稀释1:250的TBS在高湿度室中补充有1％（体积/体积）正常山羊血清为至少1小时。该复合物的稀释取决于批量。 </li><li>用TBS洗涤载玻片并转移到的Tris-HCl缓冲液pH 7.6。 </li><li>转移滑入三 -HCl缓冲液含有0.25克/升二氨基联苯胺和0.01％（体积/体积） 的 H 2 O 2的几分钟。停止孵化，当鸡心脏被染色。定期检查，在显微镜下。 </li><li>转移幻灯片的Tris-HCl中10分钟，然后以蒸馏水。 </li><li>按照项目3.13 3.14和。 注意:对于固定过程中容易被破坏抗原，培养的cryosectioning可以为免疫组化分析的替代。这在以下方案步骤描述: </li><li>用3毫升林格氏盐溶液洗活培养物以除去血清蛋白。 </li><li>加入一滴包埋剂的预冷（-16°C）标本冷冻切片机的持有人。从玻璃盖玻片边缘转移培养的组织，以这种下降的顶部之前它被完全冻结。 </li><li>酷文化到-16℃，切6微米厚的冰冻切片，并收集他们在明胶包覆载玻片上。 </li><li>固定在丙酮中的幻灯片，在4℃下储存于4℃之前10分钟。 </li><li>按照项目4.5至4.15。 </li></ol> 5.评价试验结果注意:在本测定中，侵入被定义为逐行占领PHF由面对测试电池。所有连续部分从一个面对培养的微观分析允许测试细胞聚集和在三维空间中的PHF之间的相互作用的重建。 <ol><li>观察交互并根据以下标准等级的不同的模式（参见图5）。 0级:只有PHF观察。没有面对可观察细胞。 I级:该面对测试细胞附着到PHF，但不占用的心脏组织，甚至没有最外的成纤维细胞的细胞层。 等级IIa族:所述PHF的职业是仅限于外成纤维细胞样细胞和成肌策LL层。 级防爆:本PHF已包围的细胞聚集体，但有占领的迹象。 III级:面对的细胞已经占领了住房公积金，但超过一半的心脏组织完好的原始金额留下更多的。 IV级:该面对细胞已超过一半的PHF的原体积的占用更多。 注:等级I和II与无创性细胞群观察到，而三年级，四是典型的入侵。为了评估不同的时间框架内发展，组织学分析应适用于文化面临不同的固定培养时间之后。 </li></ol><img alt="图5" src="/files/ftp_upload/52792/52792fig5.jpg" /> 图相互作用5.实施例面临癌细胞和的PHF之间 。面对培养物的组织切片染色或者用苏木精 - 曙红（左小图）或immunohistochemically随鸡对心脏（右图）的抗体。互动牌号在协议文本中定义。 （H:心脏组织，T:肿瘤细胞）。 <a href="https://www.jove.com/files/ftp_upload/52792/52792fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 6.毒性评估后的药物治疗<ol><li>转移在最小体积到24孔组织培养multidishes用巴斯德吸移管的所面临的文化。 </li><li>通过加入500μl的不含药物的培养基中洗涤培养物，和6小时后刷新。 </li><li>每天检查所有孔从使用倒置显微镜（物镜40×）的外植体的细胞生长。除以增长超过培养由外植培养物的总数，得到的相面对培养物存活的索引的数目。比较药物处理的培养物与溶剂处理的那些的结果。 注:使用免疫组织化学Ki67表达的（细胞prolif累加器）和TUNEL法的（凋亡）的建议作为互补技术的植试验评估毒性。 </li></ol>面对文化7.成长性评价<ol><li>使培养物的黑与白的照片只是固定前，用配有数字photocamera显微镜（物镜50X） </li><li>在毫米测量较大（a）和小（B）直径考虑到放大率的培养物。 </li><li>通过式16计算出近似体积（V）在毫米3 </li><li> V = 0.4 xaxb 2 </li><li>通过在对峙的开始比较与PHF和细胞聚集体的组合体积这个最终体积计算的培养物的生长。 注意:因为孤的PHF倾向于培养过程中减少其体积，相面对成对的生长是依赖于肿瘤细胞。 </li></ol>

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The authors declare that they have no competing financial interests.

