我们感谢技术援助和博士Shivraj Tyagi埃梅卡Ifedigbo宝贵的专业知识。我们也感谢哈佛NeuroDiscovery中心与显微镜的帮助。这项工作是支持部分由美国心脏协会授予09PRE2250120 Predoctoral林Hilaire指出，与美国国立卫生研究院拨款，以R01 - HL60234，R01 - HL55330，R01 - HL079904，授予AMK的财。

整个协议需要2天从动物组织中的细胞，细胞增殖的5-10天，并在气 - 液界面的细胞分化的一个额外的10-14天的隔离。一个额外的一天所需的细胞暴露和样品的收获。 1。鼠标气管，支气管上皮细胞（MTEC）的分离。 注意：下面描述的所有程序进行了审查和批准机构的动物护理和使用布里格姆与妇女医院/哈佛医学院区委员。 在开始之前： <ul><li>准备含有抗生素火腿的F12媒体。到250毫升火腿的F12（Cellgro）基底媒体添加2.50毫升的100 x青霉素/链霉素（10 2 U/mL-10 2毫克/毫升）溶液和250μL一个1000 X Fungizone解决方案。在4 ° C至4个星期的商店。 </li><li>准备0.15％链霉蛋白酶的解决方案。添加15毫克链霉蛋白酶至10毫升火腿的F12含有抗生素的媒体。做出新的，使用之前保持冰。 </li><li>准备一个干净的工作表面和无菌手术器械，适合于小动物手术，以及层状组织文化细胞隔离罩。所有其他设备被认为是动物细胞培养，包括加湿的CO 2孵化器，寒冷的房间，桌上细胞离心机，倒置显微镜，和一次性塑料吸管和文化制品的标准。 </li><li>准备Ⅰ型胶原溶液（0.02 N醋酸50微克/毫升）。移液器1.0毫升到每12 Transwell小板（康宁）以及胶原蛋白的解决方案。包装在封口膜，让站在一个平坦的表面在室温下过夜。 </li></ul><ol><li>使用市售的小鼠品系（即，C57BL / 6，男性6-8周龄），安乐死批准安乐死的方法，如CO 2，诱导麻醉的小鼠。通常情况下，6小鼠产生足够的细胞（1.5-2.0 × 10 5细胞/鼠标）种子12孔Transwell小板。 </li><li>喷雾用70％乙醇溶液，消毒领域的动物尸体。 </li><li>用干净的手术剪，手术刀，取出气管周围地区的皮肤，暴露气管。打开腹部，切沿胸骨，取出肋骨。删除组织，直到气管年底暴露。 </li><li>到50 mL锥形管含有30毫升火腿的F12媒体含有抗生素，在冰上放置气管。 </li><li>在无菌的板层流罩，气管组织转移到100毫米的无菌培养皿中含有10毫升火腿的F12媒体含有抗生素。 </li><li>用无菌镊子和手术剪，轻轻地剖析结缔组织。 </li><li>气管组织转移到一个新的100毫米的培养皿中含有10毫升火腿的F12媒体含有抗生素冲洗。暴露管腔沿垂直轴切气管。 </li><li>气管转移到0.15％链霉蛋白酶溶液10毫升50毫升试管中孵育4℃过夜° C。 </li><li>第二天，有准备： <ul><li>准备一个DNA酶I溶液。火腿的F12媒体含有抗生素10 mg / mL的牛血清白蛋白（BSA）原液，加2 ml和原油胰腺DNA酶一10毫克设为1毫升分装保存在-20 ° C至18毫升（解冻上使用前的冰）。 </li><li>准备含有20％胎牛血清（FBS），抗生素火腿的F12媒体。至200 ml火腿的F12（Invitrogen）的基底媒体添加50毫升热灭活胎牛血清，2.5毫升的100 x青霉素/链霉素（10 2 U/mL-10 2毫克/毫升）的解决方案，和250μL一个1000 X Fungizone解决方案。 </li><li>准备MTEC基本培养基中含有抗生素。 475.5毫升的DMEM/F12（Cellgro）基本培养基中添加7.5毫升1 M羟乙基解决方案，10毫升200毫米谷氨酰胺的解决方案，2毫升7.5％的碳酸氢钠溶液，5毫升的100 x青霉素/链霉素溶液，500μL，一个1000 X Fungizone解决方案</li><li>准备MTEC medium/10％胎牛血清。 