这项研究是由T32（CA 9686-18）和TL1（TL1TR001431）博士后培训给予奖励（LT），DOD PC140268（CA），W81XWH-13-1-0327（CA）TR000102-04（CA），以及支持U01 PAR-12-095（库马尔）和P30 CA051008-21（韦纳）。样品固定，切片和染色是在隆巴迪综合癌症中心的组织学与组织共享资源进行。我们感谢理查德·施莱格尔有益的讨论。内容完全是作者的责任，并不一定代表美国国家癌症研究所和美国国立卫生研究院的官方意见。

1.三维细胞培养建立插入系统<ol><li>放置在倒置方向（滤波器侧上）的6孔板（ 图1A）向下2聚碳酸酯细胞培养插入。 </li><li>在水施加0.1％明胶的薄层于刀片的底侧并晾干（10-20分钟）在生物安全柜以保持无菌。 </li><li>重复步骤1.2，共上刀片0.1％明胶的3应用程序两次以上。 注:明胶涂层将有助于室之间的限制扩散。 </li><li>为了收集从标准培养条件的主要社区康复中心，加入5毫升的PBS洗培养瓶中。 <ol><li> Trypsinize使用细胞（1毫升为一个T-25和2mL的T-75）0.25％胰蛋白酶-EDTA在37℃5分钟。轻轻拍打烧瓶侧面中撞出的细胞提供帮助。然后继续以停止胰蛋白酶反应，并通过添加完全DMEM的5毫升收集细胞。 </li><li>搜集<html>将细胞在15毫升的管中。离心管中，在4℃和188 XG和使用自动细胞计数器或血细胞计数器计数。 </li><li>重悬在250微升的条件培养基（CM）60万的CRC，并添加到正确定向（滤波器面朝下）培养插入物（ 图1B）。这被称为内室。 注意:CM是F-媒体通过照射J2细胞的条件和包含10微米Y-27632如前所述7，8，9，11。 </li></ol></li><li>应用2毫升的CM到6孔板的外室，并在37℃下孵育过夜（至少18小时）。 </li><li>小心地用一个1毫升或200微升吸管除去在CM上的内室和缓慢分化培养基（DM）中添加用1毫升吸移管内室满为止。小心不要扰乱细胞层。 </li></ol>乐"&gt; 2，3D文化的维护<ol><li>检查文化的每一天。健康的细胞出现， 如图2。 </li><li>改变每天或每隔一天取决于细胞的代谢中的外室中的CM。 </li><li>当内腔室改变颜色（黄色）的媒体，小心地取出在内部过滤器室用1毫升或200微升吸管介质。 </li><li>吸媒体在外6孔板室与吸气提示。 </li><li>用1毫升吸管，新鲜DM慢慢地应用到内室满为止。要小心，不要刮或以其他方式打跑细胞层。 </li><li>用2mL新鲜的CM代替在外部腔室中的介质。 </li><li>维持培养2周，然后进行处理所需的双分子隔离。 注意:在6孔板中的每一行，共六个刀片（ 图1B），应每实验处理，以获得足够的细胞DNA，RNA或蛋白质进行分析。 </li><li>在外部腔室抽吸介质，并用2毫升磷酸盐缓冲盐水（PBS）冲洗。 </li><li>小心地从用1毫升或200微升吸管内腔取出介质，并添加500微升PBS中。避免刮擦或以其它方式干扰在膜上的细胞。 </li><li>从外室抽吸PBS中并小心地从内室除去的PBS与1毫升或200微升吸管。一旦PBS已被去除，直接进行以下详述的适当的应用程序。不要让膜干燥。文化可以保持简单的PBS，直到应用程序已经准备好开始。 </li></ol> 3.加工球团银行业的过滤器<ol><li> 100μL的0.25％胰蛋白酶-EDTA添加到每个内室，并在37℃下孵育5分钟。 </li><li>简单地说，仔细搅拌/刮膜表面的细胞用200微升吸管吸取，同时向上和向下（避免PUNC图灵膜）。在37℃下孵育1分钟。 </li><li>添加的CM 250微升到第一内室和吸取上下轻轻冲洗过滤器。 </li><li>转移悬浮细胞至包含相同的媒体的条件并重复上下吹打相邻过滤室。 </li><li>重复此过程为每个单一条件的刀片。收集并转移细胞到1.5mL离心管中。 </li><li>每次冲洗内腔与CM额外100-200微升收集任何剩余的细胞。添加到1.5毫升离心管中，在步骤3.5。 </li><li>旋在423 xg离心所述细胞在微量在4℃下5分钟。 </li><li>吸出上清液并用1000微升的PBS洗一次细胞沉淀。 </li><li>在423 xg离心再次旋在微量在4℃下5分钟。 </li><li>抽吸PBS中并在-80℃以备后用冷冻。 </li></ol> 4.处理RNA或DNA分离过滤器<ol&gt; <li>添加硫氰酸胍 - 苯酚 - 氯仿或类似的提取试剂的200μL到内室和通过上下吹打简要地搅动膜表面上的细胞。 </li><li> 孵育在提取试剂，在室温下5分钟与外室干。 <ol><li>作为过滤器可以从插入件分离，通过上下吹打提取液洗解除关联过滤膜。通过倾斜6孔板并且吸取残留液体收集尽可能多的提取试剂成为可能。 </li></ol></li><li>按照制造商的净化和DNA，RNA或蛋白回收协议。 </li></ol> 5.处理蛋白质分离过滤器<ol><li>放置在冰上6孔板的过滤器插入件。到内室中，添加10微升补充有1mM的氟化钠（氟化钠），1mM的钒酸钠（NAV），100毫dithiothre蛋白质裂解缓冲液itol醇（DTT）和100微升抗蛋白酶鸡尾酒。 </li><li>搅动/刮去膜表面上的细胞用20微升吸管而上下吹吸和。避免在裂解液产生气泡。 </li><li>孵育滤芯在冰上6孔板10分钟。 </li><li>重复刮，然后用清水冲洗裂解液膜表面。 </li><li>从6插入收集裂解物并转移到1.5mL离心管在冰上。 </li><li>与另外的10μL的裂解缓冲液并转移到下一个过滤器冲洗所述第一膜。 </li><li>重复步骤5.6所有插入，收集任何残留的裂解缓冲液，并加入到1.5mL管中（步骤5.5）。 </li><li>在冰上孵育样品额外5分钟。 </li><li>自旋以最大速度（16873 XG在台式离心机）在4℃下15分钟。 </li><li>吸取裂解上清液到一个新的1.5毫升管。 </li><li>继续蛋白浓度或申通快递的分析再在-80℃的裂解液。 </li></ol> 6.处理H＆E和免疫染色<ol><li> 500微升和1毫升10％NBF（中性缓冲的福尔马林）添加到内和外室，并让在4℃孵育过夜。 </li><li>从外室吸出NBF并添加1毫升羟乙基琼脂糖（HEA）处理凝胶到1.5mL离心。使用低功率慢慢融化琼脂糖在微波炉和反复10-20小号脉冲直到琼脂糖融化。 </li><li>保持HEA在37℃的温水澡，防止凝固，直到准备使用。 </li><li>从内腔中取出的NBF和应用熔融HEA的25μL到内室，并允许琼脂糖固化2-5分钟。 </li><li>在10％NBF湿2泡沫垫组织学和放一个垫在包埋盒（ 见图3D）。 </li><li>使用＃11刀片的手术刀（其具有非常尖锐的，细尖头刀）从刀片的底侧得分滤波器腔室，部分地从塑料插入腔室释放它。 </li><li>放置NBF少量（100-200微升）在培养皿中并浸泡在NBF（ 图3A）的部分移出过滤器。 </li><li>使用＃10刀片的手术刀，轻轻压靠在过滤器的中间，从插入室的内部，以充分去除从插入筒（ 图3B）的滤波器。如果存在仍连接到筒的过滤器的任何部分，用手术刀断绝它。 </li><li>切HEA涂层过滤器的一半与＃10手术刀刀片（ 图3C）。 </li><li>过滤器的每一半放置到在步骤6.4（ 图3D）中制备的组织学暗盒中泡沫垫。 </li><li>第二个10％NBF浸泡海绵添加到盒，夹在中间的滤光器。 </li><li>捕捉封住NBF纸盒，地点和孵育过夜。 </li><li>流程过滤器石蜡块切片和染色以前12描述。 </li></ol>