期间的PHF的制备，所述片段可以不留在悬浮液中，但附着于血管壁;这可以通过增加培养基的体积来克服。如果的PHF的数目太低，其大小过大，降低培养基的体积。测试电池的故障聚集，可能是由于在该温度或微生物感染的波动。可替代地，一个无法聚集可能是该细胞的固有特性。期间附着聚集到PHF的，附着力差可以由延伸在半固体琼脂培养基的顶部的潜伏期或通过吸收滤纸装置除去周围的培养多...

侵袭是恶性肿瘤的标志。此活动不仅导致周围组织的破坏，但也牵连转移形成。由于癌症患者从浸润和转移，以及高效抗侵入性治疗死仍然很少，实验室化验模仿的肿瘤细胞的侵袭已经开发出来。小鸡心脏检测的目标是提供一个相关的器官培养的方法来研究肿瘤浸润在三个维度。该分析方法可侵入性和非侵入性的细胞之间的区别，并允许研究试验化合物对肿瘤的侵袭的影响。 使用该测定的背后的基本原理是实际的概念，即肿瘤是生态系统，其中的肿瘤细胞不断地与他们的基质（宿主细胞和细胞外基质）相互作用，并通过这些分子间的相互作用的入侵是微调1。所以，在测定肿瘤细胞所面临的LIVI纳克胚胎鸡心脏片段2，它不仅作为由肿瘤细胞侵袭基板，还可以作为不同类型的基质细胞和矩阵元素的来源。小鸡心脏包含肌细胞，成纤维细胞和内皮细胞，以及细胞外基质由层粘连蛋白，纤连蛋白和不同类型的胶原蛋白的。以这种方式，三维器官培养技术涵盖牵连侵袭患者肿瘤的许多细胞和分子的相互作用。 小鸡心脏测定的主要优点是对基质的实施效果。这方面比体外其他入侵检测是基于肿瘤细胞侵入到非生物凝胶基底膜3或间质基质4分子构成更加完整。在器官培养实验发现肿瘤细胞和正常的生活宿主组织之间对抗的概念已经推出几个作者包括沃尔夫和施奈德在法国5，Easty和Easty在英国6和施莱希德国7。以上所引用的方法的鸡心脏侵袭测定两种技术优点是，片段的体积很容易被标准化，并且它们保持收缩，这允许功能完整性监视器官培养期间。此外，禽类胚胎是优选的，因为它们可以容易地从蛋的无菌内容解剖。该测定具有通过提供周围的肿瘤细胞的复合基质概念相似小鸡尿囊绒膜膜试验8。 该测定法已成功应用于从同一人肿瘤侵入性和非侵入性的细胞的变体之间进行区分，例如，在MCF-7（乳房）9和HCT-8（结肠）10细胞系的家庭。该技术可用于检测潜在的抗侵入性化合物以及<suP&gt; 11,12。如进一步说明的，它可以用于建立有机小分子的结构 - 活性关系。该试验确实，但是，没有考虑到免疫细胞对癌症侵袭的贡献。应当强调，该技术不能被认为是因为高数操作的，试验运行的有限数目（最多30培养物）和长的周转时间（约1个月），为高通量分析系统。

