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 border="0" cellpadding="0" cellspacing="0" width="1024">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Name of Reagent/ Equipment</td>
			<td style="width:187px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:365px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Ethanol</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">A405-P</td>
			<td>prepare a 70% solution in Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">18 M&#937;-cm water, sterile filtered</td>
			<td style="width:187px;"></td>
			<td style="width:119px;"></td>
			<td>sterile filtered, Type 1 reagent grade water</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10cc plastic disposable syringe, sterile</td>
			<td style="width:187px;">BD</td>
			<td style="width:119px;">305482</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.2&#181;m polyethersulfone (PES) syringe filter, sterile</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td style="width:119px;">194-2520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">15ml polypropylene conical tubes, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>14-959-49B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">50ml polypropylene conical tubes, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>05-539-6</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">1.5ml low retention microcentrifuge tubes,sterile</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">02-681-331</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">nutrient medium, DMEM-F12/HEPES</td>
			<td style="width:187px;">Invitrogen</td>
			<td style="width:119px;">11330-032</td>
			<td>with L-glutamine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Insulin, human recombinant</td>
			<td style="width:187px;">Roche</td>
			<td style="width:119px;">11376497001</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal Growth Factor (EGF), human recombinant</td>
			<td style="width:187px;">Millipore</td>
			<td style="width:119px;">GF144</td>
			<td>100&#181;g/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin sulfate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">S1567</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gentamicin sulfate</td>
			<td style="width:187px;">Sigma-Aldrich</td>
			<td style="width:119px;">G19114</td>
			<td>10mg/ml stock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filtration flask and filter top, sterile</td>
			<td style="width:187px;">Millipore</td>
			<td>SCGPU02RE</td>
			<td>0.22&#181;m PES membrane</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">25ml sterile, disposable pipettes</td>
			<td style="width:187px;">Fisher</td>
			<td>4489</td>
			<td>paper-plastic wrapped</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">10ml sterile, disposable pipettes</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">4488</td>
			<td>paper-plastic wrapped</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:353px;">Tissue marking dyes (black, blue, red, green, yellow and orange)</td>
			<td style="width:187px;">CDI</td>
			<td style="width:119px;">MD2000</td>
			<td style="width:365px;">after opening use only with single-use, sterile cotton tipped applicators, or use once and discard</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Cotton tipped applicators, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>23-400-115</td>
			<td>single use only</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Gauze pads, 10x10cm, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">2187</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic transfer pipettes, sterile, disposable</td>
			<td style="width:187px;">Samco</td>
			<td>202-20S</td>
			<td></td>
		</tr>
		<tr height="43">
			<td height="43" style="height:43px;">Vinegar, white distilled</td>
			<td style="width:187px;">household use</td>
			<td></td>
			<td style="width:365px;">5% acetic acid; after opening use only with sterile pipettes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">#10 scalpels, sterile, disposable&#160;</td>
			<td style="width:187px;">Thermo Scientific</td>
			<td>31-200-32&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">petri dish, sterile</td>
			<td style="width:187px;">Fisher</td>
			<td>FB0875713A</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">TPP 115cm2 flask, with removable lid</td>
			<td style="width:187px;">MidSci</td>
			<td style="width:119px;">90652</td>
			<td>screw cap with filter</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:353px;">CO2 incubator</td>
			<td style="width:187px;">Fisher</td>
			<td>13-998-074</td>
			<td>5% CO2, 37 oC, humidified chamber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">inverted light microscope</td>
			<td style="width:187px;">Olypmus</td>
			<td>IX51</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">8M urea</td>
			<td style="width:187px;">Fisher</td>
			<td style="width:119px;">BP169-500</td>
			<td>optional, for mass spectrophotometric analysis of cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">2X SDS tris-glycine buffer</td>
			<td style="width:187px;">Life Technologies</td>
			<td>LC2676</td>
			<td>optional, for proteomic analysis of cultured cells</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:353px;">Cytocentrifuge</td>
			<td style="width:187px;">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

12.5K 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

Mouse Mammary Epithelial Cells form Mammospheres During Lactogenic Differentiation

