我们感谢教师，研究员，居民和布里格姆与妇女医院的助理医师，病理科组织为这些研究提供。这项工作是支持由美国国立卫生研究院/国立癌症研究所（P50，U01，K08 CA108748 CA105009 CA152990），卵巢癌研究基金，研究资助的5月凯基金会，诺华制药，罗伯特和Deborah第一基金，兰迪和乔尔卡特勒卵巢癌研究基金，玛莎里夫金先生基金会 - 科学学者奖，美国癌症研究学会 - 乔治和帕特里夏Sehl癌症遗传学研究奖学金，并在以色列的美国医学医师奖学金 - 克莱尔和Emmanuel G.罗森布拉特基金会的资助。

1。胶原蛋白的制备和筛选涂层。 <ol><li>为了准备人类胎盘胶原蛋白，tweeze人胎盘胶原蛋白的30毫克和50毫升的蒸馏水（DH 2 O）的顶部，在一个500毫升烧杯中搅拌棒。 </li><li>直接添加到胶原蛋白100冰醋酸μls，使其更容易溶解。预热至37 ° C，搅拌溶解的胶原蛋白。它应该溶解在20-30分钟。如果胶原蛋白链留在溶液中，过滤除菌将是困难的。 </li><li>稀与450毫升DH 2 O和一个0.2微米的膜过滤消毒，50毫升股票。这是最终的工作方案。保存在4 ° C。 </li><li>准备涂层他们与胶原蛋白（50-100μL）一夜之间Transwell小滤膜。冰鲜胶原蛋白必须加热到RT，然后添加到上层舱一夜之间覆盖膜，在室温下。 Transwell小过滤器上的电镀前游离原发性输卵管（FT）上皮（见下文），清洗过滤器3次1X磷酸缓冲液（PBS）至少1个小时之前使用，以除去多余的胶原蛋白。 </li></ol> 2。组织收集和分离。 <ol><li>新鲜的输卵管（FT）的伞应收集在无菌1X PBS 50 mL离心管中，并保持在冰上前提示在无菌组织培养罩的加工。 </li><li>组织样品洗净，一次20毫升冷冻1X PBS（如果样品已经被收集在另一种类型的媒体或至少3X得到尽可能多的血液和国外媒体报道，尽可能摆脱的，因为这可能会干扰电镀效率）的血腥清洗。 </li><li>使用无菌镊子传输英尺至10厘米的一点点PBS和组织培养皿中，用消毒的手术刀和无菌镊子，切纵向最大化的上皮细胞暴露。打开伞，以便它不再是一管，但组织几乎平板。然后切成块较小，但仍检索（直径约3毫米）。 </li><li>件转移至45毫升50毫升管冷冻分解媒体（MEM含1.4毫克/毫升链霉蛋白酶和0.1毫克/毫升DNA酶）。 </li><li>岩轻轻在4 ° C为24至72小时。 （从经验来看，48小时是最佳）。要检查的解离适量，放幻灯片上下降解离媒体和检查的聚集量和组织活力（殴打纤毛细胞）。您想看到单个细胞和一些小团块。 </li></ol> 3。输卵管电镀。 <ol><li>当上皮细胞分解量是最优的，灭活通过添加10％胎牛血清体积的媒体。颠倒几次打跑聚合成更小的细胞悬液。 </li><li>允许大的组织团块（间质组织）在底部定居管。取出介质，它包含了第二个50毫升管上皮细胞。 </li><li>添加50毫升冷的MEM（无FBS），原来的50毫升管。反转，以收集更多的细胞。收集到第三个50毫升管（如果样品是非常大的，高的细胞数量预计的，可重复此步骤，以最大限度地提高细胞采集）的媒体。基质/外组织的大块现在可以被丢弃。你现在有两个50 ml的细胞悬液管。离心5分钟，从细胞颗粒吸分解媒体1000 RPM。 </li><li>重悬在约5-10毫升媒体副秘书长（辅以2％的副秘书长和1％钢笔链球菌的DMEM：F12）预热至37 ° C移液器轻轻。要小心不要吸管太大力，因为这可能会破坏上皮细胞，并减少的文化素质。应该有单细胞和小团块（图1）。 </li><li>转移到Primaria培养皿，孵育至少1小时或长达3个小时。成纤维细胞和红细胞，将继续坚持以塑料，但“金融时报”上皮细胞不会。一个小时后，来看看什么是坚持要在板的外观。注意到跳动的纤毛，纤毛细胞的存在可以看出。 </li><li>在此孵化，胶原蛋白涂层过滤器可水洗和细胞电镀（见步骤1.4） </li><li> Primaria板孵化1-3小时后，细胞悬液转移至15毫升管和冲洗用5毫升副秘书长媒体Primaria板，管这个媒体传输，使终体积为15毫升。离心5分钟1000转。 </li><li>小心吸媒体和避免破坏细胞沉淀。颗粒大小来看，重悬在副秘书长媒体适量。 （通常在1-3毫升）是更好的开始，让您可以稀释进一步，如果你需要。重悬轻轻。 </li><li>加入10μL的悬浮细胞hemacytometer确定细胞密度。提示：您有一个明显的暗的细胞膜和非柱状上皮细胞。您也可以肯定是移动的纤毛细胞计数。