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原代细胞系是癌症研究的一个重要的，迅速发展的平台。基于插入培养三维培养系统支持原发性前列腺CRC的为期两周的时间内的分化。该CRC滤波方法代表前列腺研究一个新的，中等吞吐量方法。现有鼠标PDX模型是耗时且极其昂贵，并且许多对PDX样品不能在培养物中生长，限制研究者发起实验和它们的用途。此外，同时建立PDX的成功率相差很大13中，CRC方法具有成功的建立细胞系3的比率很高。我们认为，我们的系统是基于R-脊椎前列腺类器官的做法互补;然而，关于建立从小学前列腺癌组织体缺乏成功的已报告14。此外，媒体，建立方法，和CRC的维护比对R-脊椎基于前列腺类器官的方法显著简单。作为一个额外的好处，两个CRC可以建立和用于测试直接分子比较匹配的正常和肿瘤培养物。 一些技术问题仍然建立这一系统时予以解决。首先，明胶包衣施加到过滤器插入件帮助的下侧，但并没有消除，穿过膜的媒体的扩散。频繁更换培养基用于维持分化（上部或顶电池层） 与增长的各梯度（细胞较低或基础层）的室中的条件。第二，成功的3D CRC培养所需要的相对高的细胞接种密度。下接种密度，得到差的结果与问候，该过滤器表面上发展了细胞层的完整性。这是一种常见的考虑培养社区康复中心的时候，当保持着他们成长最大力T高细胞密度。最后，合适的去除和完整细胞层的随后石蜡包埋是用于限定细胞分化的程度是至关重要的。加入羟乙基琼脂糖（HEA）的均降低信元丢失和改进相比，非嵌入式滤波器单元的整体形态（未示出）。一旦正确嵌入，将滤膜切片并在从典型的细胞和组织样品没有区别的方式进行处理。 我们才刚刚开始效仿采用这种滤波方法前列腺上皮的结构。其他修改的培养条件下，向媒体和基底是必要的，这可以容易地解决。例如，重新分化媒体，可能会更好地促进管腔VS基础分化可以快速测试。由于CRC的慢病毒感染已与商业癌细胞7和2D的CRC进行的轻松拥有人这样被转染CAS9 / CRISPR基因编辑技术向量永久修改细胞基因组中（数据未示出），的突变，缺失或修复基因可以被测试的效果。同样，谱系追踪可以通过引入谱系特异性的报告基因成为可能。 除了改变介质或细胞，修改到过滤器表面也可以进行测试。不同的过滤膜的基材是可商购。此外，我们已发现当施加到过滤器的内部，商业细胞外基质提供合适的生长环境。作为插入系统的目标之一是前列腺微环境更好的模型，也许是最有趣的修改可能是引入正常或肿瘤衍生的基质细胞，无论是在共培养与插入的限制或范围内的CRC在底室以调节生长培养基。修改插入很好地部分Ø˚F脱细胞的前列腺组织也是可能的。在这种方法中，基质/上皮相互作用可能允许初级细胞使用去细胞的组织作为用于生长的支架。 建立的初级细胞并随后提供其分化的条件的能力，对于多种生物相关的研究进入正常腺发育和用于癌症研究的理想格式。本文所描述的系统提供了一个平台，由该细胞类型和修改的数量不受限制，可以合并到该系统调整向各个实验的需要和可靠地收集试样进行分析。