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Ringer&#39;s salt solution</td>
			<td>Braun</td>
			<td>CEO123</td>
			<td></td>
		</tr>
		<tr>
			<td>Bacto-agar</td>
			<td>Becton Dickinson</td>
			<td>214010</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris-(hydroxymethyl)-aminomethaan Analar</td>
			<td>VWR</td>
			<td>103157P</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA : albumin from bovine serum, cohn V fract.</td>
			<td>Sigma</td>
			<td>A4503-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM-Rega 3</td>
			<td>Life Technologies&#160;</td>
			<td>19993013</td>
			<td></td>
		</tr>
		<tr>
			<td>Gyrotory shakers</td>
			<td>New Brunswick Scientific</td>
			<td>G10 and G33</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 50 ml</td>
			<td>Novolab</td>
			<td>92717</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Petri dishes</td>
			<td>Novolab</td>
			<td>68516</td>
			<td></td>
		</tr>
		<tr>
			<td>Iridectomy scissors</td>
			<td>Rumex</td>
			<td>11-0625</td>
			<td></td>
		</tr>
		<tr>
			<td>Macroscope with calibrated ocular grid</td>
			<td>Wild</td>
			<td>157702</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin wax</td>
			<td>International Medical products</td>
			<td>8599956</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin Y</td>
			<td>Sigma</td>
			<td>230251-25g</td>
			<td></td>
		</tr>
		<tr>
			<td>Harris&#39; hematoxylin</td>
			<td>Sigma</td>
			<td>HHS32-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Coverslipping resin (Tissue-Tek)</td>
			<td>Sakura</td>
			<td>4494</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraffin melting apparatus</td>
			<td>GFL</td>
			<td>1052</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome for paraffin sectioning</td>
			<td>Reichert</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Stppers for Erlemeyer flasks with gas in- and outlets</td>
			<td>Novolab</td>
			<td>2602421 and 260243</td>
			<td></td>
		</tr>
		<tr>
			<td>Diaminobenzidine</td>
			<td>Sigma</td>
			<td>D8001</td>
			<td></td>
		</tr>
		<tr>
			<td>REAX rocking table</td>
			<td>Heidolph</td>
			<td>54131</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well tissue dishes</td>
			<td>VWR</td>
			<td>NUNC142475</td>
			<td></td>
		</tr>
		<tr>
			<td>Ophtalmological enucleation spoon</td>
			<td>Rumex</td>
			<td>16-060</td>
			<td></td>
		</tr>
		<tr>
			<td>Sharp forceps</td>
			<td>Rumex</td>
			<td>4-111T</td>
			<td></td>
		</tr>
		<tr>
			<td>Blunt forceps</td>
			<td>Nickel-Electro LTD</td>
			<td>7112</td>
			<td></td>
		</tr>
		<tr>
			<td>Erlenmeyer flasks 5 ml</td>
			<td>Novolab</td>
			<td>211901</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slides washed and degreased</td>
			<td>International Medical products</td>
			<td>8613908</td>
			<td></td>
		</tr>
	</tbody>
</table>

chick heart invasion assay for testing the invasiveness of cancer cells and the activity of potentially anti-invasive compounds

小鸡心脏检测的目标是提供一个相关的器官培养的方法来研究肿瘤浸润在三个维度。该分析方法可侵入性和非侵入性的细胞之间的区别，并允许试验化合物对肿瘤侵袭的影响研究。癌细胞 - 无论是作为聚集体或单个细胞 - 都面临着小鸡胚胎心脏的片段。在悬浮器官培养几天或几周后，将面对培养物固定并包埋在石蜡中用于组织学分析。癌细胞和正常组织之间的三维相互作用，然后从用苏木精 - 曙红或免疫组化染色的表位在心脏组织或对抗癌细胞后的连续切片重建。该测定是用最近的概念，癌浸润是癌细胞和它们相邻的基质主元件（肌成纤维细胞，endoth之间的分子相互作用的结果是一致的elial细​​胞，细胞外基质成分等）。在这里，这种基质环境提供给癌细胞作为活体组织片段。配套方面来测定的相关性是多方面的。侵袭测定中是按照癌症侵袭的标准:在时间渐进占领和更换和宿主组织的空间，以及侵袭和非侵袭性的对抗细胞体内通常与该测定的结果。此外，细胞在体内的侵入图案，由病理学家所定义，是反映在组织学图像中测定。定量结构活性关系（QSAR）分析与众多潜在抗侵入有机同类化合物所得到的结果所允许的在临床中使用的类黄酮和查耳酮，和已知的抗转移药的结构-活性关系（ 例如，微管抑制剂的研究）抑制侵袭测定中也是如此。 HoweveR，该法没有考虑到癌症侵袭账户免疫学的贡献。

在图5中呈现的组织切片作为显示一些成功试验的最终结果。培养物的声音组织学表明活细胞，并允许解释肿瘤细胞和正常组织之间的相互作用。此外，从正常组织无免疫反应可以观察到，这证实使用如小鸡胚胎的正确年龄，免疫排斥反应系统的开发之前。有没有细菌可见，这表明在培养期间不存在（毛）的污染。最后部分的圆形周边印证文化悬浮没有（临时）坚持锥形瓶中墙上的迹...