较小的细胞，具有晕appearanCE是红血细胞和不应该算。会有一些结块，所以你需要估计，到你的细胞计数。播种的目标是1.65 × 10 5细胞/膜，或至少75％的过滤器覆盖（图1）。如果镀过于稀疏的细胞，细胞将无法形成一个完整的上皮细胞层。 </li><li>加入500μL的副秘书长媒体以及一个24孔板底部插入Transwell小。然后可以添加到每个膜FT细胞悬液（见上文）的要求量，这通常是100μL。 </li><li> 1-2天的孵化和成长过程中没有令人不安的顶部膜。在那些日子里，您可以更改基础的媒体，如果有必要。 1-2天之后，你可以仔细冲洗副秘书长媒体的顶部和媒体对膜的顶部添加少量（50-100μL）。这是比较容易，以确定细胞培养汇合，如果媒体是从心尖侧镜前的过滤器除去。下格应始终包含媒体。一旦文化完全融合（通常在5天之内），媒体传播通过过滤器从基底到心尖双方应是微不足道的。 </li><li>更改第10天，每两天一次的基础媒体。下格必须始终保持细胞存活的媒体。一旦细胞已经形成了严密的上皮细胞层（图1，图2），他们可以在进一步的研究，如免疫荧光（IF）或免疫组织化学（IHC），使用。 </li></ol> 4。膜处理。 <ol><li>膜处理是依赖于类型的研究，将要执行的，但通常涉及从Transwell小插入过滤器去除。 </li><li>该过滤器通常是在清除之前的1X PBS 200μL洗3次。 </li><li>从心尖和基底侧的过滤器，吸PBS。这项工作应在1井一次，以防止膜干燥。 </li><li>从板卸下插入，翻转，倒置，并用无菌手术刀切断周围的膜。尝试删除周围的膜边缘的直线切割，而不是周围的边缘拖动手术刀在一块膜。 </li><li>使用镊子取出插入过滤器，跟踪细胞对哪一方。该过滤器，然后可以酌情处理的IF，IHC等。 </li><li>例如：如果过滤器（固定，通透，阻塞和相应抗体的潜伏期后，如果程序标准）的研究，应放在幻灯片上，细胞朝上，用DAPI Vectashield下降到过滤器和一个覆盖玻璃盖玻片。 </li></ol> 5。代表性的成果： Transwell小过滤器可以很容易地清除到体外上皮细胞免疫组织化学（IHC）和免疫荧光（IF）的研究。谱系特异性标志物的使用，可以形象和量化的分泌（Pax8正）和纤毛（Sall2阳性）细胞，在这些文化室（图3）。此外，使用这些标记，可以监控不同的细胞类型如何响应不同的生理线索 10 。该系统已用于表征secretome的FT上皮细胞内磷酸化的变化，在各种刺激的反应。 <img src="/files/ftp_upload/2728/2728fig1.jpg" alt="图1" /> 图1。插图描绘体外文化是如何从初级人类输卵管组织的产生 。输卵管菌毛组织是从手术套房和肉末产生小片段 1，洗净，与解离媒体孵育24-72 小时 2 。解离完成后，组织碎片是可以解决的潜伏期管的底部，并含有游离的上皮细胞的媒体 ，收获3。离解效率可以监视通过考试，下一个相衬显微镜 4 。游离的上皮细胞，然后培养Primaria板，以帮助消除成纤维细胞和造血干细胞，无不与5上皮细胞掺。一旦有足够的拆除，非上皮细胞的上皮细胞接种于Transwell小涂人胎盘胶原蛋白5的过滤器。媒体扩散，通过从下面的Transwell小。文化是培养24-48小时，然后删除根尖媒体。 体外培养，然后成长为5-8天，形成一个全面，完整的草坪Transwell小过滤器。 体外培养，可以保持在这种状态下viably至4个星期。卡通5显示了一个完全成长的体外培养与从插入和染色与造血删除一个过滤器的一个例子的代表性xylin和伊红（H＆E） 证明体内上皮细胞的极性和架构。 <img src="/files/ftp_upload/2728/2728fig2.jpg" alt="图2" /> 图2。明场显微镜的FT 体外培养。Transwell小插入涂人胎盘胶原0.4μm毛孔可见（一）。金融时报上皮细胞培养的胶原蛋白涂层刀片（B），在那里形成的上皮细胞层（C）。坚持细胞在上皮文化（用箭头表示），最常见的纤毛细胞，通常是观察到一些碎片。 <img src="/files/ftp_upload/2728/2728fig3.jpg" alt="图3" /> 图3。金融时报前体外培养免疫荧光（IF）。如果固定体外培养的图像，并与抗体染色对（A）分泌（Pax8）和（D）纤毛细胞（Sall2）标记。 DAPI是用来作为细胞核（蓝色）的位置（B和E）和合并后的抗体和DAPI染色也显示（C和F）的控制。细胞的数量取决于细胞培养（Pax8染色，7天; Sall2染色，3天）的时间长度。