这是个性化的个人识别和使用的癌症疗法在癌症研究的一个主要目标。最近，新的方法已经开发了允许在建立原代细胞培养物的更易于，潜在地同时提供识别和检验的个性化疗法的方法。例如，在基于R-脊椎前列腺类器官的方法1可用于三维（3D）正常和转移性前列腺癌的细胞在商业细胞外基质的培养（ 例如 ，基质胶），而细胞的有条件重新编程（CRC）的方法在乔治城2开发，利用3以上标准的2D培养条件。具体而言，Rho激酶抑制剂（Y-27632）和辐照J2小鼠成纤维细胞饲养细胞的组合导致角质形成细胞的CRC 2的无限期培养。在CRC的方法是极为坚固，与原代细胞系成功建立，并从前列腺和其他许多正常和恶性上皮组织3无限期地保持下去。重要的是，允许在谁没有一些以前的药物治疗的患者复发性呼吸道乳头状病原学基础的快速识别我们的CRC技术。此外，使用正常和肿瘤来源的CRC，FDA的成功鉴定批准的药物，伏立诺他，二周初始组织活检的内作出。病人被放置在伏立诺他，导致他们的疾病4的成功治疗。 正常的前列腺是由鲁米那，基底和罕见的神经内分泌细胞5。管腔细胞形成腺体的柱状上皮层和表达雄激素受体（AR），以及其他管腔标记物如角蛋白8和18和亲状态特定抗原（PSA）6。相反，基底细胞是局部管腔层下方，表达细胞角蛋白5和p63，但AR 5的低水平。我们7，8，9和其他10已成功地应用于前列腺康复中心在临床前机械药敏调查。然而，当标准的2D组织培养条件下生长，这些细胞不能完全啮合AR信令10。重要的是，当免疫缺陷小鼠的肾包膜下放置两个CRC恢复正常前列腺腺体结构和功能说明放置在一个宽容的环境，当前列腺康复中心保留其血统的承诺。此处所描述的基于插入滤波器的细胞培养系统的发展允许快速（2周） 在体外的前列腺CRC的分化所证明by中的Ar和Ar靶基因的表达增加以及降低p63的水平。 使用的过滤器插入件包含聚碳酸酯膜（孔径0.4微米），可支持的哺乳动物细胞的培养。该系统，为开发，利用正常的和恶性的前列腺康复中心，6孔培养皿和滤芯的。通过J2单元11调节的细胞培养基被放置在底室和前列腺区分媒体在顶室中。本文描述的技术支持2周的时间内前列腺管腔细胞分化，与个性化药物的目标相一致。它也必须开发出允许培养物的全面分子，遗传和细胞分析的方法。用于从过滤表面释放的细胞中分离DNA，RNA和蛋白质的方法已被开发并简化为准确重复样品的处理。菲娜LLY，需要取下过滤网，使嵌入，切片和H＆E，免疫组化及免疫荧光染色的方法，充分说明。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Corning Costar Snapwell Culture Inserts</td>
			<td>Corning</td>
			<td>3801</td>
		</tr>
		<tr>
			<td>Millicell Cell Culture Inserts</td>
			<td>Millipore</td>
			<td>PIHP01250</td>
		</tr>
		<tr>
			<td>Multiwell 6 Well</td>
			<td>Falcon</td>
			<td>353046</td>
		</tr>
		<tr>
			<td>Gelatin 0.1% in water</td>
			<td>Stemcell Technologies</td>
			<td>7903</td>
		</tr>
		<tr>
			<td>Sterile Saftey Scapel 10 Blade</td>
			<td>Integra Miltex</td>
			<td>4-510</td>
		</tr>
		<tr>
			<td>Sterile Standard Scalpel 11 Blade</td>
			<td>Integra Miltex</td>
			<td>4411</td>
		</tr>
		<tr>
			<td>RIPA Lysis and Extraction Buffer</td>
			<td>Thermofisher Scientific</td>
			<td>89900</td>
		</tr>
		<tr>
			<td>Sodium Fluoride</td>
			<td>Fishcer Scientific</td>
			<td>S299-100</td>
		</tr>
		<tr>
			<td>Sodium Vanadate</td>
			<td>Fishcer Scientific</td>
			<td>13721-39-6</td>
		</tr>
		<tr>
			<td>Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td>3483123</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail (AEBSF, Aprotinin, Bestatin, E-64, Leupeptin and Pepstatin A)</td>
			<td>Sigma-Aldrich</td>
			<td>P8340</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA (1x)</td>
			<td>Thermofisher Scientific</td>
			<td>25200-056</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermofisher Scientific</td>
			<td>11965-092</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma-Aldrich</td>
			<td>F2442</td>
		</tr>
		<tr>
			<td>Glutamine</td>
			<td>Thermofisher Scientific</td>
			<td>25030081&#160;</td>
		</tr>
		<tr>
			<td>Penicillin Streptomycin</td>
			<td>Thermofisher Scientific</td>
			<td>15140-122</td>
		</tr>
		<tr>
			<td>F-12 Nutrient Mix Media</td>
			<td>Thermofisher Scientific</td>
			<td>11765054</td>
		</tr>
		<tr>
			<td>Y-27632 dihydrochloride Rock inhibitor</td>
			<td>Enzo</td>
			<td>ALX-270-333-M025</td>
		</tr>
		<tr>
			<td>Hydrocortisone</td>
			<td>Sigma-Aldrich</td>
			<td>H0888</td>
		</tr>
		<tr>
			<td>EGF</td>
			<td>Thermofisher Scientific</td>
			<td>PHG0315</td>
		</tr>
		<tr>
			<td>Insulin</td>
			<td>Thermofisher Scientific</td>
			<td>12585-014</td>
		</tr>
		<tr>
			<td>Cholera Toxin</td>
			<td>Sigma-Aldrich</td>
			<td>C3012</td>
		</tr>
		<tr>
			<td>Gentamicin</td>
			<td>Thermofisher Scientific</td>
			<td>15710-064</td>
		</tr>
		<tr>
			<td>Fungizone</td>
			<td>Fisher Scientific</td>
			<td>BP264550</td>
		</tr>
		<tr>
			<td>Trizol Reagent</td>
			<td>Invitrogen</td>
			<td>15596026</td>
		</tr>
		<tr>
			<td>Histogel Specimen Processing Gel (hydroxyethyl agarose (HEA))</td>
			<td>Thermo Scientific</td>
			<td>HG-4000-012</td>
		</tr>
		<tr>
			<td>Non-Adherent Dressing</td>
			<td>Telfa</td>
			<td>KDL2132Z</td>
		</tr>
		<tr>
			<td>Cell Culture Dish</td>
			<td>Sigma</td>
			<td>SIAL0167</td>
		</tr>
		<tr>
			<td>HISTOSETTE II Tissue Processing / Embedding Cassettes</td>
			<td>Crystalgen</td>
			<td>CG-M492</td>
		</tr>
	</tbody>
</table>