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of dome shaped cell structures referred to as mammospheres 1. The HC11 cell line has been employed as a model system for the study of regulation of mammary lactogenic differentiation both in vitro and in vivo 2. The HC11 cells differentiate and synthesize milk proteins in response to treatment with lactogenic hormones. Following the growth of HC11 mouse mammary epithelial cells to confluence, lactogenic differentiation was induced by the addition of a combination of lactogenic hormones including dexamethasone, insulin, and prolactin, referred to as DIP. The HC11 cells induced to differentiate were photographed at times up to 120 hours post induction of differentiation and the number of mammospheres that appeared in each culture was enumerated. The size of the individual mammospheres correlates with the degree of differentiation and this is depicted in the images of the differentiating cells.

HC11 lactogenic differentiation can be characterized by the formation of domed structures referred to as mammospheres. The structures can be enumerated by phase contrast microscopy to aid in quantifying lactogenic differentiation.

小鼠乳腺上皮细胞形成过程中Lactogenic分化的微球体

A phenotypic measure commonly used to determine the degree of lactogenic differentiation in mouse mammary epithelial cell cultures is the formation of ...

科研

生物学

Generation of Single-Cell Suspensions from Mouse Neural Tissue

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a repertoire of cell surface molecules, or molecular determinants, through which it can be identified and characterized. Currently, robust cell identification and separation technologies require single-cell preparations to be generated while simultaneously limiting cell death and destruction of characteristic surface protein. The gentleMACS Dissociator, when used in combination with trypsin or papain-based dissociation kits, can effectively and gently dissociate brain tissue while preserving antigen epitopes and limiting cell loss. Standardized preparation of single-cell suspensions is achieved using C Tubes and optimized, preset gentleMACS Programs. Once generated, single-cell suspensions can be treated with monoclonal conjugates like Anti-Prominin-1 MicroBeads, which identify neural progenitors, or purified further using Myelin Removal Beads.

Dissociating cells from specific tissue types requires specific parameters for tissue aggitation to obtain a high volume of viable, culturable cells. The Miltenyi gentleMACS Dissociator optimizes this task with a simple, practical protocol. In this publication the use of this apparatus on nerual tissue is explained.

从小鼠神经组织产生单细胞悬浮液

Within the nervous system, hundreds of neuronal and glial cell types have been described. Each specific cell type in the brain or spinal cord has a ...

Preparation of Pooled Human Platelet Lysate (pHPL) as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for mesenchymal stromal cells (MSCs), fibroblasts and endothelial colony-forming cells with platelets activated by thrombin3-5 or lysed by freeze/thaw cycles6-14 before the platelet releasate is added to the cell culture medium. The trophic effect of platelet derived growth factors has already been tested in several trials for tissue engineering and regenerative therapy.1,15-17 Varying efficiency is considered to be at least in part due to individually divergent concentrations of growth factors18,19 and a current lack of standardized protocols for platelet preparation.15,16 This protocol presents a practicable procedure to generate a pool of human platelet lysate (pHPL) derived from routinely produced platelet rich plasma (PRP) of forty to fifty single blood donations. By several freeze/thaw cycles the platelet membranes are damaged and growth factors are efficiently released into the plasma. Finally, the platelet fragments are removed by centrifugation to avoid extensive aggregate formation and deplete potential antigens. The implementation of pHPL into standard culture protocols represents a promising tool for further development of cell therapeutics propagated in an animal protein-free system.

Preparation of Pooled Human Platelet Lysate pHPL as an Efficient Supplement for Animal Serum-Free Human Stem Cell Cultures

Human platelet lysate is a rich source of growth factors and a potent supplement in cell culture. This protocol presents the process of preparing a large pool of human platelet lysate by starting from platelet rich plasma, performing several freeze-thaw cycles and depleting the platelet fragments.

汇集人类血小板裂解液作为一种有效的补充（pHPL）的动物血清自由人干细胞培养制备

Platelet derived growth factors have been shown to stimulate cell proliferation efficiently in vivo1,2 and in vitro. This effect has been reported for ...