<ol>
	<li>Jemal, A., Siegel, R., Xu, J., Ward, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+statistics.">Cancer statistics.</a> CA Cancer J. Clin. 60, 277-300 (2010).</li><li>Markman, M., Walker, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intraperitoneal+chemotherapy+of+ovarian+cancer:+a+review,+with+a+focus+on+practical+aspects+of+treatment.">Intraperitoneal chemotherapy of ovarian cancer: a review, with a focus on practical aspects of treatment.</a> J. Clin. Oncol. 24, 988-994 (2006).</li><li>Liu, J., Matulonis, U. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+advances+in+ovarian+cancer.">New advances in ovarian cancer.</a> Oncology (Williston Park). 24, 721-728 (2010).</li><li>Cannistra, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+of+the+ovary.">Cancer of the ovary.</a> N. Engl. J. Med. 351, 2519-2529 (2004).</li><li>Kurman, R. J., Shih, I. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+origin+and+pathogenesis+of+epithelial+ovarian+cancer:+a+proposed+unifying+theory.">The origin and pathogenesis of epithelial ovarian cancer: a proposed unifying theory.</a> Am. J. Surg. Pathol. 34, 433-443 (2010).</li><li>Fong, M. Y., Kakar, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+mouse+models:+a+summary+of+current+models+and+their+limitations.">Ovarian cancer mouse models: a summary of current models and their limitations.</a> J Ovarian Res. 2, 12-12 (2009).</li><li>Shan, W., Liu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+ovarian+cancer:+focus+on+genetics+and+animal+models.">Epithelial ovarian cancer: focus on genetics and animal models.</a> Cell Cycle. 8, 731-735 (2009).</li><li>Levanon, K., Crum, C. P., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+pathogenesis+of+serous+ovarian+cancer+and+its+clinical+impact.">New insights into the pathogenesis of serous ovarian cancer and its clinical impact.</a> J. Clin. Oncol. 26, 5284-5293 (2008).</li><li>Karst, A. M., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+pathogenesis:+a+model+in+evolution.">Ovarian cancer pathogenesis: a model in evolution.</a> J Oncol. 2010, 932371-932371 (2010).</li><li>Levanon, K., Ng, V., Piao, H. Y., Zhang, Y., Chang, M. C., Roh, M. H., Kindelberger, D. W., Hirsch, M. S., Crum, C. P., Marto, J. A., Drapkin, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ex+vivo+cultures+of+human+fallopian+tube+epithelium+as+a+model+for+serous+ovarian+carcinogenesis.">Primary ex vivo cultures of human fallopian tube epithelium as a model for serous ovarian carcinogenesis.</a> Oncogene. 25, 1103-1113 (2010).</li><li>Landen, C. N., Birrer, M. J., Sood, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+events+in+the+pathogenesis+of+epithelial+ovarian+cancer.">Early events in the pathogenesis of epithelial ovarian cancer.</a> J. Clin. Oncol. 26, 995-1005 (2008).</li><li>Vermeer, P. D., Einwalter, L. A., Moninger, T. O., Rokhlina, T., Kern, J. A., Zabner, J., Welsh, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+receptor+and+ligand+regulates+activation+of+epithelial+growth+factor+receptor.">Segregation of receptor and ligand regulates activation of epithelial growth factor receptor.</a> Nature. 422, 322-326 (2003).</li></ol>

