这项工作得到了部分支持（1）国防部乳腺癌研究发展计划（美国陆军医学研究收购活动）奖＃W81XVVH-10-1-0781到LAL和VE，和（2）的苏珊科曼基金会资助IR122224446拉尔和VE。病理学的支持和组织卖座被好心Inova公司费尔法克斯病理科，哈桑Nayer博士，博士Geetha A.梅内塞斯和查尔斯Bechert博士提供。患者的知情同意和样品采购是通过熟练的INOVA的费尔法克斯医院临床研究协调员加利莫尔冬青，石楠Huryk和埃米尔卡玛指导。

人体乳腺组织是由患者参加一项研究，以书面的知情同意书收集起来，下面国防部乔治·梅森大学中文系，和INOVA的卫生系统机构审查委员会批准的协议。 1.准备营养丰富的培养基与生长因子和抗生素<ol><li>制备储备溶液胰岛素，表皮生长因子（EGF），硫酸链霉素和庆大霉素硫酸盐。 <ol><li>重组胰岛素的无菌过滤的水，以10毫克/毫升的最终浓度。吸液加入10ml 1型试剂级水在10ml无菌一次性注射器。附加0.22微米聚醚砜过滤器，注射器。分配所述无菌过滤的水进入无菌的15毫升锥形管中。 </li><li>加入10毫升的无菌过滤的水，以胰岛素100mg的小瓶中。简要地混合在涡流混合器上的内容。置于冰上胰岛素药瓶。分装450微升胰岛素股票的SOLU化入标，无菌的离心管中，并储存在-20℃。胰岛素储备溶液是稳定的，直到在管形瓶中的到期日期。 </li><li>准备表皮生长因子（EGF）的储备液:加入500μl无菌水500微克EGF（股票1微克/微升）。简要地混合在涡流混合器上的内容。置于冰上的EGF小瓶。 <ol><li>准备EGF的工作储备液:将100μl股票EGF溶液900微升培养基（工作股价为0.1微克/微升）。简要地混合在涡流混合器上的内容。分配50μl的在-80℃的EGF的工作原液入标记，无菌微量离心管中并储存的。 注:EGF原液（1微克/微升）是稳定1年，在-80℃;工作储备溶液（0.1微克/微升）稳定2个月，在-80℃下。 </li></ol></li><li>称量在分析天平上硫酸链霉素40毫克。将硫酸链霉素INTOA无菌的15 mL锥形管。添加4毫升营养培养基。简要地混合在涡流混合器上的内容。解决的办法应该是淡粉红色。储存在4℃下避光保存。硫酸链霉素溶液是稳定7天。 </li><li>称在分析天平上20毫克的硫酸庆大霉素。将硫酸庆大霉素到无菌的15毫升锥形管中。加2 ml培养基中。简要地混合在涡流混合器上的内容。该溶液应为黄色。储存在4℃下避光保存。硫酸庆大霉素溶液是稳定7天。 </li></ol></li><li>准备两个200毫升分批补充有重组人表皮生长因子的营养培养基（10毫微克/毫升的最终浓度），胰岛素（10μg/ ml的终浓度），硫酸链霉素（100μg/ ml的最终浓度）和硫酸庆大霉素（20微克/ ml终浓度）。 <ol><li>添加200毫升营养培养基中于真空过滤烧瓶配有0.2微米的聚醚砜过滤器烧瓶中，并在250ml接收瓶中。添加20＃181;升工作原液EGF，200微升的库存胰岛素，2- ml储备硫酸链霉素，和400μl的股票硫酸庆大霉素到200毫升营养培养基。附加的过滤烧瓶中的真空度。过滤介质。丢弃过滤器和标签的瓶作为"营养丰富的培养基中。"储存在4℃下长达14天。 </li></ol></li></ol> 2.组织采集和卖座<ol><li>在手术室，采购乳房组织后维持无菌技术。将乳腺组织在无菌纸盒，盖上无菌塑料包装托盘。 <ol><li>按要求通过体制运输组织中的覆盖托盘放射科/病理。不要打开托盘。机构内部的运输时间有所不同。组织可以在RT这个盖纸盒可容纳45分钟后切除仍然是可行的。