笔者要感谢莫里斯·史密斯和玛丽亚Talikka对稿件的审查。

在器官组织支气管文化模式1.文化注：再造组织模型购买的刀片是准备使用。备选地，培养物可以从原代细胞发展成为由卡普和同事16描述的例子。在收到在无菌包装组织，总是处理在无菌条件下的人体器官组织。 <ol><li>添加0.7预热（37℃）测定培养基到一个新的无菌的24孔板引擎盖下的每个孔中的溶液中。 </li><li>取出组织的包装罩下，并在组织培养插入物转移到新制备的24孔板（在步骤1.1中描述。上文）。 </li><li>维持和培养在培养箱中在组织中，在37℃（5％CO 2，90％湿度）。 </li><li>更换培养基，每2天。 </li><li>在一个介质改变，检查在光学显微镜下的组织，以确保它们它们contaminati在无和有没有泄漏介质中。 </li></ol> 2.香烟烟雾（CS）曝光用体外曝光系统<ol><li>在曝光之前实验三天中，组织培养的顶侧用200μl培养基洗涤。 </li><li>在暴露的一天，如果睫状跳动测量曝光之前计划，如在第3节中所述处理该组织为睫状打浆测量。 </li><li>预暖体外曝光系统的气候室和培养基本模块到37℃。与17.5毫升每行中的填充种植基地模块。 </li><li>从培养箱中取出的组织，并将其放置在通风橱下从培养板转移的组织培养插入到体外曝光系统的培养基的模块。此外，盖上玻璃盖的种植基地模块。 </li><li>转移组织在体外接触SYSTE的气候室米暴露于主流的CS。 </li><li>如果在体外暴露系统装有一个微量天平，设置了石英晶体微天平上运行的曝光来测量使用微量天平软件石英晶体烟雾的实时颗粒沉积之前。 <ol><li>更换晶体连接到稀释/分发系统中的每一行微量天平模块。 </li><li>微量天平模块连接到微量天平软件。在软件设置测量的刻度为零。 </li></ol></li><li>根据实验过程露出的组织（如在图1中如 ）。 注意：在此之前的曝光，参考香烟（3R4F）之间7至21天在受控条件下根据ISO标准3402 17调节至22±1℃和60±3％的相对湿度。 <ol><li>使用一个30端口转盘吸烟马赫生成香烟烟雾（ 例如 ，从3R4F香烟）国家统计局根据加拿大卫生部强烈的政权：抽香烟到一个标准的对接长度， 即约35毫米55毫升粉扑超过2秒，两次分8秒泵的排气时间。 注意：如果需要，稀释主流的CS，以使剂量没有毒性太大。在此处报道的结果，烟稀释至16％（体积/体积）。 </li><li>使用60％的加湿空气的对照样品（空气暴露）。 </li><li>暴露组织为6-7分钟（ 即 ，1香烟或空气暴露）在体外曝光系统</li><li>把组织放回培养箱中，在37℃（5％CO 2，90％湿度）1小时。 </li><li>重复步骤2.7.3和2.7.4三次。 </li></ol></li><li>在曝光实验结束时，停止微量天平软件。该软件将生成包含随着颗粒沉积的图形表示的所有测量值的文件。 </li><li>传回从种植B组织ASE模块到培养板中。将组织放回培养箱中，在37℃（5％CO 2，90％湿度），直到所述测量结果为在该特定曝光后的时间点的不同端点。 </li></ol> 3.测量睫状跳动注意：根据实验方案（图1），记录睫状打浆前曝光和后直接曝光。 <ol><li>从培养箱中取出的组织培养板中，并让它们在引擎盖下在室温30分钟以稳定该纤毛跳动。 </li><li>放置在组织中的光显微镜连接到CiliaMetrix软件下。 </li><li>记录睫状打浆的电影和频率（以赫兹为单位）。测量频率每3秒1分钟。 </li><li>返回组织回到培养箱中在37℃（5％CO 2，90％湿度）。 </li></ol> 4.跨上皮电阻（TEER）测量注意：TEER的测定进行3天前，用筷子电极（STX-2），连接到一个voltohmmeter无菌条件下罩下曝光后的48小时。 <ol><li>加入200μl培养基对人类支气管组织的顶端面。 </li><li>打开EVOMX机器上;用70％的乙醇溶液洗涤电极。 </li><li>用培养基清洗电极。 </li><li>通过不接触组织插入电极插入组织培养的根尖侧的介质测量的电阻（Ω）。 </li><li>重复步骤4.4 5倍，以获得一式五份的测量。 </li><li>测量后，轻轻地从使用抽吸的组织培养物的顶侧除去介质。 </li><li>返回的组织培养培养箱在37℃（5％CO 2，90％湿度）中培养。 </li></ol> 5.细胞色素P450（CYP）1A1 / 1B1 ActiviTY <ol><li>在研究开始前，被瓶的适当重建缓冲液中的整个内容转移到含有（按照制造商的说明）的荧光素检测试剂的琥珀色瓶中制备的荧光素检测试剂。 2毫升，在-20℃下等份商店最多1个月。 </li><li>为48小时后的曝光时间，孵育之后的CS暴露在37℃下将组织48小时。 </li><li>暖荧光素检测试剂至RT。 </li><li>组织孵育插入3小时或O / N与荧光素CEE基板（稀释至1:50）在基础培养基。 </li><li>转移50微升来自各组织培养基插入到一个96孔不透明白色光度计板在RT。 </li><li>添加50微升荧光素检测试剂至各孔以启动发光反应。 </li><li>孵育板在RT下20分钟。 </li><li>阅读使用发光的发光。 注：对于光度计，使用每孔0.25-1秒的积分时间为指针。 </li></ol> 6. RNA提取。 <ol><li>之前收集三维器官组织的插入，制备1）的盒用干冰，2）足够量的叶片（每3D刀片1），3）冷PBS无钙和氯化镁，4）1个25毫升玻璃移液管，和5 ）无菌玻璃针抽吸。 <ol><li>标签管充满了陶瓷珠均质固体细胞样本，并加入700μlQiazol的。 </li><li>打开吸机上。 </li><li>收集从培养箱三维器官组织插入物的板。 </li><li>用冷PBS在板洗每个插入的3倍。在洗涤之间，吸的PBS与玻璃针。在第三次洗涤后，吸用PBS和盖板，以防止插入件的干燥。 </li><li>用于从所述板的每个刀片，切出的插入物膜（3D组织刀片所在的），直到membrANE平躺下来的刀片。 </li><li>从叶片到均质管适当标记的转移组织（连同插入膜）并拧盖子上的管中，摇动和涡流它。 </li><li>冷冻在干冰和存储的样品在-80℃或继续萃取液中的RNA。 注意：在此之前的RNA的提取，根据随机设计（如果适用的话）标记所有管和列。准备根据制造商的建议的所有试剂，并采取了从-80℃冷冻样品并解冻它们在冰上直到它们被完全解冻。 </li></ol></li><li>同质化<ol><li>研磨样品均质仪对6000赫兹，持续45秒。如果样本不是正确匀浆并离开样品5分钟，在室温重复。 </li><li>加入140微升氯仿样品和旋涡10-15秒。 </li><li>从均质管转移上清到锁相管和动摇上thermoshaker为以1400rpm和RT 2分钟。 </li><li>在4℃下进一步温育在室温和样品2分钟离心样品在12000×g离心15分钟。 </li></ol></li><li> RNA沉淀，洗净，悬挂和净化QIAcube工作站。 <ol><li>传送在新的2ml管中的上层水相。 </li><li>加载所有必需的试剂提取机器人根据制造商的协议表，选择相应的协议，将洗脱体积（30微升），并运行提取的机器人。 </li><li>当运行完成后，取出转子适配器和立即保持洗脱管在4℃用于进一步的分析或在-80℃长期储存。 </li></ol></li><li>分离的RNA的质量控制<ol><li>确定根据制造商的说明，使用的是紫外线读取器的分离的RNA的量。 </li><li>检查RNA的质量和使用的RNA完整性微流体 - 凝胶为基础的芯片实验室和紫外线读取器，ACC奥尔丁制造商的说明。 </li></ol></li></ol> 7.芯片工作流程注意：下面描述的方法着重于方法Affymetrix基因芯片的高通量3&#39;IVT快车套件，其用于目标合成，用于对从所述制剂中的RNA，以使用一个复杂的起始的Affymetrix 3&#39;的基因表达盒的杂交液体处理系统（ 如 ，移液，稀释，分装，或整合）。 <ol><li>准备<ol><li>随机样本，以避免批量的影响。分批处理的样本的数目为从1到96，其中包括一个正（商业人类普遍参照RNA）和一个负（无RNA酶的水）每批次的控制。 </li><li>开始使用100ng的总RNA在3微升的无RNA酶的水的目标合成。对于100毫微克的总RNA，使用16小时的培养时间。 </li></ol></li><li> RNA扩增<ol><li>准备POLY-A RNA穗通过串行稀释聚-A RNA股票与聚-A控制稀释液控制解决方案： <ol><li>如需稀释1，准备2微升聚腺苷酸控制库存36微升稀释液。 </li><li>如需稀释2，准备2微升稀释1的98微升的稀释液。 </li><li>如需稀释3，准备4微升稀释2的196微升的稀释液。 </li><li>如需稀释4，在180微升的稀释液准备20微升稀释3。 </li></ol></li><li>淡化RNA酶自由水的RNA得到33.3纳克/微升的浓度。 </li><li>添加2微升的polyA对照溶液到3微升总RNA样品的直接在96孔板中。这个步骤是通过所述液体处理系统进行的。 </li><li>制备所述液体处理系统和目标合成。放置在机器人上的印版。将所有必需的试剂及实验室用品在甲板上。