Watch this Scientific Journal Video about Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds at JoVE.com

Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

在这里，我们提出了一个协议入侵肿瘤细胞研究到生活的正常组织碎片在三个方面。这种器官培养技术主要应用于体外测试潜在抗侵袭药物。

鸡心脏侵袭试验试验癌细胞的侵袭和潜在的反侵入性化合物的活性

The goal of the chick heart assay is to offer a relevant organ culture method to study tumor invasion in three dimensions. The assay can distinguish ...

chick-heart-invasion-assay-for-testing-invasiveness-cancer-cells

Research

JoVE Journal

Medicine

9.0K 次观看.  University of Ghent. 在这里，我们提出了一个协议入侵肿瘤细胞研究到生活的正常组织碎片在三个方面。这种器官培养技术主要应用于体外测试潜在抗侵袭药物。 

视频: Chick Heart Invasion Assay for Testing the Invasiveness of Cancer Cells and the Activity of Potentially Anti-invasive Compounds

Modeling and Imaging 3-Dimensional Collective Cell Invasion

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy tissue is the root cause of disease and death. For other cancers (e.g. breast, lung, etc.), it is the process of metastasis, in which tumor cells move from a primary tumor mass, colonize distal sites and ultimately contribute to organ failure, that eventually leads to morbidity and mortality 3. It has been estimated that invasion and metastasis are responsible for 90&#37; of cancer deaths 4. As a result, there has been intense interest in identifying the molecular processes and critical protein mediators of invasion and metastasis for the purposes of improving diagnosis and treatment 5.
A challenge for cancer scientists is to develop invasion assays that sufficiently resemble the in vivo situation to enable accurate disease modeling 6. Two-dimensional cell motility assays are only informative about one aspect of invasion and do not take into account extracellular matrix (ECM) protein remodeling which is also a critical element. Recently, research has refined our understanding of tumor cell invasion and revealed that individual cells may move by elongated or rounded modes 7. In addition, there has been greater appreciation of the contribution of collective invasion, in which cells invade in strands, sheets and clusters, particularly in highly differentiated tumors that maintain epithelial characteristics, to the spread of cancer 8.
We present a refined method 9 for examining the contributions of candidate proteins to collective invasion 10. In particular, by engineering separate pools of cells to express different fluorescent proteins, it is possible to molecularly dissect the activities and proteins required in leading cells versus those required in following cells. The use of RNAi provides the molecular tool to experimentally disassemble the processes involved in individual cell invasion as well as in different positions of collective invasion. In this procedure, mixtures of fluorescently-labeled cells are plated on the bottom of a Transwell insert previously filled with Matrigel ECM protein, then allowed to invade "upwards" through the filter and into the Matrigel. Reconstruction of z-series image stacks, obtained by confocal imaging, into three-dimensional representations allows for visualization of collectively invading strands and analysis of the representation of fluorescently-labeled cells in leading versus following positions.

Models of tumor cell invasion into three-dimensional extracellular matrix better reflect the in vivo situation than two-dimensional motility assays. Using matrix invasion assays combined with confocal imaging of fluorescently-labeled cells, detailed information on invasion modes and the distinct contributions of leading versus following cells can be obtained.

3维建模和成像集体细胞的侵袭

A defining characteristic of cancer malignancy is invasion and metastasis 1. In some cancers (e.g. glioma 2), local invasion into surrounding healthy ...

科研

医学

Minimal Erythema Dose (MED) Testing

Ultraviolet radiation (UV) therapy is sometimes used as a treatment for various common skin conditions, including psoriasis, acne, and eczema. The dosage of UV light is prescribed according to an individual's skin sensitivity. Thus, to establish the proper dosage of UV light to administer to a patient, the patient is sometimes screened to determine a minimal erythema dose (MED), which is the amount of UV radiation that will produce minimal erythema (sunburn or redness caused by engorgement of capillaries) of an individual's skin within a few hours following exposure. This article describes how to conduct minimal erythema dose (MED) testing. There is currently no easy way to determine an appropriate UV dose for clinical or research purposes without conducting formal MED testing, requiring observation hours after testing, or informal trial and error testing with the risks of under- or over-dosing. However, some alternative methods are discussed.

