作者感谢杜兰大学细胞和分子生物学系的 Dr. 罗伯特. Dotson 协助编辑杜兰大学化学系的手稿和 Dr. 詹姆斯. 布林协助采用质谱法设计。作者进一步承认杜兰大学细胞和分子生物学系和杜兰大学化学系的支持和使用空间和设备。这项工作得到了烟草产品管理科学研究金和杜兰大学科学与工程学院安德森的支持。

1. 装置的装配
<ol><li>将100毫升傅书礼烧瓶 (图 1, #4) 固定在钢制圆环架上, 并通过填充50克氯化钙来形成一个真空阱, 作为干燥剂。用橡胶通孔塞子密封烧瓶, 用石蜡膜包裹塞子接合处, 并在孔中运行吸管。</li>
	<li>使用乙烯基油管, 将吸管从塞子延伸到 t 形交叉软管连接器。</li>
	<li>使用乙烯基油管, 连接两个器 (图 1, #2 & #3), 并将第二个器的输出连接到 t 形交叉管接头。</li>
	<li>使用乙烯基油管, 连接第一个器的输入端口作为吸入端口 (图 1, #1)。</li>
	<li>使用乙烯基油管, 将傅书礼烧瓶的侧臂连接到气流调节器 (图 1, #7) 的输入端口和气流调节器的出口端到真空泵。</li>
	<li>根据图 2A中的示意图组装电路。</li>
	<li>通过串行适配器和制造商的软件, 将 PBASIC 程序诚 bs1 (图 2B, 也可在 https://github.com/ChastainAnderson/SVL) 上载到 BS1 微控制器 (图 1, #6)。</li>
	<li>将510螺纹底座 (图 1 #8) 置于环形支架夹具中。</li>
	<li>使用乙烯基油管, 将电磁阀 (图 1, #5) 连接到 t 型交叉管接头的自由端。 注: 该设备应完整, 并准备运行, 检查所有接头, 以确保他们是空气紧张, 并应用软管钳和真空润滑脂的需要。</li>
</ol><img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56709/56709fig2.jpg"> 图 2: 电气示意图和 PBASIC 代码.图 2A显示用于装配所需电路的电气示意图, 以激活正常打开的电磁阀和按钮激活电子烟的加热线圈 (通过510螺纹电子烟罐底座)。加热线圈的电气参数 (P: 功率;R: 电阻;和 I: 电流) 的预测, 并应经验验证用万用表后组装。图 2B显示了在图 2A中控制电路所需的 PBASIC 计时程序 (也可在 https://github.com/ChastainAnderson/SVL 中使用)。定时常数 SVT & 间隙 (#5 & #6) 是以 ms 为单位, 并设置为2秒的激活时间, 并在二十八年代停机.<a href="//cloudflare.jove.com/files/ftp_upload/56709/56709fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
2. 样品贮存和制备
<ol><li>在室温下, 将所有未开封的传统和电子卷烟样品存放在密闭的塑料袋中。</li>
	<li>一旦打开, 在 4 c 的密闭塑料袋中储存样品, 用纸巾吸收多余的水分。</li>
	<li>预平衡所有样品在室温下盒60% 湿度至少30分钟前使用。</li>
</ol>3. 卷烟烟雾/电子烟气溶胶萃取装置的一般操作
<ol><li>使用分析天平确定每个电子烟 cartomizer/罐前 vaping 的质量。前/后 vaping 重量的差异将用于确定适当的剂量。 注: 3R4F 参考卷烟被假定含有0.7 毫克的尼古丁, 而商业卷烟品牌的尼古丁含量可以用传统的分析方法13来确定。</li>
	<li>对于样品应用 1, 用4.3 毫升内皮细胞培养基填充原器的储层。样品应用 2, 用5毫升丙酮填充油层。</li>
	<li>
准备用于提取的电子香烟或常规香烟:
	<ol>
<li>如果使用传统的卷烟, 在过滤器周围应用一条清晰的胶带, 并在卷烟纸加入过滤器的地方放置一个容易看到的标记。</li>
		<li>如果使用香烟, 如电子烟, 确保电池是很好的充电和 cartomizer 紧拧到电池。</li>
		<li>如果使用电子香烟罐, 确保在油箱内装上适当数量的电子香烟液体, 并将油箱拧到510螺纹底座上。</li>
	</ol>
</li>