Drapkin博士是诺华制药公司的顾问。没有其他作者申报任何潜在利益冲突。

“金融时报”作为SOC的原产地标识候选网站旨在破译连接已知的危险因素和实际的浆液性致癌过程的机制的基础和转化研究提供了机会。这个关键是听话的模型系统的发展，使我们开始测试的假设FTSEC是一个盆腔浆液性癌的细胞的原产地。此处所述的体外培养模型是一种新型的系统允许的方式，在保留本地金融时报“上皮细胞的形态和生物学的主要金融时报”的分泌和纤毛细胞的分离和文化合作。使用这个系统，我们最近的特点secretome本上皮上皮细胞如何响应机械和遗传毒性损伤10。排卵与卵巢肿瘤发生有关的主要危险因素，其特点是结合组织损伤，炎症介质，生长因子和激素11。该系统可用于研究的分泌和纤毛细胞的反应和生存能力的影响排卵环境。此外，其他类型的细胞（即炎症细胞）的影响，可以检查，以便更好地了解一个事件驱动型细胞分化和促进这些细胞的恶性转化的因素。类似的模型已被极化上皮细胞是目前在其他组织。例如，在呼吸道上皮细胞极化小学文化，对细胞生长和细胞损伤的效果，heregulin评估12。这些类型的模型系统研究的启动和从上皮组织产生的肿瘤的发病机制提供了一个新的和有用的方式，这是至关重要的组织中，如无细胞系目前存在的“金融时报，那里是一个伟大的需要确定的主要途径和发展新的治疗策略。

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Collagen from human placenta (Bornstein and Traub Type IV)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C7521</td>
<td>&nbsp;</td>
<tr>
<td>DMEM:F12 media</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>15-090-CV</td>
<td>&nbsp;</td>
<tr>
<td>Ultroser G</td>
<td>&nbsp;</td>
<td>Crescent Chemical Company</td>
<td>67042</td>
<td>Add 20 ml of DMEM:F12 to stock bottle and use 10mls per 500 ml of media (2% final concentration)</td>
</tr>
<tr>
<td>Pen Strep</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15140-122</td>
<td>&nbsp;</td>
<tr>
<td>BD Primaria culture plates</td>
<td>&nbsp;</td>
<td>BD Bioscience</td>
<td>353802</td>
<td>&nbsp;</td>
<tr>
<td>24 well Transwell Permeable supports</td>
<td>&nbsp;</td>
<td>Corning (Costar)</td>
<td>3470</td>
<td> Clear, 6.5 mm insert, 0.4 &mu;m pore size, treated polyester </td>
</tr>
<tr>
<td>MEM (Minimal Essential Media)</td>
<td>&nbsp;</td>
<td>Cellgro</td>
<td>10-010-CV</td>
<td>&nbsp;</td>
<tr>
<td>Pronase</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>11459643001</td>
<td>&nbsp;</td>
<tr>
<td>DNAse</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>DN25</td>
<td>&nbsp;</td>
<tr>
<td>Sall2 antibody</td>
<td>&nbsp;</td>
<td>Dr T. Benjamin, Harvard</td>
<td>Gift</td>
<td>1:20 dilution</td>
</tr>
<tr>
<td>Pax8 antibody</td>
<td>&nbsp;</td>
<td>Proteintech</td>
<td>10336-1-AP</td>
<td>1: 1000 dilution</td>
</tr>		</tbody>
		</table>
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ex vivo culture of primary human fallopian tube epithelial cells