Minimal Erythema Dose MED Testing

This article describes how to conduct minimal erythema dose (MED) testing in order to determine the lowest dose of ultraviolet radiation that will cause erythema (burning) when administered to an individual.

最小红斑剂量（MED）测试

Ultraviolet radiation (UV) therapy is  sometimes used as a treatment for various common skin conditions, including  psoriasis, acne, and eczema. The ...

Osmotic Drug Delivery to Ischemic Hindlimbs and Perfusion of Vasculature with Microfil for Micro-Computed Tomography Imaging

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to stimulate microcirculation. The choice of delivery method for these agents is important because the route of administration profoundly affects the bioactivity and efficacy of these agents1,2. In this article, we demonstrate how to locally administer a substance in ischemic hindlimbs by using a catheterized osmotic pump. This pump can deliver a fixed volume of aqueous solution continuously for an allotted period of time. We also present our mouse model of unilateral hindlimb ischemia induced by ligation of the common femoral artery proximal to the origin of profunda femoris and epigastrica arteries in the left hindlimb. Lastly, we describe the in vivo cannulation and ligation of the infrarenal abdominal aorta and perfusion of the hindlimb vasculature with Microfil, a silicone radiopaque casting agent. Microfil can perfuse and fill the entire vascular bed (arterial and venous), and because we have ligated the major vascular conduit for exit, the agent can be retained in the vasculature for future ex vivo imaging with the use of small specimen micro-CT3.

We show here the in vivo insertion of an osmotic pump for constant local drug delivery and the creation of hindlimb ischemia in a mouse model. Moreover, the hindlimb vasculature is perfused with Microfil, a silicone radiopaque agent, to prepare for micro-computed tomography (micro-CT) imaging.

药物输送到微型计算机断层扫描成像缺血后肢血管灌注与微滤渗透

Preclinical research in animal models of peripheral arterial disease plays a vital role in testing the efficacy of therapeutic agents designed to ...

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the actual molecules towards which the cells migrate remain elusive. This modified invasion assay has been developed to identify targets which drive cell migration and invasion. This technique compares the invasion index under three conditions to determine whether a specific hormone, growth factor, or cytokine plays a role in mediating the invasive potential of a cancer cell. These conditions include i) normal fetal bovine serum (FBS), ii) charcoal-stripped FBS (CS-FBS), which removes hormones, growth factors, and cytokines and iii) CS-FBS + molecule (denoted &#8220;X&#8221;). A significant change in cell invasion with CS-FBS as compared to FBS, indicates the involvement of hormones, cytokines or growth factors in mediating the change. Individual molecules can then be added back to CS-FBS to assay their ability to reverse or rescue the invasion phenotype. Furthermore, two or more factors can be combined to evaluate the additive or synergistic effects of multiple molecules in driving or inhibiting invasion. Overall, this method enables the investigator to determine whether hormones, cytokines, and/or growth factors play a role in cell invasion by serving as chemoattractants or inhibitors of invasion for a particular type of cancer cell or a specific mutant. By identifying specific chemoattractants and inhibitors, this modified invasion assay may help to elucidate signaling pathways that direct cancer cell invasion.

A Modified In vitro Invasion Assay to Determine the Potential Role of Hormones, Cytokines and/or Growth Factors in Mediating Cancer Cell Invasion

This video article describes a modified in vitro method to examine the role of hormones, cytokines and/or growth factors in driving cancer cell invasion.

一种改进<em&gt;在体外</em&gt;入侵检测，以确定激素，在调节肿瘤细胞侵袭的细胞因子和/或生长因子的潜在作用

Blood serum serves as a chemoattractant towards which cancer cells migrate and invade, facilitating their intravasation into microvessels. However, the ...