	<li>将常规或电子卷烟的尖端插入吸入式端口 (图 1, #1), 并用软管钳固定。</li>
	<li>打开真空泵。</li>
	<li>调整流量计, 以拉动1.65 升/分钟, 以确保55毫升粉扑超过2秒。</li>
	<li>打开微控制器。如果使用传统的香烟, 点燃香烟的第一个粉扑。</li>
	<li>运行, 直到预计的预期浓度 (在部分每百万或% 重量/体积) 达到。</li>
	<li>
使用分析天平确定每个电子烟 cartomizer/罐在汽化后的质量。将此测量与步骤3.1 中所采取的测量值进行比较, 以确定所消耗的总质量。计算溶剂的消耗质量/体积的浓度。使用所消耗的尼古丁的摩尔浓度, 使产品正常化。
	<ol>
<li>如果消耗的质量不足, 则将电子卷烟退回设备并进一步消耗。</li>
		<li>如果消耗了足够或过剩的质量, 请继续。</li>
	</ol>
</li>
</ol>4. 过滤和贮存
<ol><li>如果提取物是用于细胞培养, 过滤通过0.22 µm PES 注射器过滤器。</li>
	<li>立即使用提取物或存储在-80 c。作为准备安德森的一部分, et al.8, 电子烟气雾剂被证明是稳定的至少两个星期, 和两年的香烟烟雾的稳定性已建立由骗子, et al.13。</li>
</ol>5. 清洗设备
<ol><li>每次提取后, 用70% 乙醇和去离子水冲洗该装置的油管和储油层, 以防止样品之间的携带。</li>
	<li>在漂洗之后, 简要地运行空的设备允许气流帮助烘干线。</li>
</ol>6. 样品应用 1: 中性红吸收细胞活力测定
<ol><li>以4.3 毫升的内皮细胞生长培养基进行提取。</li>
	<li>一天前, 种子人脐静脉内皮细胞进入96井板, 密度为 1 x 104细胞/在100µL 内皮细胞生长培养基。</li>
	<li>用100µL 的新鲜内皮细胞培养基取代原有的内皮细胞培养基来治疗细胞, 以作为对照或75µL 的内皮细胞生长培养基, 混合25µL 2 毫米消耗的尼古丁浓度提取物 (1.4 毫克将尼古丁消耗到4.3 毫升内皮细胞生长培养基中), 最终浓度为500µM, 作为治疗。</li>
	<li>由于香烟烟雾和电子烟气溶胶中的许多成分都是挥发性的, 所以使用铝箔密封来保持油井的密闭。</li>
	<li>孵育板 24个 h 在37° c 和 5% CO2。</li>
	<li>
准备中性红染色溶液:
	<ol>
<li>创建一个100x 中性红股票解决方案, 溶解33毫克的中性红色染料到10毫升的缓冲盐溶液。</li>
		<li>使用前不久, 稀释100x 库存溶液1:100 在细胞培养基中创建1x 中性红染色液。</li>
		<li>孵育中性红染色液在37° c 至少30分钟之前使用和立即使用。 注意: 有些晶体在孵化过程中沉淀是正常的。应注意避免将这些晶体应用于细胞培养井。如果需要, 22 µm 过滤器可用于过滤中性红色股票和染色解决方案。</li>
	</ol>
</li>
	<li>去除提取物, 并添加100µL 中性红染色溶液每井, 使用过量创造至少三空白井为适当的量化。</li>
	<li>在37° c 和 5% CO2的 2-4 h 处孵育盘。</li>
	<li>去除中性红染色液, 在 PBS 中浸泡3x 冲洗。</li>
	<li>应用中性红脱污液 (50% 去离子水, 49% 乙醇, 1% 乙酸)。</li>
	<li>室温下至少孵育10分钟。</li>
	<li>读取 540 nm 的吸光度。</li>
	<li>通过将空白井的平均值减去平均值和正则化到空白调整控制井值的平均值来分析数据。</li>
</ol>7. 样品应用 2: 气相色谱质谱
<ol><li>在5毫升丙酮中进行提取。</li>
	<li>运行的设备, 以达到最终的浓度为100分之每百万 (重量的 e 液体消耗/丙酮体积) 的样品。</li>
	<li>使用精密玻璃注射器, 注入1µL 到注射器的 GC-MS 装置。在250° c 的条件下注入1:20 分裂比例的气相色谱/四极谱仪系统, 配有带有以下烤箱协议的 ZB-5 柱: 1 分钟在50° c;斜坡10° c/分至140° c;舷梯20° c/分钟到300° c 并且举行10分钟。</li>
	<li>将产生的质量谱与目标库相匹配, 以确定气溶胶成分。</li>
</ol>