上皮性卵巢癌是女性癌症死亡率在美国的首要原因。相比之下其他女性特定癌症，如乳腺癌和子宫癌，死亡率在最近几年下降，卵巢癌的治愈率保持相对不变，在过去二十年 1 。这主要是由于缺乏适当的筛检工具，用于检测疾病早期手术和化疗是最有效的2， 3。因此，大多数患者目前晚期疾病和弥漫性腹部参与。这是进一步复杂化的事实，卵巢癌是一种异质性疾病与多个组织学亚型4，5。浆液性卵巢癌（SOC）是最常见的和积极的亚型，并与BRCA基因突变有关的最常见的形式。目前的实验模型，在这一领域涉及肿瘤细胞株和小鼠模型的使用，以便更好地了解发起的遗传事件和发病机制的疾病6，7。近日，作为SOC的起源小说网站，已经出现输卵管输卵管（FT）的分泌上皮细胞（FTSEC）作为建议的起源8，9细胞。目前没有任何细胞系或文化系统研究“金融时报上皮或FTSEC。在这里，我们描述了一种新型的体外培养系统，其中首要人权FT上皮细胞培养的方式，可以保留他们的体系结构，极性，免疫，生理和遗传毒性应激。这种体外模型SOC的研究提供了一个有用的工具，允许更好地了解如何从这个组织的肿瘤可以产生，并参与肿瘤启动和进展的机制。

Watch this Scientific Journal Video about Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells at JoVE.com

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

输卵管（FT）是一种新兴的原产地为浆液性卵巢癌（SOC）的另一幅土地。本协议描述了一种新方法和隔离<em&gt;体外</em&gt;文化输卵管上皮细胞。此系统概括了<em&gt;在体内</em&gt;上皮和允许SOC的发病机制的研究。

前初级人类输卵管上皮细胞的体外培养

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

ex-vivo-culture-of-primary-human-fallopian-tube-epithelial-cells

Research

JoVE Journal

Biology

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

19.5K 次观看.  Dana-Farber Cancer Institute. 输卵管（FT）是一种新兴的原产地为浆液性卵巢癌（SOC）的另一幅土地。本协议描述了一种新方法和隔离体外文化输卵管上皮细胞。此系统概括了在体内上皮和允许SOC的发病机制的研究。

视频: Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Method for Culture of Early Chick Embryos ex vivo (New Culture)

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying  the formation and patterning of brain, neural tube, somite and heart primordia. Applications of chick embryo culture include electroporation of DNA or RNA constructs in order to analyze gene function, grafts of growth factor coated beads such as FGFs and BMPs , as well as whole mount in situ hybridization and immunohistochemistry. This video demonstrates the different steps in chick embryo culture; First, the embryo is explanted in saline. Then, the embryo is centered on a glass ring. The membranes surrounding the embryo are lifted along the walls of the ring. The ring is then placed in a culture dish containing a pool of albumine. The culture dish is sealed and placed in a humid chamber, where the embryo is cultured for up to 24 hrs. Finally, the embryo is removed from the ring, fixed and processed for further applications. A troubleshooting guide is also presented.

Method for Culture of Early Chick Embryos ex vivo New Culture

This video demonstrates New culture, a method by which chick embryos are cultured outside the egg for up to 24 hr. This method enables one to study early development (primitive streak to 14 som.), a period corresponding to E7-9 in mouse. Applications of this technique include electroporation, in situ hybridization and immunohistochemistry.