Electrospinning Fibrous Polymer Scaffolds for Tissue Engineering and Cell Culture

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address the requirements (e.g., mechanics and vascularity) of more intricate organs and tissues. Electrospinning is a popular technique to create fibrous scaffolds that mimic the architecture and size scale of the native extracellular matrix. These fibrous scaffolds are also useful as cell culture substrates since the fibers can be used to direct cellular behavior, including stem cell differentiation (see extensive reviews by Mauck et al. and Sill et al. for more information). In this article, we describe the general process of electrospinning polymers and as an example, electrospin a reactive hyaluronic acid capable of crosslinking with light exposure (see Ifkovits et al. for a review on photocrosslinkable materials). We also introduce further processing capabilities such as photopatterning and multi-polymer scaffold formation. Photopatterning can be used to create scaffolds with channels and multi-scale porosity to increase cellular infiltration and tissue distribution. Multi-polymer scaffolds are useful to better tune the properties (mechanics and degradation) of a scaffold, including tailored porosity for cellular infiltration. Furthermore, these techniques can be extended to include a wide array of polymers and reactive macromers to create complex scaffolds that provide the cues necessary for the development of successful tissue engineered constructs.

The process of electrospinning polymers for tissue engineering and cell culture is addressed in this article. Specifically, the electrospinning of photoreactive macromers with additional processing capabilities of photopatterning and multi-polymer electrospinning is described.

静电纤维聚合物支架用于组织工程和细胞培养

As the field of tissue engineering evolves, there is a tremendous demand to produce more suitable materials and processing techniques in order to address ...

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

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

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

长期培养的人类乳腺癌标本，并利用光学投影层析成像分析

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

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

鼠胚体外发育无血清全胚胎培养系统

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...

A Tissue Culture Model of Estrogen-producing Primary Bovine Granulosa Cells

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca and, in particular, of the granulosa cell layer profoundly change their morphological, physiological, and molecular characteristics and form the progesterone-producing corpus luteum that is responsible for maintaining pregnancy. Cell culture models are essential tools to study the underlying regulatory mechanisms involved in the folliculo-luteal transformation. The presented protocol focuses on the isolation procedure and cryopreservation of bovine GC from small- to medium-sized follicles (&#60; 6 mm). With this technique, a nearly pure population of GC can be obtained. The cryopreservation procedure greatly facilitates time management of the cell culture work independent of a direct primary tissue (ovaries) supply. This protocol describes a serum-free cell culture model that mimics the estradiol-active status of bovine GC. Important conditions that are essential for a successful steroid-active cell culture are discussed throughout the protocol. It is demonstrated that increasing the plating density of the cells induces a specific response as indicated by an altered gene expression profile and hormone production. Furthermore, this model provides a basis for further studies on GC differentiation and other applications.

A long-term culture model of bovine granulosa cells under serum-free conditions is described. This model allows researchers to study the effects of diverse factors and conditions as different plating densities on the characteristics of estrogen-producing bovine granulosa cells.

雌激素原代牛颗粒细胞的组织培养模型

Ovarian granulosa cells (GC) are the major source of estradiol synthesis. Induced by the preovulatory luteinizing hormone (LH) surge, cells of the theca ...

Tissue Collection of Bats for -Omics Analyses and Primary Cell Culture

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of these methods to non-model organisms. A series of protocols to optimize tissue collection from bats has been developed for a series of high-throughput sequencing approaches. Outlined here are protocols for the capture of bats, desired demographics to be collected for each bat, and optimized methods to minimize stress on a bat during tissue collection. Specifically outlined are methods for collecting and treating tissue to obtain (i) DNA for high molecular weight genomic analyses, (ii) RNA for tissue-specific transcriptomes, and (iii) proteins for proteomic-level analyses. Lastly, also outlined is a method to avoid lethal sampling by creating viable primary cell cultures from wing clips. A central motivation of these methods is to maximize the amount of potential molecular and morphological data for each bat and suggest optimal ways to preserve tissues so they retain their value as new methods develop in the future. This standardization has become particularly important as initiatives to sequence chromosome-level, error-free genomes of species across the world have emerged, in which multiple scientific parties are spearheading the sequencing of different taxonomic groups. The protocols outlined herein define the ideal tissue collection and tissue preservation methods for Bat1K, the consortium that is sequencing the genomes of every species of bat.