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L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+Reactivity+and+Respiratory+Toxicity+of+the+-Diketone+Flavoring+Agents:+2,3-Butanedione,+2,3-Pentanedione,+and+2,3-Hexanedione.">Chemical Reactivity and Respiratory Toxicity of the -Diketone Flavoring Agents: 2,3-Butanedione, 2,3-Pentanedione, and 2,3-Hexanedione.</a> Toxicol. Pathol. 44 (5), 763-783 (2016).</li><li>Cooperation Centre for Scientific Research Relative to Tobacco. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CRM No. 84 - Determination of Glycerin, Propylene Glycol, Water, and Nicotine in the Aerosol of E-Cigarettes by Gas Chromatographic Analysis. , (2017).</li></ol>

由杜兰大学管理的烟草产品管理科学研究金项目由奥客户服务管理事务处提供资金。

本协议最关键的元素是确保设备在每次提取的开始和结束时都是干净的, 并确保所有的密封都保持稳定, 以使气流保持一致。如果设备没有被正确地清洗, 有风险在样品之间运载。此外, 如果设备在长时间内不洁, 凝结气溶胶和干溶剂就会堵塞系统。请注意, 这是正常的, 有一个压力下降时, 膨化一个传统的卷烟和气流表应调整, 以提供所需的气流期间的粉扑, 而不是当设备是拉动房间空气。该方法的?...

尽管保健组织作出了集中的努力, 但烟草产品的使用仍然是全世界可预防死亡的主要原因, 其中大多数死亡归因于吸烟的1。自2003年进入市场以来, 电子卷烟在烟草产品用户中的知名度越来越高。目前, 电子香烟是最流行的替代传统香烟在美国成年人 (〜 5%)2和最流行的尼古丁传递系统中间 (〜 5.3%) 和高中生 (〜 16%)3。如果目前的趋势继续下去, 电子卷烟有望取代传统的香烟, 为子孙后代。然而, 电子卷烟使用的健康后果仍不清楚。
电子卷烟的研究直到 2013年3,4的电子卷烟普及程度迅速增加, 才开始认真。自那时以来, 采用了一些不同的模式来解决其毒性问题。然而, 许多研究的结果是相互矛盾的, 虽然电子卷烟的毒性一般低于传统卷烟, 但目前没有就电子香烟的健康后果达成共识使用5,6,7. 我们以前的研究表明, 电子卷烟对血管内皮的毒性要比传统的香烟低得多, 尽管它们有能力引起 DNA 损伤和诱导氧化应激和细胞死亡,8.然而, 在我们得出关于电子卷烟使用的健康后果的确切结论之前, 需要进行更多的研究。
由于常规卷烟是可预防的血管疾病的主要病因9, 因此对电子卷烟使用的血管健康风险的兴趣与日俱增, 如10、11、12。为了研究电子香烟对血管系统的影响, 我们的实验室开发了一个单片机操作的冒烟/vaping 设备 (图 1)8。该装置能够在水或有机溶剂中产生传统卷烟烟雾或电子香烟气溶胶的液体萃取物。由于气流由可调式气流调节器和 PBASIC 定时程序的组合控制, 因此该装置可根据任意数量的用户定义的协议来生成提取物。在这里, 我们详细介绍了该设备的组装和操作以及两个潜在的应用:体外细胞活力评估和气相色谱质谱。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56709/56709fig1.jpg"> 图 1: 吸烟/Vaping 设备.为吸烟/vaping 装置的物理装配示意图在香烟或香烟象电子香烟 (电子烟) 配置 (A) 和坦克电子香烟配置 (B)。组件键: 1) 吸入口;2) 初级收藏器;3) 溢流器;4) 傅书礼瓶真空疏水阀;5) 一般开式电磁阀;6) BS1 单片机;7) 气流调节器;8) 510 螺纹电子烟罐底座。<a href="//cloudflare.jove.com/files/ftp_upload/56709/56709fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

