我们感谢迈阿密大学的生命器官联盟追回署提供的肺部。我们也感谢丽莎昆兹博士用于图2B和3-D，本Gerovac博士和丽莎·诺瓦克在图3A至C.用于实验的mCherry病毒所示的实验DNA准备，我们也感谢加布里埃尔Gaidosh从成像设备，眼科在迈阿密大学。 这项研究是由来自美国国立卫生研究院，在CF基金会和乘务员医学研究所的马蒂亚斯Salathe博士助学金资助。

1.第一阶段:HEK 293T文化<ol><li>制备HEK细胞培养基中添加10％（V / V）胎牛血清（FBS）和1％（体积/体积）青霉素/链霉素，以高糖的Dulbecco氏改良Eagle氏培养基（DMEM）（简称为HEK介质）。 </li><li>涂布一10厘米组织培养皿胶原I.混合30胶原微升我在2ml蒸馏水中。扩散混合到培养皿孵育在37℃下2小时和5％ 的 CO 2。 </li><li>取出混合物，并让在层流柜的菜干燥15分钟。 </li><li>板HEK细胞上以在10毫升的HEK媒体1×10 6个细胞的密度涂布培养皿孵育在37℃和5％的CO 2。 </li><li>当HEK细胞50-60％融合（ 图1a，电镀后2-3天），进行第2阶段的Visual汇合的评估就足够了。不需要定量。 注意:HEK细胞是在HEK培养基（见上文）。当他们到达汇合，T哎可以分割（ 例如 1/5）或冷冻下来以后使用。 </li></ol> 2.第二阶段:在HEK细胞生产和慢病毒（LVS）的浓度注:本协议应与机构和政府规章线以下的BSL-2在美国+（增强BSL-2）的条件下执行。 <ol><li>使用钙沉淀过程转染（脂质体和其它转染剂不如钙沉淀步骤，有效）。市售的转染试剂盒的用途，建议以保证再现性，虽然该过程修改如下。将所有试剂置于室温。这为0的一天。 </li><li>对于HEK293细胞每10 cm培养皿，搭配4.5微克包膜DNA PMD2-VSVG的，7.5微克包装DNA pMDLg / pRRE，3.75微克DNA PRSV-REV，慢病毒表达载体的10微克DNA，86微升的2M钙溶液和无菌水在一15ml离心管，以700微升的最终体积（在商业转染试剂盒中提供的后两个解）。 </li><li>逐滴添加2×HEPES-缓冲盐水的700微升（HBS，在转染试剂盒中提供），而涡旋（在层流柜）并在室温下温育30分钟（执行步骤4在等待）。 </li><li>在早期的孵化时间，定金5微升幻灯片上的搭配。检查液滴是10X的目标，在显微镜下，重点慢慢向上和向下。一种细的沉淀物应该是可见的，证实了磷酸钙沉淀法（ 图1b）。 </li><li>通过加入10ml的HEK细胞介质和移液器上下的混合停止30分钟后的沉淀。 </li><li>以HEK细胞的培养皿步骤2.1-2.5，并取出介质（0天）。小心缓慢地添加来自步骤2.5的沉淀溶液沿着盘的边缘。 </li><li>次日（第1天），除去并丢弃培养基含有沉淀并更换瓦特第i个10毫升HEK培养基。检查在显微镜下精细沉淀物:它应该是在细胞（ 图1C和D）之间的空白空间的菜可见。 </li><li>第2天，收集培养基用注射器，过滤器通过0.45μm针筒过滤器进含有3.8毫升40％聚乙二醇（PEG）溶液15毫升离心管中。倒置5倍以混合并储存在4℃下至少24小时（对于PEG沉淀形成，但超过5天不再），直到所有的病毒集合是完整的。 </li><li>天3和4。第4天，重复步骤2.8不HEK中等取代但10％的漂白粉消毒和丢弃的菜。 </li><li>离心机所有收集管（通常在第5天在一起的所有集合）于1650×g离心20分钟，在4℃以沉淀病毒。除去并弃去上清液，并在4℃下于1650 xg离心再次旋转5分钟以收集和去除的PEG上清的任何剩余。 </li><li> Resus挂起在200μl支气管上皮生长培养基（BEGM）的每个管的无两性霉素B 8沉淀。池他们在一起，并在200微升等分试样。在-80°C储存病毒。分装额外的10微升进行HIV-1加格（P24）抗原ELISA检测。 </li><li>使用商业ELISA试剂盒，根据制造商的协议来执行一个p24抗原测定。该试验给出了以pg / ml的病毒的p24的估计。 </li></ol> 3.第三阶段:培养初HBE细胞<ol><li>在10ml BEGM介质板HBE细胞（用100μl两性霉素B，如果通道0细胞用于消除可能的真菌污染）在我涂覆10厘米组织培养皿胶原一个1×10 6个细胞的密度。在37℃和5％的CO 2。 </li><li>次日，除去培养基并用10ml BEGM取代（用100μl两性霉素B为通道0细胞），共4天。返回到孵化器。 </li><li>经过4天AYS，只使用不BEGM两性霉素B更改媒体隔日1次。 </li><li>当细胞为80-90％汇合（ 图2a;〜铺板后7天），则继续转导（注意3.5用于制备转导之前）。再次，汇合的视觉评估是足够的。不需要定量。 </li><li>前转导，外衣用IV型胶原溶液的透气性支承插入物稀释1/10用无菌蒸馏水。加入200μl到每个插入，并让在层流柜干燥过夜。 </li></ol> 4.第四阶段:支气管上皮细胞转导<ol><li>除去介质，用磷酸盐缓冲盐水（EDTA / PBS）中加入3毫升0.1N％胰蛋白酶在1mM的乙二胺四乙酸和孵育在37℃5分钟和5％ 的 CO 2。检查显微镜（10X物镜）下，看看是否所有单元被分离。如果没有，返回到培养箱中另外5分钟。 </li><li>当所有的细胞分离，加入3毫升大豆胰蛋白酶抑制剂（SBTI 1毫克/毫升），混合并转移至15毫升离心管中。通过在460 xg离心纺丝在室温下5分钟沉淀细胞。 </li><li>除去上清液，重悬沉淀于3ml BEGM并具有计数继续。 </li><li>悬浮HBE细胞（现在通道1或P1）以2×10 5个细胞每112毫米透气性支承插入于400μlBEGM的（ 见表2）的比率。推断基于将要转导的透气性支承插入物的量这些数字。 </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 表面 </td><td> 表面积 </td><td> 细胞计数 </td><td> 媒体 </td><td> 体积（媒体） </td></tr><tr><td> 10cm皿</td><td> 52.7厘米2 </td><td> 1×10 6个 </td><td> BEGM </td><td> 10毫升</td> &lt;/ TR&gt; <tr><td> 12毫米过滤器</td><td> 1.13厘米2 </td><td> 2×10 5个 </td><td> BEGM </td><td> 400微升</td></tr><tr><td> 24毫米过滤器</td><td> 4.52厘米2 </td><td> 8×10 5个 </td><td> BEGM </td><td> 800微升</td></tr></tbody></table> 表2:每个细胞计数媒体推断。 <ol start="5"><li>使用的4感染复数（MOI）因子的多个，添加病毒的适当体积的细胞悬浮液。 </li><li>聚凝胺添加至2微克/毫升的最终浓度。 </li><li>免除400微升每透水支持插入混合（BEGM，HBE细胞，病毒和聚凝胺）进入顶部隔室，独自加入1ml BEGM到基底仓。在37℃和5％的CO 2总是板HBE细胞孵育过夜无病毒作为对照和测试嘌呤选择（如果适用）。 </li><li>第二天，对物OVE根尖和基底外侧培养基，用PBS洗，并替换为空气-液体界面（ALI）媒体8两个室。 </li><li>当细胞汇合，除去心尖流体（称为建立气 - 液界面）。继续隔日舱底部改变介质（ALI），直到他们达到完全成熟;通常为3至4周后。 注意:如果慢病毒载体含有一个选择基因，如嘌呤霉素，细胞可以通过根据选择浓度曲线加入嘌呤霉素进行选择。对于HBE细胞，1微克/ ml的浓度被确定为理想的转导的细胞的一致的选择。然而，这个浓度可能会有所不同其他细胞或条件，应由杀死曲线来确定。媒体已经更换后ALI媒体或更高版本（2-3天），允许选择基因充分表达嘌呤霉素的加入可以开始，早一天。一定要嘌呤霉素添加到一个插入使得h因为没有被转导。可以加入嘌呤霉素直至细胞完全分化。 </li></ol>