MTEC基本培养基含有抗生素要45毫升，加入5毫升热灭活胎牛血清。 </li></ul></li><li>轻轻摇动管（步骤1.8）10-12次，然后让它站立30-60分钟，在4 ° C。 </li><li>添加10毫升火腿的F12媒体含20％胎牛血清和抗生素管和岩石的12倍。 </li><li>准备3月15日毫升含有10毫升火腿的含有20％胎牛血清和抗生素的F12媒体的锥形管。 </li><li>从链霉蛋白酶溶液中取出气管，撇开这个解决方案上的冰。传输气管含火腿的F12键的第一个锥形管和仰拱管的12倍。重复此过程两次。 </li><li>结合三个步骤1.13上清到一个50 mL试管链霉蛋白酶的解决方案。丢弃余下的组织。 </li><li>在1400rpm离心10分钟在4 ° C，并弃上清（390 XG的Eppendorf 5810R离心机配备了一架A - 4 - 62转子）。 </li><li>轻轻重悬颗粒（100〜200μL/气管在1毫升DNA酶溶液）在冰上孵育5分钟。 </li><li>在1400rpm（390 XG）离心5分钟，4 ° C，弃上清。 </li><li> 8毫升MTEC含10％胎牛血清的介质中，重悬细胞沉淀</li><li>盘上Primaria板的细胞悬液（猎鹰）。在37 ° C 95％的空气气氛中，5％CO 2为5小时（注：这是为成纤维细胞的负选择步骤）。 </li><li>从板收集细胞悬液和冲洗，用4毫升MTEC含10％胎牛血清板的两倍。池细胞悬液和洗涤，在50 mL锥形管。 </li><li>节约1毫升cytospin和细胞计数。在台式离心机旋转5分钟5000转（Eppendorf公司5415D）。在剩余的上清液中取出500μL和重新悬浮颗粒。使用100μL的细胞计数采用台盼蓝活体染色方法。保护cytospin分析4等分100μL </li><li>转速1400rpm（390 XG）余下的15 mL细胞悬液，在4 ° C为10分钟。 </li></ol> 2。在气 - 液界面的繁殖和分化的MTEC 准备维甲酸股票方案。原液答：称取在黑暗中的视黄酸（议员300.44克/摩尔）在95％乙醇使一个5毫米的原液。商店在铝箔包裹管在-80 ° C根据需要，准备通过增加库存为50μL，500μL，BSA溶液（100毫克/毫升）和49.5毫升汉克的平衡盐溶液（HBSS）股票溶液B（5微米）。箔包裹于-80 ° C至4个星期的管店。 准备MTEC增殖培养基中含有维甲酸。为45.7毫升MTEC含有抗生素的基础介质，添加2.5毫升热灭活胎牛血清，1毫升维甲酸B股，250μL胰岛素溶液（2 mg / ml的4毫米盐酸胰岛素），250μL的表皮生长因子的解决方案（5微克/ mL的表皮生长因子在含有1 mg / mL的BSA哈佛商学院），200μL，牛垂体提取物（15毫克/毫升含1 mg / mL的BSA在哈佛商学院），50μL，转铁蛋白溶液（5 mg / mL的转含1 mg / mL的BSA在哈佛商学院），50μL霍乱毒素溶液（100毫克/毫升含1 mg / mL的BSA在哈佛商学院）。过滤消毒媒体的最后准备和使用在2天的准备。 <ol><li>取出Transwell小板的胶原溶液，让引擎盖下站立5分钟，用PBS洗涤2次。 </li><li>离心后，悬浮细胞沉淀，以方便在适当的音量增殖媒体（500μL）7.5 × 10 4 -1.0 × 10 5细胞，每孔电镀。移液器500μL到Transwell小聚碳酸酯膜插入根尖表面细胞悬液。加入1.5毫升扩散介质基底的Transwell小仓。 </li><li>淹没MTEC文化在37 ° C中含有95％的空气加湿孵化器，5％CO 2为7-10天。 </li><li>等待在不断变化的媒体首次前3天。更改媒体隔日。通过目测和跨上皮细胞电阻测量电极（EVOM Ohmvoltometer，世界精密仪器，萨拉索塔，佛罗里达州）的监视器文化。 </li><li>当细胞出现融合和上皮抵抗力达到1000Ω/ cm 2的细胞是准备在ALI的区分。 </li><li>准备MTEC含有2％NuSerum的基础媒体。 MTEC基本培养基，添加NuSerum到终浓度为2％V / V维甲酸B股加入到终浓度为1 × 10-7中号后方可使用。准备好新鲜，并使用2天之内。 </li><li>允许的细胞分化为10-14天，通过消除根尖媒体和750μL含有2％Nuserum和维甲酸的MTEC基底媒体取代基媒体，。更改基础的媒体和MTEC含2％NuSerum隔日用根尖方。 </li></ol> 3。卷烟烟气中培养上皮细胞中的应用<ol><li>揭露Transwell小文化的心尖的一面主流香烟烟雾中使用CS细胞曝光系统（EMI仪器，匹兹堡，宾夕法尼亚州）。仪器的照片，请参阅图1。请参阅特定的试剂和设备的技术规格表。简言之，该仪器由吸烟〜7-8喷香烟，主流烟绘制成一个聚丙烯生产线，是连接到一个通风曝光室蠕动泵。曝光室保持在37℃，循环水，气行，并保持在5％的CO 2的气氛。细胞可以接触到的烟雾使用肯塔基3R4F研究参考的过滤嘴卷烟（烟草研究所，肯塔基大学，肯塔基州列克星敦）。通常情况下，细胞暴露在1-2香烟的烟雾，总颗粒物（TPM）的高产100-200毫克/米3。控制文化暴露于室内空气相当于暴露期。 </li><li> TPM是根据制造商的协议，与TE提供- 10C吸烟机（蒂格企业）。简单地说，一个过滤器（Pallflex）被放置在一个行内领先的吸烟腔，一个恒流泵（抽样单位）从取样口取样管上的不锈钢架。流出管连接到燃气表（干式煤气表，ONM61，67，美国设备制造商协会）的抽样单位。该过滤器是权衡前后气体取样。总材料沉积在毫克的过滤器是空气采样量正常化。 </li><li>收获细胞，然后可以在任何时间点（即0-24小时）暴露后的标准单元分析程序（即，西方免疫印迹分析，基因分析等）或显微镜（即焦或电子显微镜）。培养基中还可以分析细胞因子或LDH的释放细胞毒性检测。 </li></ol> 4。代表性的成果扬智成功上皮分离，增殖，并分化产生一个完整的单层与鹅卵石形态。 Transepithelilal细胞性健康文化应约1000-2000Ω/厘米2。图2描绘了一个单层的上皮细胞和纤毛标记染色控制的情况下，香烟烟雾暴露后的小鼠呼吸道上皮细胞。图3显示了健康MTEC文化代表性的电子显微照片，描绘超微结构和纤毛形态。 <img src="/files/ftp_upload/2513/2513fig1.jpg" alt="图1" /> 图1。上皮细胞通过三个阶段，隔离一步，传播阶段，并在气-液界面（计划）的分化阶段所得的制备。种子，在那里他们深层培养增殖到transwells，从小鼠气管上皮细胞分离，然后转换成气 - 液界面，他们完全区分。实验通常是进行连续培养24 日 。画面描绘了一个模型系统主流香烟烟雾中的培养细胞暴露。一个Transwell小阿里文化系统被放置在一个自定义的模块化香烟烟雾中的输送系统，这是温度，湿度和CO 2控制。 <img src="/files/ftp_upload/2513/2513fig2.jpg" alt="图2" /> 图2上皮细胞培养，原子核固定和染色（Hoechst 33258荧光），F -肌动蛋白（绿色; Alexa的福陆公司的488 -标记的鬼笔环肽） 和纤毛标记的乙酰化的α-微管蛋白（红色; Cy3标记标记的二抗）。较低的面板显示图像相当于4小时后，暴露在香烟烟雾（2支香烟，150 毫克 /立方米）的上皮细胞。 （二）测定乳酸脱氢酶（LDH）释放在24小时后暴露在香烟烟雾中的不同剂量表示的培养基。 <img src="/files/ftp_upload/2513/2513fig3.jpg" alt="图3" /> 图3。上皮细胞从小鼠气管上皮分离气-液界面培养区分几个亚型，通过透射电子显微镜（TEM）有纤毛，基底和非纤毛细胞的特点。这些细胞典型假的呼吸道上皮。图标签对应：BB：基体，轴丝，M：线粒体，N：细胞核，S：基板（聚碳酸酯膜），MV：microvili