然而，组织成营养丰富的培养基中的提示处理提供用于随后的组织体培养的最佳条件。 </li></ol></li><li>毛的组织解剖，以确定导管原位癌的区域内的乳房组织:使用无菌手套，刀片，解剖刀，组织标记染料和醋组织解剖期间保持组织无菌。 <ol><li>清洁用70％乙醇或1％的漂白粉的工作区。打开无菌手套，并放置在工作表面上的手套包装。地方的手套包装正面朝上的无菌室内。戴上无菌手套。 <ol><li>清洗，用70％乙醇的检体容器的表面上。取下组织样本容器的保鲜膜，然后将试样在手套包装。 </li></ol></li><li>浸二棉拭子插入组织标记染料。通过在组织表面上滚动的拭子应用的染料到组织的表面上。 注:每个病理科，建有标准化的染料颜色/组织定向协议。组织标记有染料定向相对于所述组织其在患者中的位置。染料被吸干或涂在组织上。不要倒在组织中的染料。浇注的染料会引起染料运行/点滴到组织中的缝隙从而混淆手术切缘的方向。组织的取向提供了外科医生和病理学家与解剖学界标，以a）描述的组织标本，以及b）确定与所述肿瘤的手术切缘的位置。 </li><li>倾蒸馏白醋（5％乙酸）到无菌纱布垫。吸干用醋浸泡过的纱布染成组织。丢弃的纱布。醋是用来修复组织的标记染料。 </li><li>切乳房组织成垂直切片约5毫米厚。切不要通过组织（ 图2）的所有道路。观察/触诊组织的钙化区。鉴别原位癌病灶的特点事务所，面色苍白的外观由微红包围，橡胶边框的坚韧不拔的感觉（因为钙骨针）。 注意:在某些情况下，粉刺（疙瘩状）的区域可以是视为白点，表示从大直径的DCIS病灶坏死物质。 </li><li>切出的DCIS /钙化的乳腺组织的区域，包括少量周围的乳房组织。放置在乳房组织进入无菌50ml管中含有20-30毫升，在步骤1中制备的营养丰富的培养基中。 <ol><li>轻轻颠倒试管几次混合组织和平台。弃去培养基，加入新鲜培养基。将装有组织和培养基中的管插入一个绝缘容器和输送到组织的组织培养室。 </li></ol></li></ol></li></ol> 3.组织培养<ol><li>工作在生物安全柜中，倒入组织和少量的营养培养基到无菌培养皿中。用无菌解剖刀切开并丢弃纤维组织。切开的乳房组织成块（组织体）约为3mm 2（ 图1C-D）。试着切割组织，使得每个器官oid的含有至少一种可辨别的管段与周围的基质。 </li><li>打开该组织培养瓶的盖子。用无菌镊子或镊子，将组织体到烧瓶中。合上盖子。丢弃的培养皿。 </li><li>添加11毫升无血清的营养丰富的培养基（在步骤1中制备）于组织培养烧瓶中。收烧瓶中，摇动烧瓶使组织体和介质被均匀地分布在整个烧瓶表面（ 图1E-F）。 </li><li>孵育烧瓶中，在37℃，在湿润的5.0％的CO 2气氛中进行2天。在此期间，不要移动烧瓶中。 </li><li>后第2天培养后，从培养箱取出烧瓶，以检查是否有潜在的细菌/真菌的污染。避免了瓶突然，剧烈运动，或旋涡。将烧瓶放置在倒置显微镜载物台上。 <ol><li>观察对细菌，酵母菌和/或真菌的培养基。如果没有污染注意，返回烧瓶培养箱为additional一天。如果污染注意，丢弃在适当的容器中的烧瓶和内容。 </li></ol></li><li>后第3天培养后，更换新鲜培养基的条件培养基。 <ol><li>放置11毫升介质中的无菌管中，在37℃下进行20-30分钟。请从孵化器的组织培养瓶。不干扰类器官，除去并丢弃使用无菌血清移液管将烧瓶中的介质。 </li><li>在预热的新鲜培养基的使用新的无菌血清移液管中，添加11毫升。非常轻轻地旋转烧瓶，以分布到烧瓶表面的平台。 </li><li>孵育烧瓶中，在37℃，在湿润的5.0％的CO 2气氛中。 </li></ol></li></ol> 4.维修建立类器官/上皮细胞集落<ol><li>制备新鲜的培养基，每2周，并更换介质中的组织培养烧瓶中，每周3次。 <ol><li>放置11毫升培养基在无菌容器中，在37°℃，20〜30分钟。请从孵化器的烧瓶中。用无菌吸管血清，捞出从烧瓶中丢弃的调理液，注意不要打扰类器官。 </li><li>在预热的新鲜培养基的使用新的无菌血清移液管中，添加11毫升。非常轻轻地旋转烧瓶，以分布到烧瓶表面的平台。孵育烧瓶中，在37℃，在湿润的5.0％的CO 2气氛中。 </li></ol></li><li>经过10 - 在培养14天，去除任何组织片在培养瓶中所没有粘附。 <ol><li>定期收获细胞和/或从烧瓶中用于传播到新的培养瓶中组织体，对异种移植物移植，或用于表型和/或分子的分析。 </li><li>请从孵化器的组织培养瓶。喷雾用70％乙醇的烧瓶中。擦去用干净的纸巾已经用70％的乙醇的烧瓶中加入过量的乙醇。 </li><li>类器官繁殖:&lt;OL&gt; <li>打开烧瓶的盖子，将盖面朝上，在生物安全柜中。在显微镜下可视化，找到组织体（S）收获。用无菌镊子或钳子回升的类器官。 </li><li>传播在新的培养瓶中的组织体，将组织体中的新瓶。添加11毫升清新，温暖的媒介。 3.6.3 -如在步骤3.3中所述孵育在37℃下，在潮湿的5％的CO 2气氛中。替换在组织培养细胞培养基烧瓶中，每周3次。 </li></ol></li><li>收获细胞: <ol><li>在直接镜检vixualization，轻轻刮去并吸出细胞和微球体使用1000微升吸管用无菌一次性吸头。吸出细胞和周围介质。分配所述细胞/培养基到无菌微量离心管中。 </li></ol></li><li>短暂离心细胞在微型离心机在12100×g离心5秒。取出并丢弃的媒介。 &lt;OL&gt; <li>用于DNA分析，立即冻结细胞沉淀在干冰上，在小体积的培养基中（10微升），具有长期贮存于-80℃。 </li><li>用于蛋白组学分析，溶解于10μl的8M尿素进行质谱法或免疫印迹/反相蛋白质微阵列的2×SDS tris-甘氨酸缓冲液中的细胞。另外，旋转细胞的cytocentrifuge使细胞涂片进行免疫组化分析。 </li></ol></li><li>后从瓶中收获细胞，取出并丢弃剩余的媒介。添加11毫升温暖，新鲜营养丰富的培养基中。孵育烧瓶中，在37℃，在潮湿的5％的CO 2气氛中。 </li></ol></li></ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Attacking+breast+cancer+at+the+preinvasion+stage+by+targeting+autophagy.">Attacking breast cancer at the preinvasion stage by targeting autophagy.</a> Women's Health. 9 (2), 1-14 (2013).</li><li>D'amonte, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammary+carcinoma+behavior+is+programmed+in+the+precancer+stem+cell.">Mammary carcinoma behavior is programmed in the precancer stem cell.</a> Breast Cancer Res. 10 (3), (2008).</li></ol>