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The authors have nothing to disclose.

这里所概述的协议保证接近100％转导效率。但是，也有在此过程中是实现这一目标的重要步骤。例如，在转染过程中，析出物中的转导结构的存在（下降），或在培养皿后转导（ 图1b和d）该天是一个良好的转染都相关。没有沉淀物或附聚物的存在可能是由于在转染试剂中的一个的遗漏，pH值的问题，或坏质粒制备。如果沉淀缺失，有很高的可能性，没有将可见的第二天。...

复制缺陷型，假慢病毒的发展（LV）也为研究人员提供一个安全和有效的方法， 在体外 1提供转基因。由于它们的长期表达，这些LV也可以用于治疗剂的体内 2,3的输送。生产的LV需要人胚胎肾（HEK）细胞的几个质粒共转染:转移载体，包装的gag / POL，包装转速 ，和包络水泡性口炎病毒糖蛋白（VSV-G）的质粒。第三代包装系统被认为比第二代系统更安全，因为转速基因被编码在一个单独的包装质粒，从而降低重组的可能性，以产生具有复制能力的慢病毒。虽然第二代包装系统收率五倍高的总转导单元，原病毒基因组的分割以创建第三代系统已降低转导的水平仅最低限度3,4。 而细胞系可更容易和有效地转导，研究人员已经将重点转向主细胞，因为这些是更具有代表性的体内组织中5,6。然而，原代细胞是难治转导。而完全分化呼吸道上皮细胞的转导已经报道，整体效率还没有超过15％的7。 这里描述的是一个协议，允许生产高滴度的LV的。一个一步一步的方法概述实现近100％的转导效率分为原发性支气管上皮细胞。更具体地，该协议描述器乐成功3的最佳培养条件。简言之，HEK 293T细胞使用的是带有增强型绿色荧光的EF-1启动子驱动表达慢病毒载体转染PCDH蛋白（EGFP）或mCherry，红色荧光蛋白，和一个第三代包装系统。释放到介质的LV被收集24至72小时后。病毒颗粒集中用聚乙二醇（PEG），病毒浓度的方便和简单的方法，采用了p24抗原的酶联免疫吸附测定（ELISA）试剂盒滴度估计之前。随后，未分化的初级HBE细胞被转导在增殖培养基（支气管上皮生长培养基或BEGM）8，而安装（被胰蛋白酶处理后），在感染复数的4（MOI）因子，加入聚凝胺和温育16小时（过夜）。然后允许细胞分化。由于病毒的转基因被表达长期（使用适当的启动子，见讨论），表达将在整个分化维持成假呼吸道上皮或可以（使用诱导型启动子）的分化后诱导。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEK293T/17 cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)&#160;</td>
			<td>Sigma</td>
			<td>12306C</td>
			<td>use at 10%</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (100x)</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD Biosciences</td>
			<td>354231</td>
			<td>dilute 1:75 in distilled water</td>
		</tr>
		<tr>
			<td>Calphos</td>
			<td>Clontech</td>
			<td>631312</td>
			<td>Transfection kit containing 2M Calcium solution, 2X HEPES-Buffered solution (HBS) and sterile H2O</td>
		</tr>
		<tr>
			<td>Polythylene glycol 8000</td>
			<td>VWR scientific</td>
			<td>101108-210</td>
			<td>stock of 40%</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A9528</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell 12mm</td>
			<td>Corning</td>
			<td>3460</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma</td>
			<td>C7521</td>
			<td>dilute 1:10 in distilled water</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>stock of 2mg/ml</td>
		</tr>
		<tr>
			<td>Puromycin (10.000x)</td>
			<td>Thermo Scientific</td>
			<td>A11138-03</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Alliance HIV-1 p24 ELISA</td>
			<td>PerkinElmer</td>
			<td>NEK050001KT</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Thermo Scientific</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>pCDH-EF1-MCS-IRES-Puro</td>
			<td>System Biosciences</td>
			<td>CD532A-2</td>
			<td>lentiviral expression vector</td>
		</tr>
		<tr>
			<td>pMD2-VSVG</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pRSV-rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td>packaging DNA</td>
		</tr>
	</tbody>
</table>

optimal lentivirus production and cell culture conditions necessary to successfully transduce primary human bronchial epithelial cells

在使用空气-液体界面条件下提供了一个有用的模型来研究气道细胞分化和功能的处理原代人支气管上皮（HBE）细胞的体外培养。在过去的几年中，为转基因递送中使用的慢病毒载体的成为普遍的做法。虽然有完全分化的呼吸道上皮细胞与某些非HIV伪类型的慢病毒转导的报道，整体转导效率通常低于15％左右。这里介绍的协议提供了可靠和有效的方法，以产生慢病毒和转导原代人支气管上皮细胞。用未分化的支气管上皮细胞，支气管上皮生长培养基转导，而细胞附着，具有4感染因子的多个提供接近100％的效率。本协议描述的，一步一步的，高滴度的慢病毒载体及日的准备和浓度Ë传导过程。它讨论了确定的最佳培养条件，以实现初级人支气管上皮细胞的高效转导的实验。

图1描绘了在转染过程评估细胞和溶液的不同的关键步骤。 图1A示出的HEK293T细胞的转染病毒的产生之前最佳汇合。重要的是，将细胞在整个培养皿均匀分布。 图1b中揭示了在使用明视野光学系统和一个10X物镜的转染混合物一滴沉淀。越的析出物像砂粒，而不是附聚物，更有效的转染将。在这张图片中，沉淀物是有点粗糙。该沉淀物是?...

Watch this Scientific Journal Video about Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells at JoVE.com

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce 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 ...

optimal-lentivirus-production-cell-culture-conditions-necessary-to

Research

JoVE Journal

Biology

19.1K 次观看.  University of Miami, Miller School of Medicine. 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.

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

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

人类胚胎干细胞：启动文化冷冻细胞

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

科研

生物学

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

慢病毒生产

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

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 ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

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 uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

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 ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

增强生物安全与聚合物复合物Ecotropic慢病毒的人体细胞的转导

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

诱导上皮和分析间质转化

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

大鼠肾上腺皮质细胞的原代培养及非甾体功能的测定

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

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, ...