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协议描述小鼠气管上皮细胞的分离，是改编自你等的协议 ，1，和其他修改2-3。至于任何协议，描述细胞的隔离，最关键的环节，以避免细菌或真菌病原体的污染，采用严格的无菌操作技术。第二个关键步骤，以避免成纤维细胞污染的文化，可避免的气管仔细解剖，和阴性选择步骤1.19。文化监控，洗净，和文化传媒补充隔日最初的72小时孵化后，应充分区分文化在大约两个?...

*吸烟机是一个定制的设计和制造14“X14”X20“双墓室和水套光色调清楚地证明1 / 2”厚的聚碳酸酯的Lexan室温度控制，水位传感器香烟烟雾暴露控制关闭系统。一个吸烟/膨化机是安装一个变量的膨化率的香烟。当单位在使用时，它模仿孵化器中的温度，湿度和二氧化碳控制系统中的意义。该系统包括：（一）一个定制的双室/水套单元，保持一个控制的环境中进行组织培养实验。 （II）数字重型，高精度双泵水温水位传感器和温度控制（三​​）香烟与吸烟单位膨化泵循环体系。 （四）泵循环传感器控制率循环仪（IV）不锈钢高精密线滤波器持有。 （五）安装一个可拆卸的盖子“大小的轴向均匀香烟烟雾混合风扇（六）一个中等大小的水的循环与安装支架水浴（七）11 / 2 1 / 2”厚的有机玻璃托盘的括号粉扑泵和烟灰收藏家持有。 这描述的机器可以取代类似市售一种从谢系统（如吸烟<a href="http://www.tse-systems.com">机www.tse -</a> systems.com） 。

isolation of mouse respiratory epithelial cells and exposure to experimental cigarette smoke at air liquid interface

肺上皮细胞可以分离出小鼠的呼吸道及气 - 液界面（ALI）的培养作为一个差异化的呼吸道上皮模型。协议是描述卷烟主流烟雾（CS）隔离和揭露这些细胞，上皮细胞的反应，以研究为CS暴露。该协议由三部分组成：隔离的呼吸道上皮细胞从小鼠气管，气 - 液界面（ALI）作为完全分化的上皮细胞，这些细胞的培养，这些细胞在文化和校准主流CS交付。 ALI的文化系统允许的条件下，更接近于他们比普通的液体培养系统的生理设置的呼吸道上皮细胞的文化。接触到CS分子和肺癌细胞反应的研究是了解环境空气污染对人体健康的影响的关键组成部分。最终，在这方面的研究结果可能有助于了解慢性阻塞性肺疾病（COPD），及其他与烟草有关的疾病，这​​代表了重大的全球性健康问题的病因。

Watch this Scientific Journal Video about Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface at JoVE.com

Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

从小鼠的呼吸道和肺上皮细胞可以分离，气液界面的差异化的呼吸道上皮模式培养。协议是分离，培养，这些细胞暴露卷烟主流烟雾，为了研究这种环境毒素的分子反应。

小鼠呼吸道上皮细胞和实验香烟烟雾暴露在空气分离液界面

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated ...

isolation-mouse-respiratory-epithelial-cells-exposure-to-experimental

Research

JoVE Journal

Medicine

33.8K 次观看.  Harvard Medical School. 从小鼠的呼吸道和肺上皮细胞可以分离，气液界面的差异化的呼吸道上皮模式培养。协议是分离，培养，这些细胞暴露卷烟主流烟雾，为了研究这种环境毒素的分子反应。

视频: Isolation of Mouse Respiratory Epithelial Cells and Exposure to Experimental Cigarette Smoke at Air Liquid Interface

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

人体实验肺炎球菌运输

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

科研

医学

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

分离，冻存和文化的人羊膜上皮细胞的临床应用

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Automated Measurement of Pulmonary Emphysema and Small Airway Remodeling in Cigarette Smoke-exposed Mice