作者什么都没有透露。

本文所描述的培养体系构成的基础和转化研究工作产生的生活浸润前乳腺肿瘤细胞的新模式。在过去，恶变前乳房癌的进展已经典型地使用了三种不同的方法学。第一种方法是病理组织学和显微切割冷冻或固定的人体标本12-14的遗传分析。第二种方法利用了含有增生的肺泡结节（韩病灶）被认为是与人类相似的预浸润性乳腺癌病灶15小鼠模型。第三个模型使用建立的乳腺癌细胞系?...

乳腺导管和腺泡（ 导管原位癌 ）的范围内上皮细胞的增生被认为是一种专为前驱体浸润性导管和乳腺小叶癌。然而，分子机制和细胞群的动力学允许进展为浸润性癌是知之甚少。阐明所使用的预浸润性乳腺癌细胞中，或任何预先侵入肿瘤的生存机理，揭示可能的治疗策略进行查杀，甚至预防，预侵入肿瘤1。然而，对于功能性研究人类浸润前病变简单的低成本方法已经缺乏。虽然在体外单层转化细胞系的培养是一个既定的实验室方法，这些永生化细胞系的表型和基因型未能概括原代人肿瘤细胞2的分子状态。此外，即使在非致瘤性乳腺癌MCF-10A细胞系，其recA启动pitulates三维乳腺架构，未能充分代表了功能表型和个体病人的预浸润性乳腺癌病灶3,4的分子特征。 以确定是否能够入侵干细胞样肿瘤细胞的原位 （DCIS）的细胞群的导管癌中的存在，我们开发了一种方法，用于收集和在外科手术时进行培养的无菌人类乳腺组织（ 图1）5。我们的体外乳腺组织体培养系统不依赖于酶的组织破坏，基底膜提取物基质，或成纤维细胞的耗竭，用于分离和从新鲜人乳腺导管癌组织6-8传播乳腺球形成细胞。我们的新系统是基于细胞的流/迁移5的原理。的可辨别乳房导管，和周围的基质浸没在无血清培养基中（只是电子的最小体积交流或覆盖管的片段），以最大限度地提高气体交换，以暴露于培养基中的管的切割表面，但在没有在该烧瓶中（ 图1E-F）的特定方位。该培养系统允许细胞迁移出导管并进入/到自体基质和培养瓶。营养培养基中，只用表皮生长因子（EGF），胰岛素，和抗生素补充的，支持混合的细胞群从组织体发出的增长。在组织培养瓶有一个可拆卸的，可重复密封的盖子，其允许组织体和/或细胞，而不会中断整个烧瓶或邻近组织体收获，同时保持无菌的湿润环境。

<table><tbody><tr><td>Ethanol</td><td>Fisher</td><td>A405-P</td><td>prepare a 70% solution in Type 1 reagent grade water</td></tr><tr><td>18 M&amp;Omega;-cm water, sterile filtered</td><td></td><td></td><td>sterile filtered, Type 1 reagent grade water</td></tr><tr><td>10 cc plastic disposable syringe, sterile</td><td>BD</td><td>305482</td><td></td></tr><tr><td>0.2 &amp;micro;m polyethersulfone (PES) syringe filter, sterile</td><td>Thermo Scientific</td><td>194-2520</td><td></td></tr><tr><td>15 ml polypropylene conical tubes, sterile</td><td>Fisher</td><td>14-959-49B</td><td></td></tr><tr><td>50 ml polypropylene conical tubes, sterile</td><td>Fisher</td><td>05-539-6</td><td></td></tr><tr><td>1.5 ml low retention microcentrifuge tubes, sterile</td><td>Fisher</td><td>02-681-331</td><td></td></tr><tr><td>nutrient medium, DMEM-F12/HEPES</td><td>Invitrogen</td><td>11330-032</td><td>with L-glutamine</td></tr><tr><td>Insulin, human recombinant</td><td>Roche</td><td>11376497001</td><td>10 mg/ml stock</td></tr><tr><td>Epidermal Growth Factor (EGF), human recombinant</td><td>Millipore</td><td>GF144</td><td>100 &amp;micro;g/ml stock</td></tr><tr><td>Streptomycin sulfate</td><td>Sigma-Aldrich</td><td>S1567</td><td>10 mg/ml stock</td></tr><tr><td>Gentamicin sulfate</td><td>Sigma-Aldrich</td><td>G19114</td><td>10 mg/ml stock</td></tr><tr><td>Filtration flask and filter top, sterile</td><td>Millipore</td><td>SCGPU02RE</td><td>0.22 &amp;micro;m PES membrane</td></tr><tr><td>25 ml sterile, disposable pipettes</td><td>Fisher</td><td>4489</td><td>paper-plastic wrapped</td></tr><tr><td>10 ml sterile, disposable pipettes</td><td>Fisher</td><td>4488</td><td>paper-plastic wrapped</td></tr><tr><td>Tissue marking dyes (black, blue, red, green, yellow and orange)</td><td>CDI</td><td>MD2000</td><td>after opening use only with single-use, sterile cotton tipped applicators, or use once and discard</td></tr><tr><td>Cotton tipped applicators, sterile</td><td>Fisher</td><td>23-400-115</td><td>single use only</td></tr><tr><td>Gauze pads, 10 x 10 cm, sterile</td><td>Fisher</td><td>2187</td><td></td></tr><tr><td>Plastic transfer pipettes, sterile, disposable</td><td>Samco</td><td>202-20S</td><td></td></tr><tr><td>Vinegar, white distilled</td><td>household use</td><td></td><td>5% acetic acid; after opening use only with sterile pipettes</td></tr><tr><td>#10 scalpels, sterile, disposable</td><td>Thermo Scientific</td><td>31-200-32</td><td></td></tr><tr><td>Petri dish, sterile</td><td>Fisher</td><td>FB0875713A</td><td></td></tr><tr><td>TPP 115 cm&lt;sup&gt;2&lt;/sup&gt; flask, with removable lid</td><td>MidSci</td><td>90652</td><td>screw cap with filter</td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; incubator</td><td>Fisher</td><td>13-998-074</td><td>5% CO&lt;sub&gt;2&lt;/sub&gt;, 37 &amp;deg;C, humidified chamber</td></tr><tr><td>inverted light microscope</td><td>Olypmus</td><td>IX51</td><td></td></tr><tr><td>8 M urea</td><td>Fisher</td><td>BP169-500</td><td>optional, for mass spectrophotometric analysis of cultured cells</td></tr><tr><td>2X SDS tris-glycine buffer</td><td>Life Technologies</td><td>LC2676</td><td>optional, for proteomic analysis of cultured cells</td></tr><tr><td>Cytocentrifuge</td><td>Thermo Scientific</td><td>A78300003</td><td>optional, for preparing cell smears</td></tr></tbody></table>