a rapid filter insert-based 3d culture system for primary prostate cell differentiation

有条件地重新编程细胞（CRC）的为原代细胞培养，并制定患者的细胞系广泛的"活生物银行"的能力，可持续发展的方法。对于许多类型的上皮细胞，各种三维（3D）培养的方法已经被描述了支持改进的分化状态。虽然结直肠癌保留他们的后裔承诺从他们被隔离的组织，他们无法在正常二维（2D）培养条件下生长，以表达许多与起源组织相关的分化标志。以增强患者来源CRC的前列腺癌研究中的应用，三维培养格式已被定义，它使一个快速（2周总）管腔细胞在正常和肿瘤衍生的前列腺上皮细胞的分化。这里，基于插入滤波器格式为正常和恶性前列腺CRC的培养和分化说明。一德提供的细胞收集和处理的免疫组化及免疫荧光染色所需的程序尾描述。总体描述的3D格式的文化，与主CRC线相结合，提供了重要的中期和高通量的模型系统基于生物样本前列腺的研究。

细胞培养插管的系统是用于产生支持管腔细胞分化前列腺CRC的3D培养相对简单和快速的过程。该系统的原理图（ 图1A）突出明胶涂层的过滤器插入件的底表面上的应用。该刀片只为推翻明胶的应用。在图1B中的过滤器是在用于培养适当的取向。使用透明细胞插入物，一个健康的三维前列腺CRC文化的代表图像显示（10X）（ 图2）。健康CRC中表现出的密集分层是成立后的典型，可以使用标准光学显微镜观察。在最初的建立，这是至关重要的可视化形成的，以确保该方法是与给定的细胞系相容的细胞的层，并且适当附着和拉时有发生的细胞优伶。 如上所述HEA施加到插入件的内部。的HEA的应用程序后，必须小心得分过滤器从所述插入物（ 图3A，3B）将其删除时作出。在所有的步骤（ 图3A-3D），护理也必须行使在包含细胞滤波器最小化HEA层的不必要的移动。在CRC层可以容易地破碎，并转移到H＆E盒（ 图3D）期间被损坏。从塑料桶（ 图3A-3C）的HEA涂层的过滤器的切除后，过滤器可以减半并用于切片，并使用标准嵌入盒（ 图3D）染色处理。在半分割过滤器是很重要的最大化可用表面上H＆E和IF横切。 免疫荧光染色以及明场（BF）采用与DSU（磁盘扫描单元）旋转盘共聚焦能力显微镜收集过滤层上培养的细胞的图像。用于组织学和H＆E染色的代表性结果示于滤芯和细胞（20×）（ 图4）的横截面。有了适当的照顾，我们证明了在滤芯的CRC层可以被切片，染色，因为是组织组织学的标准做法。期间切片，它是可能的CRC码来从过滤器作为细胞的完整层所证明（ 图4）成为分离。高炉图像显示在多孔膜（ 图5A）的顶部的多层细胞地层。为核的各个荧光图像（DAPI， 图5B），p63蛋白（ 图5C）和AR（ 图5D）中示出，为的是所有三种荧光标记物（ 图5E）的合并。最后，组合大ËBF和IF所示覆盖（ 图5F），建立相对细胞和过滤器的荧光信号的定位。在DAPI染色与P63的覆盖IF染色清楚地表明部分细胞仍处于增殖状态。 AR的本地化IF染色细胞核表明在前列腺细胞AR功能激活的。 我们的过滤器插入件系统的IF和一个切片前列腺组织之间的比较示出（ 图6A，6B）在我们的CRC插入层的发展的相似性表明该前列腺上皮。前列腺腺管显示P63表达，前列腺基底细胞一致。 p63的染色也可见于在插入所述的CRC。在我们插入观察到更高的百分比P63表达细胞较完整的组织部分。此外，无论是细胞核和细胞质AR被认为在前列腺炎<html>告部和，而在过滤器滤芯节目少整体的AR表达，这两种核AR表达和细胞质染色的CRC所看到的，相似于完整前列腺。 <img alt="图1" src="/files/ftp_upload/55279/55279fig1.jpg" /> 图1:6孔培养皿并插入包含3D文化系统的典型形象。 （A）的倒置插入施加明胶涂层的过滤器（箭头所示）的下侧。插入和过滤器的放大图像显示是（插图）。 （ 二 ）妥善安置插入。该系统的内部和外部室被示出（箭头）。 B中的盒装行显示的样本如何多一个给定的实验条件得到。在2周生长期结束这些刀片可以被合并，以产生足够的材料进行分析。"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/55279/55279fig2.jpg" /> 图2:健康的CRC去籽的在透明滤膜图层的代表图像显示。内和外腔中包含的条件培养基。 <a href="http://ecsource.jove.com/files/ftp_upload/55279/55279fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/55279/55279fig3.jpg" /> 图3:减半划分为两部分，并放入盒石蜡嵌入过滤器的形象代表。 （A）过滤与HEA涂层进球后和清除前插入。 （B）选自聚碳酸酯筒所释放的过滤器插入件。 （ <st荣&gt; C）的正确减半过滤器。 （D）的适当放置和包埋盒的内部的两半的取向。 <a href="http://ecsource.jove.com/files/ftp_upload/55279/55279fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/55279/55279fig4.jpg" /> 图4:过滤器滤芯和细胞的代表性H＆E染色横截面（20X）。两个膜（底部）和细胞层（顶部）被示出。 <a href="http://ecsource.jove.com/files/ftp_upload/55279/55279fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/55279/55279fig5.jpg" /> 图5:代表明场（BF）和免疫荧光（IF）的系列图像细胞切片式的过滤器（40X）。在过滤器和电池的横截面被示出。过滤表面（5A）上的细胞的未染色BF图像是伴随着对DAPI染色细胞核（5B）和免疫荧光染色为两种不同的蛋白质，p63蛋白（5C）和雄激素受体（AR，5D）图像。小区段（5E，F）的复合覆盖定义了细胞和过滤器的上下文中，IF信号的定位。 <a href="http://ecsource.jove.com/files/ftp_upload/55279/55279fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图6" src="/files/ftp_upload/55279/55279fig6.jpg" /> 图6:剖开的筛选与前列腺上皮组织从一科（20X）细胞的免疫代表（IF）的图像。我们的过滤器插入件的横截面图像中示出（图6A）相比，一个完整的前列腺（图6B）的导管上皮层。 p63蛋白的染色，AR和细胞核如图5中进行。 <a href="http://ecsource.jove.com/files/ftp_upload/55279/55279fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation at JoVE.com

A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

基于插入 - 滤池3D文化系统原发性前列腺细胞分化

Conditionally reprogrammed cells (CRCs) provide a sustainable method for primary cell culture and the ability to develop extensive &#34;living ...

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Nanyang Technological University

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Cancer Research

8.5K 次观看.  Lombardi Comprehensive Cancer Center. Here, we present a method for the establishment of a rapid in vitro system that supports the three dimensional culturing and subsequent luminal differentiation of primary prostate epithelial cells.