<table><tbody><tr><td>12 V AC/DC Wall Mount Adaptor</td><td>Digi-Key</td><td>T1099-P5P-ND</td><td></td></tr><tr><td>2.2 Ohm Resistors</td><td>Digi-Key</td><td>A105635-ND</td><td>Used in tandem to generate the 4.4 Ohm resistance in Figure 2A</td></tr><tr><td>330 Ohm Resistors</td><td>Digi-Key</td><td>330QBK-ND</td><td></td></tr><tr><td>510 Threaded Base</td><td>NJoy</td><td>N/A</td><td>Recovered by dismantalling a second generation NJoy electronic cigarette</td></tr><tr><td>Acetic Acid, Glacial</td><td>Sigma-Aldritch</td><td>A6283</td><td></td></tr><tr><td>Acetone (Chromatography Grade)</td><td>Sigma-Aldritch</td><td>34850</td><td></td></tr><tr><td>Basic Stamp Project Board</td><td>Digi-Key</td><td>27112-ND</td><td>This board contains the BS1 Microcontroller, serial adaptor, power switch, and a barrel pin connector for the AC/DC Wall Mount Adaptor</td></tr><tr><td>Basic Stamp USB to Serial Adapter</td><td>Digi-Key</td><td>28030-ND</td><td>An optional component to allow the BS1 serial adaptor to communicate through USB</td></tr><tr><td>Buchner Flask (Vacuum Flask) 250 mL</td><td>VWR</td><td>10545-854</td><td></td></tr><tr><td>Clear Tape</td><td>3M</td><td>S-9783</td><td></td></tr><tr><td>Clear Vinyl Tubing, 3/8&quot; ID</td><td>Watts</td><td>443064</td><td></td></tr><tr><td>EGM-2 Endothelial Cell Culture Medium</td><td>Lonza</td><td>CC-3162</td><td></td></tr><tr><td>Ethanol</td><td>Pharmco-Aaper</td><td>111000200</td><td></td></tr><tr><td>Flow Regulator</td><td>Dwyer</td><td>VFA-23-BV</td><td></td></tr><tr><td>Gas Chromatograph</td><td>Varian</td><td>450-GC</td><td></td></tr><tr><td>Glass Syringe, 10 mL</td><td>Sigma-Aldritch</td><td>Z314552</td><td></td></tr><tr><td>Glass Syringe, 10 &amp;micro;L</td><td>Hamilton</td><td>80300</td><td></td></tr><tr><td>High Vacuum Silicon Grease</td><td>Dow Corning</td><td>146355D</td><td></td></tr><tr><td>Hose Clamp</td><td>Precision Brand</td><td>35125</td><td></td></tr><tr><td>Human Umbilical Vein Endothelial Cells</td><td>ATCC</td><td>PCS-100-013&amp;nbsp;</td><td></td></tr><tr><td>Mass Spectrometer</td><td>Varian</td><td>300-MS</td><td></td></tr><tr><td>Midget Impinger</td><td>Chemglass</td><td>CG-1820-01</td><td></td></tr><tr><td>Neutral Red</td><td>Sigma-Aldritch</td><td>N4638</td><td></td></tr><tr><td>Paraffin Film</td><td>3M</td><td>PM-992</td><td></td></tr><tr><td>Plate Seal Roller</td><td>BioRad</td><td>MSR0001</td><td></td></tr><tr><td>Plate Seal; Foil</td><td>Thermo</td><td>276014</td><td></td></tr><tr><td>Ring Stand 20&quot;</td><td>American Educational Products</td><td>7-G15-A</td><td></td></tr><tr><td>Solenoid Valve (normally open)</td><td>US Solid</td><td>USS2-00081</td><td></td></tr><tr><td>Solid State Relay</td><td>Digi-Key</td><td>CLA279-ND</td><td></td></tr><tr><td>Stand Clamp</td><td>Eisco</td><td>CH0688</td><td></td></tr><tr><td>Syringe Filter, PES, 0.22 um</td><td>Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Syringe, 10 mL</td><td>BD Syringe</td><td>309604</td><td></td></tr><tr><td>Through Hole Stopper, Size 6</td><td>VWR</td><td>59581-287</td><td></td></tr><tr><td>Vacuum Pump</td><td>KNF Neuberger</td><td>N86KTP</td><td></td></tr></tbody></table>