non-enzymatic, serum-free tissue culture of pre-invasive breast lesions for spontaneous generation of mammospheres

原位 （DCIS）乳腺导管癌，顾名思义，是乳腺管的范围内肿瘤上皮细胞增生，不违反胶原基底膜。而原位癌是一种非专性前体浸润性乳腺癌，分子机制和细胞群，其允许进展为浸润性癌是不完全清楚。以确定是否能够入侵祖细胞的原位癌细胞群中存在的，我们开发了用于在外科手术时收集和培养无菌的人乳腺癌组织中的方法，没有组织的酶破坏。 含导管段无菌乳腺组织是由手术切除乳腺组织下面的常规病理检查收获。含原位癌组织被放置在营养丰富，含有抗生素的无血清培养基中，并输送到组织培养实验室。乳房组织是进一步dissected，来隔离钙化区域。多个乳房组织片（组织体）放置在无血清培养基中的最小体积的烧瓶中，一个可移动的盖子，并培养在加湿的CO 2培养箱中培养。上皮细胞和成纤维细胞群出现后，从10类器官 - 14天。微球体中自发形成上和​​周围的上皮细胞单层。特定的细胞群体可以直接从烧瓶中被收获，而不破坏邻近的细胞。我们的非酶组织培养系统可靠地揭示了从新鲜人原位癌病变细胞遗传学异常，侵入祖细胞。

工作流原位组织采购和无菌培养乳腺导管癌乳房组织无菌性从通过在典型的医院病理工作流小的变化（ 图1）的操作室的细胞培养室保持。组织被输送在无菌容器用塑料薄膜覆盖物，它允许放射学评估，同时保持组织无菌。用无菌手套，刀片和组织标记染料进行乳房肿瘤切除术或乳房切除术的样品总值组织处理。乳腺导管原位癌总值（GDP）的细胞形态?...

Watch this Scientific Journal Video about Non-enzymatic, Serum-free Tissue Culture of Pre-invasive Breast Lesions for Spontaneous Generation of Mammospheres at JoVE.com

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

使用完整的组织组织体原代细胞培养提供了一个模型系统，模仿的多细胞在体内微环境。我们开发了一种无血清乳腺原发上皮细胞组织培养模式，延续了混合细胞培养系，并表现出分化的形态，没有酶的组织破坏。乳房组织体保持活力&gt; 6个月。

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 ...

non-enzymatic-serum-free-tissue-culture-pre-invasive-breast-lesions

Research

JoVE Journal

Biology

12.6K 次观看.  George Mason University. 使用完整的组织组织体原代细胞培养提供了一个模型系统，模仿的多细胞在体内微环境。我们开发了一种无血清乳腺原发上皮细胞组织培养模式，延续了混合细胞培养系，并表现出分化的形态，没有酶的组织破坏。乳房组织体保持活力> 6个月。 