视频: A Rapid Filter Insert-based 3D Culture System for Primary Prostate Cell Differentiation

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma (DIPG)

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a viable treatment strategy and biopsy is not routinely performed, the availability of patient samples for research is limited. Consequently, efforts to study this disease have been challenged by a paucity of faithful disease models. To address this need, we describe here a protocol for the rapid processing of post-mortem autopsy tissue samples in order to generate durable patient-derived cell culture models that can be used in in vitro assays or in vivo orthotopic xenograft experiments. These models can be used to screen for potential drug targets and to study fundamental pathobiological processes within DIPG. This protocol can further be extended to analyze and isolate tumor and microenvironmental cells using Fluorescence-activated Cell Sorting (FACS), which enables subsequent analysis of gene expression, protein expression, or epigenetic modifications of DNA at the bulk cell or single cell level. Finally, this protocol can also be adapted to generate patient-derived cultures for other central nervous system tumors.

A Protocol for Rapid Post-mortem Cell Culture of Diffuse Intrinsic Pontine Glioma DIPG

This protocol describes a method for the rapid processing of post-mortem diffuse intrinsic pontine glioma samples for the establishment of patient-derived cell culture models or direct characterization of tumor and microenvironmental cells.

一种内在的弥漫性脑胶质瘤脑桥快速验尸细胞培养协议（DIPG）

Diffuse Intrinsic Pontine Glioma (DIPG) is a childhood brainstem tumor that carries a universally fatal prognosis. Because surgical resection is not a ...

科研

癌症研究

Establishment of a Primary Culture of Patient-derived Soft Tissue Sarcoma

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more than 50 histological subtypes of these rare solid tumors have been identified. Thus, due to their extraordinary diversity and low incidence, our understanding of the biology of these tumors is still limited. Patient-derived cultures represent the ideal platform to study STS pathophysiology and pharmacology. We thus developed a human preclinical model of STS starting from tumor specimens harvested from patients undergoing surgical resection. Patient-derived STS cell cultures were obtained from the surgical specimens by collagenase digestion and isolated by filtration. Cells were counted, seeded, and left for 14 days in standard monolayer cultures and then processed by downstream analysis. Before performing molecular or pharmaceutical analyses, the establishment of STS primary cultures was confirmed through the evaluation of cytomorphologic features and, when available, immunohistochemical markers. This method represents a useful tool 1) to study the natural history of these poorly explored malignancies and 2) to test the effects of different drugs in an effort to learn more about their mechanisms of action.

The following protocol focuses on the establishment of a primary culture of patient-derived soft tissue sarcoma (STS). This model could help us to better understand the molecular background and pharmacological profile of these rare malignancies and could represent a starting point for further research aimed at improving STS management.

病人衍生软组织肉瘤原代培养的建立

Soft tissue sarcomas (STS) represent a spectrum of heterogeneous malignancies with a difficult diagnosis, classification, and management. To date, more ...

Therapy Testing in a Spheroid-based 3D Cell Culture Model for Head and Neck Squamous Cell Carcinoma

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with or without surgery. While platinum-based chemotherapy regimens currently represent the gold standard in terms of efficacy and are given in the vast majority of cases, new chemotherapy regimens, namely immunotherapy are emerging. However, the response rates and therapy resistance mechanisms for either chemo regimen are hard to predict and remain insufficiently understood. Broad variations of chemo and radiation resistance mechanisms are known to date. This study describes the development of a standardized, high-throughput in vitro assay to assess HNSCC cell line&#39;s response to various therapy regimens, and hopefully on primary cells from individual patients as a future tool for personalized tumor therapy. The assay is designed to being integrated into the quality-controlled standard algorithm for HNSCC patients at our tertiary care center; however, this will be subject of future studies. Technical feasibility looks promising for primary cells from tumor biopsies from actual patients. Specimens are then transferred into the laboratory. Biopsies are mechanically separated followed by enzymatic digestion. Cells are then cultured in ultra-low adhesion cell culture vials that promote the reproducible, standardized and spontaneous formation of three-dimensional, spheroid-shaped cell conglomerates. Spheroids are then ready to be exposed to chemo-radiation protocols and immunotherapy protocols as needed. The final cell viability and spheroid size are indicators of therapy susceptibility and therefore could be drawn into consideration in future to assess the patients&#39; likely therapy response. This model could be a valuable, cost-efficient tool towards personalized therapy for head and neck cancer.

We describe the evolution of a spheroid-based, three-dimensional in vitro model that enables us to test the current standard of experimental therapy regimens for head and neck squamous cell carcinoma on cell lines, aiming at evaluating therapy susceptibility and resistance on primary cells from human specimens in the future.

基于球形的头颈部鳞状细胞癌3D 细胞培养模型的治疗试验

Current treatment options for advanced and recurrent head and neck squamous cell carcinoma (HNSCC) enclose radiation and chemo-radiation approaches with ...