文化早期鸡胚体外（新文化）的方法

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

科研

生物学

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

从外植体生长的首要人权支气管上皮细胞

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

成人从人体皮肤的上皮干细胞的分离和培养

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells (HSCs) differentiate into oligopotent progenitors, committed precursors and mature red blood cells 1. This process is regulated for a large part at the level of gene expression, whereby specific transcription factors activate lineage-specific genes while concomitantly repressing genes that are specific to other cell types 2. Studies on transcription factors regulating erythropoiesis are often performed using human and murine cell lines that represent, to some extent, erythroid cells at given stages of differentiation 3-5. However transformed cell lines can only partially mimic erythroid cells and most importantly they do not allow one to comprehensibly study the dynamic changes that occur as cells progress through many stages towards their final erythroid fate. Therefore, a current challenge remains the development of a protocol to obtain relatively homogenous populations of primary HSCs and erythroid cells at various stages of differentiation in quantities that are sufficient to perform genomics and proteomics experiments.
Here we describe an ex vivo cell culture protocol to induce erythroid differentiation from human hematopoietic stem/progenitor cells that have been isolated from either cord blood, bone marrow, or adult peripheral blood mobilized with G-CSF (leukapheresis). This culture system, initially developed by the Douay laboratory 6, uses cytokines and co-culture on mesenchymal cells to mimic the bone marrow microenvironment. Using this ex vivo differentiation protocol, we observe a strong amplification of erythroid progenitors, an induction of differentiation exclusively towards the erythroid lineage and a complete maturation to the stage of enucleated red blood cells. Thus, this system provides an opportunity to study the molecular mechanism of transcriptional regulation as hematopoietic stem cells progress along the erythroid lineage. 
Studying erythropoiesis at the transcriptional level also requires the ability to over-express or knockdown specific factors in primary erythroid cells. For this purpose, we use a lentivirus-mediated gene delivery system that allows for the efficient infection of both dividing and non-dividing cells 7. Here we show that we are able to efficiently knockdown the transcription factor TAL1 in primary human erythroid cells. In addition, GFP expression demonstrates an efficiency of lentiviral infection close to 90%. Thus, our protocol provides a highly useful system for characterization of the regulatory network of transcription factors that control erythropoiesis.

Lentiviral-mediated Knockdown During Ex Vivo Erythropoiesis of Human Hematopoietic Stem Cells

An ex vivo protocol to generate mature human red blood cells from hematopoietic stem/progenitors is described. Additionally we describe an efficient lentiviral-delivery method to knockdown the transcription factor TAL1 in primary erythroid cells. The efficiency of lentivirus mediated gene delivery is demonstrated using GFP expressing viruses.

慢病毒介导的击倒在体外</em人造血干细胞>红细胞

Erythropoiesis is a commonly used model system to study cell differentiation. During erythropoiesis, pluripotent adult human hematopoietic stem cells ...

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts migrate through the epidermis of the embryo where they subsequently colonize the developing hair follicles1,2. Neural crest cell migration is extensively studied in vitro but in vivo methods are still not well developed, especially in mammalian systems. One alternative is to use ex vivo organotypic culture3-6. Culture of mouse embryonic skin requires the maintenance of an air-liquid interface (ALI) across the surface of the tissue3,6. High resolution live-imaging of mouse embryonic skin has been hampered by the lack of a good method that not only maintains this ALI but also allows the culture to be inverted and therefore compatible with short working distance objective lenses and most confocal microscopes. This article describes recent improvements to a method that uses a gas permeable membrane to overcome these problems and allow high-resolution confocal imaging of embryonic skin in ex vivo culture6. By using a melanoblast specific Cre-recombinase expressing mouse line combined with the R26YFPR reporter line we are able to fluorescently label the melanoblast population within these skin cultures. The technique allows live-imaging of melanoblasts and observation of their behavior and interactions with the tissue in which they develop. Representative results are included to demonstrate the capability to live-image 6&#160;cultures in parallel.

Ex vivo Culture of Mouse Embryonic Skin and Live-imaging of Melanoblast Migration

We describe the dissection and ex vivo culture of mouse embryonic skin. The culture system maintains an air-liquid interface across the tissue surface and allows imaging on an inverted microscope. Melanoblasts, a component of the developing skin, are fluorescently labeled allowing their behavior to be observed using confocal microscopy.

小鼠胚胎皮肤和Live成像黑素细胞迁移的体外培养

Melanoblasts are the neural crest derived precursors of melanocytes; the cells responsible for producing the pigment in skin and hair. Melanoblasts ...

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of airway cell differentiation and function. In the past few years, the use of lentiviral vectors for transgene delivery became common practice. While there are reports of transduction of fully differentiated airway epithelial cells with certain non-HIV pseudo-typed lentiviruses, the overall transduction efficiency is usually less than 15%. The protocol presented here provides a reliable and efficient method to produce lentiviruses and to transduce primary human bronchial epithelial cells. Using undifferentiated bronchial epithelial cells, transduction in bronchial epithelial growth media, while the cells attach, with a multiplicity of infection factor of 4 provides efficiencies close to 100%. This protocol describes, step-by-step, the preparation and concentration of high-titer lentiviral vectors and the transduction process. It discusses the experiments that determined the optimal culture conditions to achieve highly efficient transductions of primary human bronchial epithelial cells.