COPD is projected to be the third most common cause of mortality world-wide by 2020(1). Animal models of COPD are used to identify molecules that contribute to the disease process and to test the efficacy of novel therapies for COPD. Researchers use a number of models of COPD employing different species including rodents, guinea-pigs, rabbits, and dogs(2). However, the most widely-used model is that in which mice are exposed to cigarette smoke. Mice are an especially useful species in which to model COPD because their genome can readily be manipulated to generate animals that are either deficient in, or over-express individual proteins. Studies of gene-targeted mice that have been exposed to cigarette smoke have provided valuable information about the contributions of individual molecules to different lung pathologies in COPD(3-5). Most studies have focused on pathways involved in emphysema development which contributes to the airflow obstruction that is characteristic of COPD. However, small airway fibrosis also contributes significantly to airflow obstruction in human COPD patients(6), but much less is known about the pathogenesis of this lesion in smoke-exposed animals. To address this knowledge gap, this protocol quantifies both emphysema development and small airway fibrosis in smoke-exposed mice. This protocol exposes mice to CS using a whole-body exposure technique, then measures respiratory mechanics in the mice, inflates the lungs of mice to a standard pressure, and fixes the lungs in formalin. The researcher then stains the lung sections with either Gill&#8217;s stain to measure the mean alveolar chord length (as a readout of emphysema severity) or Masson&#8217;s trichrome stain to measure deposition of extracellular matrix (ECM) proteins around small airways (as a readout of small airway fibrosis). Studies of the effects of molecular pathways on both of these lung pathologies will lead to a better understanding of the pathogenesis of COPD.

The goal of this protocol is to provide automated methods to quantify chronic lung pathologies in a murine model of COPD. The protocol includes exposing mice to cigarette smoke (CS), measuring pulmonary function, inflating the lungs, and using morphometry methods to measure emphysema and small airway remodeling in mice.

肺气肿，小气道重塑的香烟烟雾暴露小鼠自动测量

COPD is projected to be the third most common cause of mortality world-wide by 2020(1).  Animal models of COPD are used to identify molecules that ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

的肿瘤特异性内皮细胞的分离与培养扩展

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. The potential adverse health effects of electronic cigarette vapor (E-vapor) exposure are poorly defined. While several animal models of E-vapor exposure have been developed, few models expose rodents to clinically relevant quantities of nicotine and make direct comparisons to cigarette smoke within the same exposure system. Here, we present a method for constructing and operating an E-vapor chamber and cigarette smoke chamber. The chambers are constructed by outfitting anesthesia chambers with a computer controlled pumping system that delivers consistent amounts of E-vapor or cigarette smoke to rodents. Nicotine exposure is measured indirectly by quantifying pre and post-exposure serum cotinine levels. This exposure system can be modified to accommodate various types of E-cigarettes&#160;and tobacco cigarettes, and can be used to compare the effects of E-vapor&#160;and cigarette smoke in vivo.

Comparing the Effects of Electronic Cigarette Vapor and Cigarette Smoke in a Novel In Vivo Exposure System

This protocol describes a method for exposing rodents to electronic cigarette vapor (E-vapor) and cigarette smoke. Exposure chambers are constructed by modifying anesthesia chambers with an automated pumping system that delivers E-vapor or cigarette smoke to rodents. This system can easily be modified to accommodate many experimental endpoints.

比较电子烟和烟雾在小说中的影响<em&gt;体内</em&gt;曝光系统

Electronic cigarettes (E-cigarettes) are being widely used, and growing in popularity. It is estimated that more than 9 million adults use them regularly. ...

Generation of a Chronic Obstructive Pulmonary Disease Model in Mice by Repeated Ozone Exposure