Virus Delivery of CRISPR Guides to the Murine Prostate for Gene Alteration

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) for prostate cancer are hampered by tumor heterogeneity and its complex microevolution dynamics. Traditional prostate cancer mouse models include, amongst others, germline and conditional knockouts, transgenic expression of oncogenes, and xenograft models. Generation of de novo mutations in these models is complex, time-consuming, and costly. In addition, most of traditional models target the majority of the prostate epithelium, whereas human prostate cancer is well known to evolve as an isolated event in only a small subset of cells. Valuable models need to simulate not only prostate cancer initiation, but also progression to advanced disease.
Here we describe a method to target a few cells in the prostate epithelium by transducing cells by viral particles. The delivery of an engineered virus to the murine prostate allows alteration of gene expression in the prostate epithelia. Virus type and quantity will hereby define the number of targeted cells for gene alteration by transducing a few cells for cancer initiation and many cells for gene therapy. Through surgery-based injection in the anterior lobe, distal from the urinary track, the tumor in this model can expand without impairing the urinary function of the animal. Furthermore, by targeting only a subset of prostate epithelial cells the technique enables clonal expansion of the tumor, and therefore mimics human tumor initiation, progression, as well as invasion through the basal membrane.
This novel technique provides a powerful prostate cancer model with improved physiological relevance. Animal suffering is limited, and since no additional breeding is required, overall animal count is reduced. At the same time, analysis of new candidate genes and pathways is accelerated, which in turn is more cost efficient.

This protocol describes a newly established method for virus delivery to the murine prostate. Using either CRISPR/Cas9 technology, gene overexpression, or Cre recombinase delivery, the technique allows orthotopic alteration of gene expression and implements a novel mouse model for prostate cancer.

CRISPR 指南对小鼠前列腺病毒传递的基因改变

With an increasing incidence of prostate cancer, identification of new tumor drivers or modulators is crucial. Genetically engineered mouse models (GEMM) ...

3-D Cell Culture System for Studying Invasion and Evaluating Therapeutics in Bladder Cancer

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. While 75% of bladder cancers are non-invasive and unlikely to metastasize, about 25% progress to an invasive growth pattern. Up to half of the patients with invasive cancers will develop lethal metastatic relapse. Thus, understanding the mechanism of invasive progression in bladder cancer is crucial to predict patient outcomes and prevent lethal metastases. In this article, we present a three-dimensional cancer invasion model which allows incorporation of tumor cells and stromal components to mimic in vivo conditions occurring in the bladder tumor microenvironment. This model provides the opportunity to observe the invasive process in real time using time-lapse imaging, interrogate the molecular pathways involved using confocal immunofluorescent imaging and screen compounds with the potential to block invasion. While this protocol focuses on bladder cancer, it is likely that similar methods could be used to examine invasion and motility in other tumor types as well.

The processes governing bladder cancer invasion represent opportunities for biomarker and therapeutic development. Here we present a bladder cancer invasion model which incorporates 3-D culture of tumor spheroids, time-lapse imaging and confocal microscopy. This technique is useful for defining the features of the invasive process and for screening therapeutic agents.

3维细胞培养系统研究膀胱癌侵袭和评价治疗方法

Bladder cancer is a significant health problem. It is estimated that more than 16,000 people will die this year in the United States from bladder cancer. ...

Identification, Histological Characterization, and Dissection of Mouse Prostate Lobes for In Vitro 3D Spheroid Culture Models

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate cancer (PCa). Understanding the anatomy and histology of the mouse prostate is important for the efficient use and proper characterization of such animal models. The mouse prostate has four distinct pairs of lobes, each with their own characteristics. This article demonstrates the proper method of dissection and identification of mouse prostate lobes for disease analysis. Post-dissection, the prostate cells can be further cultured in vitro for mechanistic understanding. Since mouse prostate primary cells tend to lose their normal characteristics when cultured in vitro, we outline here a method for isolating the cells and growing them as 3D spheroid cultures, which is effective for preserving the physiological characteristics of the cells. These 3D cultures can be used for analyzing cell morphology and behavior in near-physiological conditions, investigating altered levels and localizations of key proteins and pathways involved in the development and progression of a disease, and looking at responses to drug treatments.

Genetically engineered mice are useful models for investigating prostate cancer mechanisms. Here we present a protocol to identify and dissect prostate lobes from a mouse urogenital system, differentiate them based on histology, and isolate and culture the primary prostate cells in vitro as spheroids for downstream analyses.

体外3D 球形培养模型小鼠前列腺叶的鉴定、组织学特征及解剖

Genetically engineered mouse models (GEMMs) serve as effective pre-clinical models for investigating most types of human cancers, including prostate ...

A Three-dimensional Thymic Culture System to Generate Murine Induced Pluripotent Stem Cell-derived Tumor Antigen-specific Thymic Emigrants

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a promising source for adoptive T cell therapy (ACT). However, classical in vitro methods for producing regenerated T cells from iPSC result in either innate-like or terminally differentiated T cells, which are phenotypically and functionally distinct from na&#239;ve T cells. Recently, a novel three-dimensional (3D) thymic culture system was developed to generate a homogenous subset of CD8&#945;&#946;+ antigen-specific T cells with a na&#239;ve T cell-like functional phenotype, including the capacity for proliferation, memory formation, and tumor suppression in vivo. This protocol avoids aberrant developmental fates, allowing for the generation of clinically relevant iPSC-derived T cells, designated as iPSC-derived thymic emigrants (iTE), while also providing a potent tool to elucidate the subsequent functions necessary for T cell maturation after thymic selection.