This is a protocol for the optimal tissue preparation for genomic, transcriptomic, and proteomic analyses of bats caught in the wild. It includes protocols for bat capture and dissection, tissue preservation, and cell culturing of bat tissue.

蝙蝠组织收集 -细胞分析和原细胞培养

As high-throughput sequencing technologies advance, standardized methods for high quality tissue acquisition and preservation allow for the extension of ...

Mucociliary Epithelial Organoids from Xenopus Embryonic Cells: Generation, Culture and High-Resolution Live Imaging

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated clearance. Many clinically relevant defects in the mucociliary epithelium are inferred as they occur deep within the body. Here, we introduce a tractable 3D model for mucociliary epithelium generated from multipotent progenitors that were microsurgically isolated from Xenopus laevis embryos. The mucociliary epithelial organoids are covered with newly generated epithelium from deep ectoderm cells and later decorated with distinct patterned multiciliated cells, secretory cells, and mucus-producing goblet cells that are indistinguishable from the native epidermis within 24 h. The full sequences of dynamic cell transitions from mesenchymal to epithelial that emerge on the apical surface of organoids can be tracked by high-resolution live imaging. These in vitro cultured, self-organizing mucociliary epithelial organoids offer distinct advantages in studying the biology of mucociliary epithelium with high-efficiency in generation, defined culture conditions, control over number and size, and direct access for live imaging during the regeneration of the differentiated epithelium.

We describe a simple protocol to develop mucociliary epithelial organoids from deep ectoderm cells isolated from Xenopus laevis embryos. The multipotent progenitors regenerate epithelial goblet cell precursors and allow live tracking of the initiation and progression of the cell transitions on the surface of organoids.

Xenopus胚胎细胞的粘膜异体器官：生成、培养和高分辨率实时成像

Mucociliary epithelium provides the first line of defense by removing foreign particles through the action of mucus production and cilia-mediated ...

Bovine Ovarian Cortex Tissue Culture

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors from somatic cells and cumulus cell-oocyte communication. Little is known about the ovarian microenvironment and how the cytokines and steroids produced in the surrounding milieu affect follicle progression or arrest. In vitro culture of ovarian cortex enables follicles to develop in a normalized environment that remains supported by adjacent stroma. Our objective was to determine the effect of nutritional Stair-Step diet on the ovarian microenvironment (follicle development, steroid, and cytokine production) through in vitro culture of bovine ovarian cortex. To accomplish this, ovarian cortical pieces were removed from heifers undergoing two different nutritionally developed schemes prior to puberty: Control (traditional nutrition development) and Stair-Step (feeding and restriction during development) that were cut into approximately 0.5-1 mm3 pieces. These pieces were subsequently passed through a series of washes and positioned on a tissue culture insert that is set into a well containing Waymouth&#39;s culture medium. Ovarian cortex was cultured for 7 days with daily culture media changes. Histological sectioning was performed to determine follicle stage changes before and after the culture to determine effects of nutrition and impact of culture without additional treatment. Cortex culture medium was pooled over days to measure steroids, steroid metabolites, and cytokines. There were tendencies for increased steroid hormones in ovarian microenvironment that allowed for follicle progression in the Stair-Step versus Control ovarian cortex cultures. The ovarian cortex culture technique allows for a better understanding of the ovarian microenvironment, and how alterations in endocrine secretion may affect follicle progression and growth from both in vivo and in vitro treatments. This culture method may also prove beneficial for testing potential therapeutics that may improve follicle progression in women to promote fertility.

In vitro culture of bovine ovarian cortex and the effect of nutritional Stair-step diet on ovarian microenvironment is presented. Ovarian cortex pieces were cultured for seven days and steroids, cytokines, and follicle stages were evaluated. The Stair-Step diet treatment had increased steroidogenesis resulting in follicle progression in culture.

牛卵巢皮质组织培养

Follicle development from the primordial to antral stage is a dynamic process within the ovarian cortex, which includes endocrine and paracrine factors ...