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

优化慢病毒生产和细胞培养必要条件，成功转导初级人支气管上皮细胞

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

由上皮细胞和原发免疫细胞组成的多细胞人类Alveal模型，用于危险评估

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

分离和富集人肺上皮祖细胞用于类器官培养

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...

Isolation, Culture, and Genetic Engineering of Mammalian Primary Pigment Epithelial Cells for Non-Viral Gene Therapy

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a multifactorial disease influenced by environmental and genetic factors, which lead to functional impairment of the retina due to retinal pigment epithelial (RPE) cell degeneration followed by photoreceptor degradation. An ideal treatment would include the transplantation of healthy RPE cells secreting neuroprotective factors to prevent RPE cell death and photoreceptor degeneration. Due to the functional and genetic similarities and the possibility of a less invasive biopsy, the transplantation of iris pigment epithelial (IPE) cells was proposed as a substitute for the degenerated RPE. Secretion of neuroprotective factors by a low number of subretinally-transplanted cells can be achieved by Sleeping Beauty (SB100X) transposon-mediated transfection with genes coding for the pigment epithelium-derived factor (PEDF) and/or the granulocyte macrophage-colony stimulating factor (GM-CSF). We established the isolation, culture, and SB100X-mediated transfection of RPE and IPE cells from various species including rodents, pigs, and cattle. Globes are explanted and the cornea and lens are removed to access the iris and the retina. Using a custom-made spatula, IPE cells are removed from the isolated iris. To harvest RPE cells, a trypsin incubation may be required, depending on the species. Then, using RPE-customized spatula, cells are suspended in medium. After seeding, cells are monitored twice per week and, after reaching confluence, transfected by electroporation. Gene integration, expression, protein secretion, and function were confirmed by qPCR, WB, ELISA, immunofluorescence, and functional assays. Depending on the species, 30,000-5 million (RPE) and 10,000-1.5 million (IPE) cells can be isolated per eye. Genetically modified cells show significant PEDF/GM-CSF overexpression with the capacity to reduce oxidative stress and offers a flexible system for ex vivo&#160;analyses and in vivo studies transferable to humans to develop ocular gene therapy approaches.

Here, a protocol to isolate and transfect primary iris and retinal pigment epithelial cells from various mammals (mice, rat, rabbit, pig, and bovine) is presented. The method is ideally suited to study ocular gene therapy approaches in various set-ups for ex vivo&#160;analyses and&#160;in vivo studies transferable to humans.

哺乳动物原色素上皮细胞的分离、培养和基因工程用于非病毒基因治疗

Age-related macular degeneration (AMD) is the most frequent cause of blindness in patients &#62;60 years, affecting ~30 million people worldwide. AMD is a ...

Efficient Dissection and Culture of Primary Mouse Retinal Pigment Epithelial Cells

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to understand the pathophysiology of ocular diseases because of their similarities with human anatomy and physiology. Alterations in the retinal pigment epithelium (RPE), including changes in morphology and function, are common features shared by many ocular disorders. However, successful isolation and culture of primary mouse RPE cells is very challenging. This paper is an updated audiovisual version of the protocol previously published by Fernandez-Godino et al. in 2016 to efficiently isolate and culture primary mouse RPE cells. This method is highly reproducible and results in robust cultures of highly polarized and pigmented RPE monolayers that can be maintained for several weeks on Transwells. This model opens new avenues for the study of the molecular and cellular mechanisms underlying eye diseases. Moreover, it provides a platform to test therapeutic approaches that can be used to treat important eye diseases with unmet medical needs, including inherited retinal disorders and macular degenerations.

This protocol, which was originally reported by Fernandez-Godino et al. in 20161, describes a method to efficiently isolate and culture mouse RPE cells, which form a functional and polarized RPE monolayer within one week on Transwell plates. The procedure takes approximately 3 hours.

原发性小鼠视网膜色素上皮细胞的有效解剖与培养

Eye disorders affect millions of people worldwide, but the limited availability of human tissues hinders their study. Mouse models are powerful tools to ...