a microcontroller operated device for the generation of liquid extracts from conventional cigarette smoke and electronic cigarette aerosol

电子卷烟是中、高中学生最受欢迎的烟草产品, 是成人中最受欢迎的替代烟草产品。对电子卷烟使用的后果进行高质量、重复性的研究, 对于了解新出现的公共卫生问题和制定基于证据的监管政策至关重要。虽然越来越多的论文讨论电子卷烟, 但在各组的方法上几乎没有一致性, 对结果的共识也很少。在这里, 我们描述一个可编程的实验室设备, 可用于创建传统卷烟烟雾和电子香烟气溶胶提取物。本协议详细说明了该设备的组装和操作, 并演示了在两个示例应用程序中使用生成的提取物: 一个体外细胞活力测定和气相色谱质谱。该方法提供了一种直接比较传统卷烟和电子卷烟的工具, 是电子卷烟研究中一个容易进入的切入点。

在24小时内, 人类脐静脉内皮细胞暴露于传统的卷烟烟雾提取物 (EAE) 或电子香烟气溶胶提取物 (的), 有一个显着的 (控制与...... 控件 vs. EAE p< 0.01;n = 6) 细胞存活率降低 (图 3A)。提取物产生的膨化轮廓为 2, 2 秒, 55 毫升喷泡每分钟和正常化的基础上的摩尔浓度尼古丁的设备。暴露于500µM 消耗的尼古丁当量的学会极大地减少了活细胞到11.06 ±...

Watch this Scientific Journal Video about A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol at JoVE.com

A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

在这里, 我们描述一个可编程的实验室设备, 可用于创建传统卷烟烟雾和电子香烟气溶胶提取物。这种方法为直接比较传统卷烟和电子卷烟提供了一个有用的工具, 是电子卷烟研究中一个容易进入的切入点。

一种单片机操作装置, 用于生成传统卷烟烟雾和电子烟气溶胶中的液体萃取物

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among ...

a-microcontroller-operated-device-for-generation-liquid-extracts-from

Research

JoVE Journal

Bioengineering

8.3K 次观看.  Tulane University. 在这里, 我们描述一个可编程的实验室设备, 可用于创建传统卷烟烟雾和电子香烟气溶胶提取物。这种方法为直接比较传统卷烟和电子卷烟提供了一个有用的工具, 是电子卷烟研究中一个容易进入的切入点。

视频: A Microcontroller Operated Device for the Generation of Liquid Extracts from Conventional Cigarette Smoke and Electronic Cigarette Aerosol

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

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface (ALI) as a model of differentiated respiratory epithelium. A protocol is described for isolating and exposing these cells to mainstream cigarette smoke (CS), in order to study epithelial cell responses to CS exposure. The protocol consists of three parts: the isolation of airway epithelial cells from mouse trachea, the culturing of these cells at air-liquid interface (ALI) as fully differentiated epithelial cells, and the delivery of calibrated mainstream CS to these cells in culture. The ALI culture system allows the culture of respiratory epithelia under conditions that more closely resemble their physiological setting than ordinary liquid culture systems. The study of molecular and lung cellular responses to CS exposure is a critical component of understanding the impact of environmental air pollution on human health. Research findings in this area may ultimately contribute towards understanding the etiology of chronic obstructive pulmonary disease (COPD), and other tobacco-related diseases, which represent major global health problems.