This article describes a novel method to generate tumor antigen-specific induced pluripotent stem cell-derived thymic emigrants (iTE) by a three-dimensional (3D) thymic culture system. iTE are a homogenous subset of T cells closely related to na&#239;ve T cells with the capacity for proliferation, memory formation, and tumor suppression.

生成毛因诱导多能干细胞衍生的肿瘤抗原特异性胸腺移民的三维胸腺培养系统

The inheritance of pre-rearranged T cell receptors (TCRs) and their epigenetic rejuvenation make induced pluripotent stem cell (iPSC)-derived T cells a ...

Rapid Generation of Primary Murine Melanocyte and Fibroblast Cultures

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation defects and cancer. Vital to the understanding and amelioration of these diseases are experiments in primary fibroblast and melanocyte cultures. Nevertheless, current protocols for melanocyte isolation require that the epidermal and dermal layers of the skin are trypsinized and manually disassociated. This process is time consuming, technically challenging and contributes to inconsistent yields. Furthermore, methods to simultaneously generate pure fibroblast cultures from the same tissue sample are not readily available. Here, we describe an improved protocol for isolating melanocytes and fibroblasts from the skin of mice on postnatal days 0-4. In this protocol, whole skin is mechanically homogenized using a tissue chopper and then briefly digested with collagenase and trypsin. Cell populations are then isolated through selective plating followed by G418 treatment. This procedure results in consistent melanocyte and fibroblast yields from a single mouse in less than 90 min. This protocol is also easily scalable, allowing researchers to process large cohorts of animals without a significant increase in hands-on time. We show through flow cytometric assessments that cultures established using this protocol are highly enriched for melanocytes or fibroblasts.

This protocol outlines a rapid method to simultaneously generate melanocyte and fibroblast cultures from the skin of 0-4 day old mice. These primary cultures can be maintained and manipulated in vitro to study a variety of physiologically relevant processes, including skin cell biology, pigmentation, wound healing and melanoma.

初级毛质素黑色素细胞和纤维细胞培养的快速生成

Defects in fibroblast or melanocyte function are associated with skin diseases, including poor barrier function, defective wound healing, pigmentation ...

A 3D Spheroid Model as a More Physiological System for Cancer-Associated Fibroblasts Differentiation and Invasion In Vitro Studies

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor stroma, host fibroblasts are stimulated by cancer cells to differentiate. Thus, they acquire a phenotype that contributes to the tumor microenvironment and supports tumor progression. By using the spheroid model, we have set up such a 3D in vitro model system, in which we analyzed the role of laminin-332 and its receptor integrin &#945;3&#946;1 in this differentiation process. This spheroid model system not only reproduces the tumor microenvironment conditions in a more accurate way, but also is a very versatile model since it allows different downstream studies, such as immunofluorescent staining of both intra- and extracellular markers, as well as deposited extracellular matrix proteins. Moreover, transcriptional analyses by qPCR, flow cytometry and cellular invasion can be studied with this model. Here, we describe a protocol of a spheroid model to assess the role of CAFs&#39; integrin &#945;3&#946;1 and its ectopically deposited ligand, laminin-332, in differentiation and in supporting the invasion of pancreatic cancer cells.

The goal of this protocol is to establish a 3D in vitro model to study the differentiation of cancer-associated fibroblasts (CAFs) in a tumor bulk-like environment, which can be addressed in different analysis systems, such as immunofluorescence, transcriptional analysis and life cell imaging.

3D球形模型作为癌症相关纤维细胞分化和入侵体内研究的更多生理系统

Defining the ideal model for an in vitro study is essential, mainly if studying physiological processes such as differentiation of cells. In the tumor ...

Evaluating the Differentiation Capacity of Mouse Prostate Epithelial Cells Using Organoid Culture

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation capacity of mouse prostate basal and luminal cells during development, tissue-regeneration and transformation. However, evaluating cell-intrinsic and extrinsic regulators of prostate epithelial differentiation capacity using a lineage tracing approach often requires extensive breeding and can be cost-prohibitive. In the prostate organoid assay, basal and luminal cells generate prostatic epithelium ex vivo. Importantly, primary epithelial cells can be isolated from mice of any genetic background or mice treated with any number of small molecules prior to, or after, plating into three-dimensional (3D) culture. Sufficient material for evaluation of differentiation capacity is generated after 7-10 days. Collection of basal-derived and luminal-derived organoids for (1) protein analysis by Western blot and (2) immunohistochemical analysis of intact organoids by whole-mount confocal microscopy enables researchers to evaluate the ex vivo differentiation capacity of prostate epithelial cells. When used in combination, these two approaches provide complementary information about the differentiation capacity of prostate basal and luminal cells in response to genetic or pharmacological manipulation.

Mouse prostate organoids represent a promising context to evaluate mechanisms that regulate differentiation. This paper describes an improved approach to establish prostate organoids, and introduces methods to (1) collect protein lysate from organoids, and (2) fix and stain organoids for whole-mount confocal microscopy.

利用有机体培养评价小鼠前列腺皮细胞的分化能力

The prostate epithelium is comprised predominantly of basal and luminal cells. In vivo lineage tracing has been utilized to define the differentiation ...