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high incidence in aging populations. The current conventional therapies for COPD focus mainly on symptom-modifying drugs; thus, the development of new therapies is urgently needed. Qualified animal models of COPD could help to characterize the underlying mechanisms and can be used for new drug screening. Current COPD models, such as lipopolysaccharide (LPS) or the porcine pancreatic elastase (PPE)-induced emphysema model, generate COPD-like lesions in the lungs and airways but do not otherwise resemble the pathogenesis of human COPD. A cigarette smoke (CS)-induced model remains one of the most popular because it not only simulates COPD-like lesions in the respiratory system, but it is also based on one of the main hazardous materials that causes COPD in humans. However, the time-consuming and labor-intensive aspects of the CS-induced model dramatically limit its application in new drug screening. In this study, we successfully generated a new COPD model by exposing mice to high levels of ozone. This model demonstrated the following: 1) decreased forced expiratory volume 25, 50, and 75/forced vital capacity (FEV25/FVC, FEV50/FVC, and FEV75/FVC), indicating the deterioration of lung function; 2) enlarged lung alveoli, with lung parenchymal destruction; 3) reduced fatigue time and distance; and 4) increased inflammation. Taken together, these data demonstrate that the ozone exposure (OE) model is a reliable animal model that is similar to humans because ozone overexposure is one of the etiological factors of COPD. Additionally, it only took 6 - 8 weeks, based on our previous work, to create an OE model, whereas it requires 3 - 12 months to induce the cigarette smoke model, indicating that the OE model might be a good choice for COPD research.

This study describes the successful generation of a new chronic obstructive pulmonary disease (COPD) animal model by repeatedly exposing mice to high concentrations of ozone.

反复臭氧暴露对小鼠慢性阻塞性肺疾病模型的产生

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and lung parenchymal destruction. It has a very high ...

Assessment of the Acute Inhalation Toxicity of Airborne Particles by Exposing Cultivated Human Lung Cells at the Air-Liquid Interface

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to gases, particles or complex atmospheres (e.g., cigarette smoke), thus providing realistic physiological exposure of the apical surface of the human alveolar region to air. In contrast to sequential exposure models with linear aerosol guidance, the modular design of the radial flow system meets all requirements for the continuous generation and transport of the test atmosphere to the cells, a homogenous distribution and deposition of the particles and the continuous removal of the atmosphere. This exposure method is primarily designed for the exposure of cells to airborne particles, but can be adapted to the exposure of liquid aerosols and highly toxic and aggressive gases depending on the aerosol generation method and the material of the exposure modules.
Within the framework of a recently completed validation study, this exposure system was proven as a transferable, reproducible and predictive screening method for the qualitative assessment of the acute pulmonary cytotoxicity of airborne particles, thereby potentially reducing or replacing animal experiments that would normally provide this toxicological assessment.

We present a robust, transferable and predictive in vitro exposure system for the screening and monitoring of airborne particles concerning their acute pulmonary cytotoxicity by exposing cultivated human lung cells at the air-liquid interface (ALI).

通过在空气-液体界面中暴露培养的人类肺细胞，评估空气中颗粒的急性吸入毒性

Here, we present a specially designed modular in vitro exposure system that enables the homogenous exposure of cultivated human lung cells at the ALI to ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

睡眠美人转座转染人视网膜色素上皮细胞氧化应激的诱导与分析

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Isolation and Profiling of Human Primary Mesenteric Arterial Endothelial Cells at the Transcriptome Level

Endothelial cells (ECs) are crucial for vascular and whole-body function through their dynamic response to environmental cues. Elucidating the transcriptome and epigenome of ECs is paramount to understanding their roles in development, health, and disease, but is limited in the availability of isolated primary cells. Recent technologies have enabled the high-throughput profiling of EC transcriptome and epigenome, leading to the identification of previously unknown EC cell subpopulations and developmental trajectories. While EC cultures are a useful tool in the exploration of EC function and dysfunction, the culture conditions and multiple passages can introduce external variables that alter the properties of native EC, including morphology, epigenetic state, and gene expression program. To overcome this limitation, the present paper demonstrates a method of isolating human primary ECs from donor mesenteric arteries aiming to capture their native state. ECs in the intimal layer are dissociated mechanically and biochemically with the use of particular enzymes. The resultant cells can be directly used for bulk RNA or single-cell RNA-sequencing or plated for culture. In addition, a workflow is described for the preparation of human arterial tissue for spatial transcriptomics, specifically for a commercially available platform, although this method is also suitable for other spatial transcriptome profiling techniques. This methodology can be applied to different vessels collected from a variety of donors in health or disease states to gain insights into EC transcriptional and epigenetic regulation, a pivotal aspect of endothelial cell biology.

The protocol describes the isolation, culture, and profiling of endothelial cells from human mesenteric artery. Additionally, a method is provided to prepare human artery for spatial transcriptomics. Proteomics, transcriptomics, and functional assays can be performed on isolated cells. This protocol can be repurposed for any medium- or large-size artery.