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

Pulmonary epithelial cells can be isolated from the respiratory tract of mice and cultured at air-liquid interface as a model of differentiated respiratory epithelium. A protocol is described for isolating, culturing and exposing these cells to mainstream cigarette smoke, in order to study molecular responses to this environmental toxin.

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 ...

科研

医学

A Protocol for Detecting and Scavenging Gas-phase Free Radicals in Mainstream Cigarette Smoke

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12. Although tobacco tars and related products in the particle phase of cigarette smoke are major causes of carcinogenic and mutagenic related diseases, cigarette smoke contains significant amounts of free radicals that are also considered as an important group of carcinogens9,10. Free radicals attack cell constituents by damaging protein structure, lipids and DNA sequences and increase the risks of developing various types of cancers. Inhaled radicals produce adducts that contribute to many of the negative health effects of tobacco smoke in the lung3. Studies have been conducted to reduce free radicals in cigarette smoke to decrease risks of the smoking-induced damage. It has been reported that haemoglobin and heme-containing compounds could partially scavenge nitric oxide, reactive oxidants and carcinogenic volatile nitrosocompounds of cigarette smoke4. A 'bio-filter' consisted of haemoglobin and activated carbon was used to scavenge the free radicals and to remove up to 90% of the free radicals from cigarette smoke14. However, due to the cost-ineffectiveness, it has not been successfully commercialized. Another study showed good scavenging efficiency of shikonin, a component of Chinese herbal medicine8. In the present study, we report a protocol for introducing common natural antioxidant extracts into the cigarette filter for scavenging gas phase free radicals in cigarette smoke and measurement of the scavenge effect on gas phase free radicals in mainstream cigarette smoke (MCS) using spin-trapping Electron Spin Resonance (ESR) Spectroscopy1,2,14. We showed high scavenging capacity of lycopene and grape seed extract which could point to their future application in cigarette filters. An important advantage of these prospective scavengers is that they can be obtained in large quantities from byproducts of tomato or wine industry respectively11,13

Spin-trapping ESR spectroscopy was used to study the effect of plant antioxidants lycopene, pycnogenol and grape seed extract on scavenging gas-phase free radicals in cigarette smoke.

卷烟主流烟气中检测和清除气相自由基议定书

Cigarette smoking is associated with human cancers. It has been reported that most of the lung cancer deaths are caused by cigarette smoking 5,6,7,12.  ...

生物工程

Bridging the Bio-Electronic Interface with Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and higher throughput. The incorporation of biological components onto biological microelectromechanical systems (bioMEMS) has shown great potential for achieving these goals. Microfabricated electronic chips allow for micrometer-scale features as well as an electrical connection for sensing and actuation. Functional biological components give the system the capacity for specific detection of analytes, enzymatic functions, and whole-cell capabilities. Standard microfabrication processes and bio-analytical techniques have been successfully utilized for decades in the computer and biological industries, respectively. Their combination and interfacing in a lab-on-a-chip environment, however, brings forth new challenges. There is a call for techniques that can build an interface between the electrode and biological component that is mild and is easy to fabricate and pattern.
Biofabrication, described here, is one such approach that has shown great promise for its easy-to-assemble incorporation of biological components with versatility in the on-chip functions that are enabled. Biofabrication uses biological materials and biological mechanisms (self-assembly, enzymatic assembly) for bottom-up hierarchical assembly. While our labs have demonstrated these concepts in many formats 1,2,3, here we demonstrate the assembly process based on electrodeposition followed by multiple applications of signal-based interactions. The assembly process consists of the electrodeposition of biocompatible stimuli-responsive polymer films on electrodes and their subsequent functionalization with biological components such as DNA, enzymes, or live cells 4,5. Electrodeposition takes advantage of the pH gradient created at the surface of a biased electrode from the electrolysis of water 6,7,. Chitosan and alginate are stimuli-responsive biological polymers that can be triggered to self-assemble into hydrogel films in response to imposed electrical signals 8. The thickness of these hydrogels is determined by the extent to which the pH gradient extends from the electrode. This can be modified using varying current densities and deposition times 6,7. This protocol will describe how chitosan films are deposited and functionalized by covalently attaching biological components to the abundant primary amine groups present on the film through either enzymatic or electrochemical methods 9,10. Alginate films and their entrapment of live cells will also be addressed 11. Finally, the utility of biofabrication is demonstrated through examples of signal-based interaction, including chemical-to-electrical, cell-to-cell, and also enzyme-to-cell signal transmission. 
Both the electrodeposition and functionalization can be performed under near-physiological conditions without the need for reagents and thus spare labile biological components from harsh conditions. Additionally, both chitosan and alginate have long been used for biologically-relevant purposes 12,13. Overall, biofabrication, a rapid technique that can be simply performed on a benchtop, can be used for creating micron scale patterns of functional biological components on electrodes and can be used for a variety of lab-on-a-chip applications.