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

Experimental Generation of Carcinoma-Associated Fibroblasts (CAFs) from Human Mammary Fibroblasts

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of extracellular matrix (ECM) and a variety of mesenchymal cells, including fibroblasts, myofibroblasts, endothelial cells, pericytes and leukocytes 1-3.
The tumour-associated stroma is responsive to substantial paracrine signals released by neighbouring carcinoma cells. During the disease process, the stroma often becomes populated by carcinoma-associated fibroblasts (CAFs) including large numbers of myofibroblasts. These cells have previously been extracted from many different types of human carcinomas for their in vitro culture. A subpopulation of CAFs is distinguishable through their up-regulation of &alpha;-smooth muscle actin (&alpha;-SMA) expression4,5. These cells are a hallmark of 'activated fibroblasts' that share similar properties with myofibroblasts commonly observed in injured and fibrotic tissues 6. The presence of this myofibroblastic CAF subset is highly related to high-grade malignancies and associated with poor prognoses in patients.
Many laboratories, including our own, have shown that CAFs, when injected with carcinoma cells into immunodeficient mice, are capable of substantially promoting tumourigenesis 7-10. CAFs prepared from carcinoma patients, however, frequently undergo senescence during propagation in culture limiting the extensiveness of their use throughout ongoing experimentation. To overcome this difficulty, we developed a novel technique to experimentally generate immortalised human mammary CAF cell lines (exp-CAFs) from human mammary fibroblasts, using a coimplantation breast tumour xenograft model.
In order to generate exp-CAFs, parental human mammary fibroblasts, obtained from the reduction mammoplasty tissue, were first immortalised with hTERT, the catalytic subunit of the telomerase holoenzyme, and engineered to express GFP and a puromycin resistance gene. These cells were coimplanted with MCF-7 human breast carcinoma cells expressing an activated ras oncogene (MCF-7-ras cells) into a mouse xenograft. After a period of incubation in vivo, the initially injected human mammary fibroblasts were extracted from the tumour xenografts on the basis of their puromycin resistance 11.
We observed that the resident human mammary fibroblasts have differentiated, adopting a myofibroblastic phenotype and acquired tumour-promoting properties during the course of tumour progression. Importantly, these cells, defined as exp-CAFs, closely mimic the tumour-promoting myofibroblastic phenotype of CAFs isolated from breast carcinomas dissected from patients. Our tumour xenograft-derived exp-CAFs therefore provide an effective model to study the biology of CAFs in human breast carcinomas. The described protocol may also be extended for generating and characterising various CAF populations derived from other types of human carcinomas.

Experimental Generation of Carcinoma-Associated Fibroblasts CAFs from Human Mammary Fibroblasts

Carcinoma-associated fibroblasts (CAFs) rich in myofibroblasts present within the tumour stroma, play a major role in driving tumour progression. We developed a coimplantation tumour xengraft model for experimentally generating CAFs from human mammary fibroblasts. The protocol describes how to establish CAF myofibroblasts that acquire an ability to promote tumourigenesis.

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

Carcinomas are complex tissues comprised of neoplastic cells and a non-cancerous compartment referred to as the 'stroma'. The stroma consists of ...

科研

医学

Processing of Human Reduction Mammoplasty and Mastectomy Tissues for Cell Culture