Assessment of Ovarian Cancer Spheroid Attachment and Invasion of Mesothelial Cells in Real Time

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not accurately model the behaviors of cancer cells within a nonadherent environment, as cancer cells inherently aggregate into multicellular structures which contribute to the metastatic process by attaching to and invading the peritoneal lining to form secondary tumors. To model this important stage of ovarian cancer metastasis, multicellular aggregates, or spheroids, can be generated from established ovarian cancer cell lines maintained under nonadherent conditions. To mimic the peritoneal microenvironment encountered by tumor cells in vivo, a spheroid-mesothelial co-culture model was established in which preformed spheroids are plated on top of a human mesothelial cell monolayer, formed over an extracellular matrix barrier. Methods were then developed using a real-time cell analyzer to conduct quantitative real time measurements of the invasive capacity of different ovarian cancer cell lines grown as spheroids. This approach allows for the continuous measurement of invasion over long periods of time, which has several advantages over traditional endpoint assays and more laborious real time microscopy image analyses. In short, this method enables a rapid, determination of factors which regulate the interactions between ovarian cancer spheroid cells invading through mesothelial and matrix barriers over time.

Ovarian cancer cell invasion into the mesothelial lining of the peritoneum is a dynamic process over time. Utilizing a real time analyzer, the invasive capacity of ovarian cancer cells in a spheroid-mesothelial cell co-culture model can be quantified over prolonged time periods, providing insights into factors regulating the metastatic process.

在实时的间皮细胞卵巢癌球体附件和入侵评估

Ovarian cancers metastasize by shedding into the peritoneal fluid and dispersing to distal sites within the peritoneum. Monolayer cultures do not ...

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 ...

Utilization of the Soft Agar Colony Formation Assay to Identify Inhibitors of Tumorigenicity in Breast Cancer Cells

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation assay (hereafter called soft agar assay), which measures the ability of cells to proliferate in semi-solid matrices, remain a hallmark of cancer research. A key advantage of this technique over conventional 2D monolayer or 3D spheroid cell culture assays is the close mimicry of the 3D cellular environment to that seen in vivo. Importantly, the soft agar assay also provides an ideal tool to rigorously test the effects of novel compounds or treatment conditions on cell proliferation and migration. Additionally, this assay enables the quantitative assessment of cell transformation potential within the context of genetic perturbations. We recently identified peptidylarginine deiminase 2 (PADI2) as a potential breast cancer biomarker and therapeutic target. Here we highlight the utility of the soft agar assay for preclinical anti-cancer studies by testing the effects of the PADI inhibitor, BB-Cl-amidine (BB-CLA), on the tumorigenicity of human ductal carcinoma in situ (MCF10DCIS) cells.

Here, we document the use of the soft agar colony formation assay to test the effects of a peptidylarginine deiminase (PADI) enzyme inhibitor, BB-Cl-amidine, on breast cancer tumorigenicity in vitro.

软琼脂集落形成实验利用识别的致瘤性乳腺癌细胞抑制剂

Given the inherent difficulties in investigating the mechanisms of tumor progression in vivo, cell-based assays such as the soft agar colony formation ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

研究胰腺癌干细胞特性开发新的治疗策略

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

从小鼠癌症模型的循环肿瘤细胞的分离和繁殖

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer ...

Cutaneous Surgical Denervation:  A Method for Testing the Requirement for Nerves in Mouse Models of Skin Disease

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies have suggested that nerves may also modulate skin disorders including atopic dermatitis, psoriasis and cancer. Here, we describe protocols for testing the requirement for nerves in maintaining a cutaneous mechanosensory organ, the touch dome (TD). Specifically, we discuss methods for genetically labeling, harvesting and visualizing TDs by whole-mount staining, and for performing unilateral surgical denervation on mouse dorsal back skin. Together, these approaches can be used to directly compare TD morphology and gene expression in denervated as well as sham-operated skin from the same animal. These methods can also be readily adapted to examine the requirement for nerves in mouse models of skin pathology. Finally, the ability to repeatedly sample the skin provides an opportunity to monitor disease progression at different stages and times after initiation.

This article includes detailed protocols for genetic labeling of mouse skin, surgical denervation, skin biopsy and visualizing labeled epithelia by whole-mount &#946;-galactosidase staining. These methods can be used to test the requirement for nerves in mouse models of normal and pathological skin.

皮肤去神经外科手术:一种用于皮肤病的小鼠模型测试要求的神经方法

Cutaneous somatosensory nerves function to detect diverse stimuli that act upon the skin. In addition to their established sensory roles, recent studies ...