This article describes a biofabrication approach: deposition of stimuli-responsive polysaccharides in the presence of biased electrodes to create biocompatible films which can be functionalized with cells or proteins. We demonstrate a bench-top strategy for the generation of the films as well as their basic uses for creating interactive biofunctionalized surfaces for lab-on-a-chip applications.

生物电子界面衔接与Biofabrication

Advancements in lab-on-a-chip technology promise to revolutionize both research and medicine through lower costs, better sensitivity, portability, and ...

Generation of Electronic Cigarette Aerosol by a Third-Generation Machine-Vaping Device: Application to Toxicological Studies

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and flavoring chemicals. The aerosol produced includes fine and ultrafine particles, and potentially nicotine and aldehydes, which can be harmful to human health. E-cig users inhale these aerosols and, with the third-generation of e-cig devices, control design features (resistance and voltage) in addition to the choice of e-liquids, and the puffing profile. These are key factors that can significantly impact the toxicity of the inhaled aerosols. E-cig research, however, is challenging and complex mostly due to the absence of standardized assessments and to the numerous varieties of e-cig models and brands, as well as e-liquid flavors and solvents that are available on the market. These considerations highlight the urgent need to harmonize e-cig research protocols, starting with e-cig aerosol generation and characterization techniques. The current study focuses on this challenge by describing a detailed step-by-step e-cig aerosol generation technique with specific experimental parameters that are thought to be realistic and representative of real-life exposure scenarios. The methodology is divided into four sections: preparation, exposure, post-exposure analysis, plus cleaning and maintenance of the device. Representative results from using two types of e-liquid and various voltages are presented in terms of mass concentration, particle size distribution, chemical composition and cotinine levels in mice. These data demonstrate the versatility of the e-cig exposure system used, aside from its value for toxicological studies, as it allows for a broad range of computer-controlled exposure scenarios, including automated representative vaping topography profiles.

Electronic cigarette (e-cig) users are increasing worldwide. Little, however, is known about the health effects induced by inhaled e-cig aerosols. This article describes an e-cig aerosol generation technique suitable for animal exposures and subsequent toxicological studies. Such protocols are required to establish experimentally reproducible and standardized e-cig exposure systems.

第三代机械 Vaping 装置生成电子烟气溶胶: 毒理学研究的应用

Electronic-cigarette (e-cig) devices use heat to produce an inhalable aerosol from a liquid (e-liquid) composed mainly of humectants, nicotine, and ...

化学

Use of Capillary Aerosol Generator in Continuous Production of Controlled Aerosol for Non-Clinical Studies