Experimental examination of normal human mammary epithelial cell (HMEC) behavior, and how normal cells acquire abnormal properties, can be facilitated by in vitro culture systems that more accurately model in vivo biology. The use of human derived material for studying cellular differentiation, aging, senescence, and immortalization is particularly advantageous given the many significant molecular differences in these properties between human and commonly utilized rodent cells1-2. Mammary cells present a convenient model system because large quantities of normal and abnormal tissues are available due to the frequency of reduction mammoplasty and mastectomy surgeries.
The mammary gland consists of a complex admixture of many distinct cell types, e.g., epithelial, adipose, mesenchymal, endothelial. The epithelial cells are responsible for the differentiated mammary function of lactation, and are also the origin of the vast majority of human breast cancers. We have developed methods to process mammary gland surgical discard tissues into pure epithelial components as well as mesenchymal cells3. The processed material can be stored frozen indefinitely, or initiated into primary culture. Surgical discard material is transported to the laboratory and manually dissected to enrich for epithelial containing tissue. Subsequent digestion of the dissected tissue using collagenase and hyaluronidase strips stromal material from the epithelia at the basement membrane. The resulting small pieces of the epithelial tree (organoids) can be separated from the digested stroma by sequential filtration on membranes of fixed pore size. Depending upon pore size, fractions can be obtained consisting of larger ductal/alveolar pieces, smaller alveolar clusters, or stromal cells. We have observed superior growth when cultures are initiated as organoids rather than as dissociated single cells. Placement of organoids in culture using low-stress inducing media supports long-term growth of normal HMEC with markers of multiple lineage types (myoepithelial, luminal, progenitor)4-5. Sufficient numbers of cells can be obtained from one individual's tissue to allow extensive experimental examination using standardized cell batches, as well as interrogation using high throughput modalities.
Cultured HMEC have been employed in a wide variety of studies examining the normal processes governing growth, differentiation, aging, and senescence, and how these normal processes are altered during immortal and malignant transformation4-15,16. The effects of growth in the presence of extracellular matrix material, other cell types, and/or 3D culture can be compared with growth on plastic5,15. Cultured HMEC, starting with normal cells, provide an experimentally tractable system to examine factors that may propel or prevent human aging and carcinogenesis.

A method to process human mammary surgical discard material is described. Processed tissue, in the form of organoids, can be stored frozen indefinitely or placed in culture for long-term growth. This method enables experimental examination of normal human epithelial cell biology, and the effects of exogenous perturbations.

处理人减少乳房整形和乳房切除术的组织细胞培养

Experimental  examination of normal human mammary epithelial cell (HMEC) behavior, and how  normal cells acquire abnormal properties, can be facilitated ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile conditions. Different tests seek to identify and quantify these hallmarks of cancerous cells; however, they often focus on a single aspect of cellular transformation and, in fact, multiple tests are required for their proper characterization. The purpose of this work is to provide researchers with a set of tools to assess cellular transformation in vitro from a broad perspective, thereby making it possible to draw sound conclusions.
A sustained proliferative signaling activation is the major feature of tumoral tissues and can be easily monitored under in vitro conditions by calculating the number of population doublings achieved over time. Besides, the growth of cells in 3D cultures allows their interaction with surrounding cells, resembling what occurs in vivo. This enables the evaluation of cellular aggregation and, together with immunofluorescent labeling of distinctive cellular markers, to obtain information on another relevant feature of tumoral transformation: the loss of proper organization. Another remarkable characteristic of transformed cells is their capacity to grow without attachment to other cells and to the extracellular matrix, which can be evaluated with the anchorage assay.
Detailed experimental procedures to evaluate cell growth rate, to perform immunofluorescent labeling of cell lineage markers in 3D cultures, and to test anchorage-independent cell growth in soft agar are provided. These methodologies are optimized for Breast Primary Epithelial Cells (BPEC) due to its relevance in breast cancer; however, procedures can be applied to other cell types after some adjustments.

This protocol provides experimental in vitro tools to evaluate the transformation of human mammary cells. Detailed steps to follow-up cell proliferation rate, anchorage-independent growth capacity, and distribution of cell lineages in 3D cultures with basement membrane matrix are described.

人类乳腺上皮细胞致癌转化的体外评价

Tumorigenesis is a multi-step process in which cells acquire capabilities&#160;that allow their growth, survival, and dissemination under hostile ...

癌症研究

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 ...

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

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

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

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

乳腺上皮和内皮细胞球状体作为乳腺癌研究的潜在功能 体外 模型

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

Quantification of Self-renewal in Murine Mammosphere Cultures

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. The role of mammary stem cells (MaSCs) is widely studied both in the physiological/developmental context and with regards to breast carcinogenesis. In this aspect, ex vivo studies focused on MaSC properties are highly sought after. Mammosphere cultures represent a surrogate of organ formation and have become a valuable tool for both basic and translational research. Here, we present a detailed protocol for the generation of murine primary mammosphere cultures and the quantitation of MaSC growth properties. The protocol includes mammary gland collection and digestion, isolation of primary mammary epithelial cells (MECs), establishment of primary mammosphere cultures, serial passaging, quantitation of mammosphere growth parameters and interpretation of the results. As an example, we present the effect of low-level constitutive Myc expression on normal MECs leading to increased self-renewal and proliferation.