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, followed by nucleation and condensation regulated through a mixture of airflow to generate aerosols, such as in an electronic cigarette (EC). The CAG is particularly useful in generating aerosols of large volumes in a continuous manner, for instances such as in vivo inhalation toxicology studies, where usage of ECs is not feasible. The thermal effects of generating aerosol from the CAG are similar in terms of temperature applied in an EC, thus allowing investigators to assess the vapors of e-liquids at scale and reproducibility. As the operation of the CAG allows users to control critical parameters such as the flow rate of e-liquid, heating temperatures and dilution air flows, it allows investigators to test various e-liquid formulations in a well-controlled device. Properties, such as aerosol particle size, are demonstrated to be regulated with the air flow rate with respect to the e-liquid flow and e-liquid composition. The CAG, however, is limited in assessing common EC-related issues, such as overheating of its elements. We seek to demonstrate that the CAG can generate aerosol that is reproducible and continuous, by assessing the chemical and physical aerosol characteristics with a chosen e-liquid formulation. The protocol describes the operating parameters of liquid flow rate, dilution air-flow rates and operating procedures needing to optimize the aerosol concentration and particle size required for an in vivo toxicology study. Presenting the representative results from the protocol and discussing the challenges and applications of working with a CAG, we demonstrate that CAG can be used in a reproducible fashion. The technology and protocol, that has been developed from prior work, serve as a foundation for future innovations for laboratory-controlled aerosol generation investigations.

The protocol describes the settings and use of a capillary aerosol generator for continuous production of controlled aerosol from a multispecies liquid solution, suitable for steady large-volume aerosol delivery (e.g., in vivo inhalation studies).

毛细管气溶胶发生器在连续生产受控气溶胶中的应用，用于非临床研究

The capillary aerosol generator (CAG) is operated with the principal of thermal liquid evaporation through heating of e-liquid in the initial phase, ...

工程学

A Versatile Kit Based on Digital Microfluidics Droplet Actuation for Science Education

This paper describes an educational kit based on digital microfluidics. A protocol for luminol-based chemiluminescence experiment is reported as a specific example. It also has fluorescent imaging capability and closed humidified enclosure based on an ultrasonic atomizer to prevent evaporation. The kit can be assembled within a short period of time and with minimal training in electronics and soldering. The kit allows both undergraduate/graduate students and enthusiasts to obtain hands-on experience in microfluidics in an intuitive way and be trained to gain familiarity with digital microfluidics.

We describe an educational kit that allows users to execute multiple experiments and gain hands-on experience on digital microfluidics.

基于数字微流体的多功能套件，用于科学教育

This paper describes an educational kit based on digital microfluidics. A protocol for luminol-based chemiluminescence experiment is reported 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 ...

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. ...

Cigarette Smoke Exposure in Mice using a Whole-Body Inhalation System

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular diseases have been widely studied, however, the impact of CS in other tissues and organs such as blood and bone marrow remain incompletely defined. Finding the appropriate system to study the effects of CS in rodents can be prohibitively expensive and require the purchase of commercially available systems. Thus, we set out to build an affordable, reliable, and versatile system to study the pathologic effects of CS in mice. This whole-body inhalation exposure system (WBIS) set-up mimics the breathing and puffing of cigarettes by alternating exposure to CS and clean air. Here we show that this do-it-yourself (DIY) system induces airway inflammation and lung emphysema in mice after 4-months of cigarette smoke exposure. The effects of whole-body inhalation (WBI) of CS on hematopoietic stem and progenitor cells (HSPCs) in the bone marrow using this apparatus are also shown.

This protocol demonstrates the study of the pathophysiologic effects of cigarette smoke (CS) with a whole-body inhalation (WBI) exposure system (WBIS) built in-house. This system can expose animals to CS under controlled repeatable conditions for research of CS-mediated effects on lung emphysema and hematopoiesis.

使用全身吸入系统在小鼠中暴露香烟烟雾

Close to 14% of adults in the United States were reported to smoke cigarettes in 2018. The effects of cigarette smoke (CS) on lungs and cardiovascular ...

免疫与感染

A Technique to Generate and Deliver Drug Aerosols Directly to Mouse Lungs

Source: Phillips, J. E. et al., <a href="https://app.jove.com/v/55454">Dry Powder and Nebulized Aerosol Inhalation of Pharmaceuticals Delivered to Mice Using a Nose-only Exposure System.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates the generation of drug aerosols and the targeted nose-only delivery of these aerosols to mice using an inhalation unit equipped with an aerosol generator. The mouse is restrained in a nose-only restrainer and connected to the inhalation unit, which delivers the drug directly into the mouse&#39;s nasal cavity. Subsequently, the drug passes through the trachea and enters the lungs.

一种产生药物气雾剂并将其直接输送到小鼠肺部的技术

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.
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