Here, we describe the implementation and interpretation of the results of an in vitro mammosphere self-renewal quantitative assay.

鼠乳层文化中自我更新的量化

The mammary gland is characterized by extensive regeneration capacity, as it goes through massive hormonal changes throughout the life cycle of a female. ...

发育生物学

Generation of Mosaic Mammary Organoids by Differential Trypsinization

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine mammary gland is composed of two distinct epithelial cell compartments, serving different functions: the outer, contractile myoepithelial compartment and the inner, secretory luminal compartment. Here, we describe a method by which the cells comprising these compartments are isolated and then combined to investigate their individual lineage contributions to mammary gland morphogenesis and differentiation. The method is simple and efficient and does not require sophisticated separation technologies such as fluorescence activated cell sorting. Instead, we harvest and enzymatically digest the tissue, seed the epithelium on adherent tissue culture dishes, and then use differential trypsinization to separate myoepithelial from luminal cells with ~90% purity. The cells are then plated in an extracellular matrix where they organize into bilayered, three-dimensional (3D) organoids that can be differentiated to produce milk after 10 days in culture. To test the effects of genetic mutations, cells can be harvested from wild type or genetically engineered mouse models, or they can be genetically manipulated prior to 3D culture. This technique can be used to generate mosaic organoids that allow investigation of gene function specifically in the luminal or myoepithelial compartment.

The mammary gland is a bilayered structure, comprising outer myoepithelial and inner luminal epithelial cells. Presented is a protocol to prepare organoids using differential trypsinization. This efficient method allows researchers to separately manipulate these two cell types to explore questions concerning their roles in mammary gland form and function.

通过微分试穿素生成马赛克乳腺有机体

Organoids offer self-organizing, three-dimensional tissue structures that recapitulate physiological processes in the convenience of a dish. The murine ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

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

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

从人乳腺癌组织及细胞系微球体形成分析

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

从人体组织中分离乳腺上皮细胞的方案

Isolation and Culture of Primary Human Mammary Epithelial Cells

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are the origin cells of breast cancer and other breast-related inflammatory diseases, have garnered considerable attention. However, isolating and culturing primary HMECs in vitro for research purposes has been challenging due to their highly differentiated, keratinized nature and their short lifespan. Therefore, developing a simple and efficient method to isolate and culture HMECs is of great scientific value for the study of breast biology and breast-related diseases. In this study, we successfully isolated primary HMECs from small amounts of mammary tissue by digestion with a mixture of enzymes combined with an initial culture in 5% fetal bovine serum-DMEM containing the Rho-associated kinase (ROCK) inhibitor Y-27632, followed by culture expansion in serum-free keratinocyte medium. This approach selectively promotes the growth of epithelial cells, resulting in an optimized cell yield. The simplicity and convenience of this method make it suitable for both laboratory and clinical research, which should provide valuable insights into these important areas of study.

作者聚焦：东西方医学结合治疗肉芽肿性乳腺炎

The present study reports an easier, time-saving, and economical protocol to efficiently isolate and grow primary human mammary epithelial cells (HMECs) from small amounts of mammary tissue. This protocol is suitable for quickly producing primary HMECs both for laboratory and clinical applications.

原代人乳腺上皮细胞的分离和培养

The mammary gland is a fundamental structure of the breast and plays an essential role in reproduction. Human mammary epithelial cells (HMECs), which are ...

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

摘要

探索更多视频

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

处理人减少乳房整形和乳房切除术的组织细胞培养

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

人类乳腺上皮细胞致癌转化的体外评价

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

鼠乳层文化中自我更新的量化

通过微分试穿素生成马赛克乳腺有机体

从人乳腺癌组织及细胞系微球体形成分析

原代人乳腺上皮细胞的分离和培养

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

摘要

探索更多视频

从人类乳腺成纤维细胞癌相关的成纤维细胞的实验发电（CAFS）

处理人减少乳房整形和乳房切除术的组织细胞培养

延时成像的主要癌前乳腺上皮细胞来源于遗传工程小鼠乳腺癌模型

人类乳腺上皮细胞致癌转化的体外评价

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

鼠乳层文化中自我更新的量化

通过微分试穿素生成马赛克乳腺有机体

从人乳腺癌组织及细胞系微球体形成分析

原代人乳腺上皮细胞的分离和培养

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