机器人将通过制备都是适宜启动过程Ëmastermixes和孵化，在自动遥控PCR块样品。 </li><li>一是质量控制检查：控制放大过程<ol><li>如果的aRNA的质量是可接受的，通过添加到每个样品8微升碎裂5X缓冲液（该步骤通过所述液体处理系统执行）执行的碎片的步骤。否则，执行前面的步骤再次从RNA开始。立即使用零散阿尔纳或储存未稀释的和零散的阿尔纳在-20°C（或-70℃下长期保存）。 </li><li>拿起随机12个样品的凝胶微流体系统的基础上盘和转让1微升检查碎片的效率进行第二次质量控制分析。 </li></ol></li></ol></li><li>杂交过程<ol><li>扫描芯片条形码，并根据指令制造商的软件注册每个样本。 </li><li>准备杂交组合为如下为一个单个反应并将其添加到33微升分散和标记的aRNA的：4.2微升对照寡核苷酸B2（为3nM），12.5微升20×真核生物杂交对照，加入25μl的100％DMSO中，125微升的2×杂交缓冲液和50微升的H 2 O代表的216.7微升的最终体积 注意：杂交，洗涤和处理样品之前扫描阳性和阴性对照。 </li><li>预湿用200μl的预杂交混合物中所含的杂交洗涤和染色试剂盒的阵列。 </li><li>放置在烘箱中于45℃杂交，芯片和60 RPM的10分钟。 </li><li>在此期间，变性在PCR块所述板（含有终鸡尾酒杂交混合物）在99℃下进行5分钟，45℃5分钟。 </li><li>从数组中取出的预杂交混合物，并将其用200μl的杂交混合液目标（每码片一个样本）取代。 </li><li>将数组复制到hybridization烘箱中在45℃下16小时（O / N）用60rpm的旋转速度。 </li></ol></li><li>洗涤和染色<ol><li>运行该软件菜单栏中的“射流”。在射流对话框中，选择感兴趣的（1 - 4）模块，然后选择Prime_450计划把所有模块。将管用于洗涤的缓冲液A中在瓶（200毫升）的瓶（400ml）中，并于洗涤缓冲液B，以及用于milliQ水中在瓶（500毫升）中。 </li><li>随后，按照液晶屏幕指示。抬起针和放置600微升染色鸡尾酒1（SAPE溶液混合）和含有微量离心管在位置＃1，＃2 600微升染色鸡尾酒2（抗体溶液混合），和900微升体列保持在位置＃3缓冲溶液。 </li><li>在O / N培养在烤箱后，从芯片吸出的鸡尾酒，并填写250微升洗涤液A.该芯片分配权芯片每个模块中，选择FS450-001协议和运行每个谟独乐寺，按照屏幕上的说明。这个过程持续90分钟。 </li><li>一旦液晶屏显示“弹出盒”的消息，取出芯片的，如果有任何气泡检查。如果存在气泡，完全填充阵列与阵列保持缓冲液中。申请贴到隔垫，以避免在扫描仪的任何泄漏和事先清洗微阵列窗口装载在扫描器。 </li></ol></li><li>扫描。 <ol><li>热身扫描仪。芯片加载到扫描仪的自动加载磁带机，并开始扫描。 注：在每个芯片〜12分钟，生成每个芯片.CEL文件。 </li><li>检查图像和对齐网格（如果需要的话），以将光点，以确定所述探针的细胞。 注：该数据集已提交Arrayexpress（登录代码= E-MTAB-1721）。 </li></ol></li></ol>对于影响评估8.基于网络的系统生物学分析<ol><li>微阵列数据的处理。 注：数据处理的ð评分方法采用R统计环境版本2.14落实。 <ol><li>打开一个R 18会话并加载AFFY 19 gcrma和affyPLM通过运行命令安装的软件包：库（AFFY）&gt;库（gcrma）&gt;库（affyPLM） </li><li>阅读运行命令的原始数据文件：data.affybatch &lt;-ReadAffy（“路径文件夹中存储的CEL文件”） </li><li>减去背景校正和位数师范使用gcrma包生成探针组的表达值，通过运行以下命令：eset.norm &lt;-gcrma（data.affybatch） </li></ol></li><li>质量控制。 <ol><li>产生RNA降解地块的命令：度&lt;-AffyRNAdeg（data.affybatch） </li><li>提取RNA降解斜率运行命令的系数：斜率= $度斜率</li><li>曲线的斜率系数，并确定可能的异常值。 </li><li>产生怒色和RLE地块运行以下命令：Pset的&lt; -fitPLM（data.affybatch）&gt; RLE值（Pset）&gt;怒色值（Pset）。 </li><li>如果确定某些生成的盒状图是异常值：一个数组被认为是离群如果至少有2定义见下文偏离其他阵列3 QC指标： - 度$斜率是由平均度坡度$不同 -怒色的情节：一个数组被视为异常，如果上四分位数低于0.95或高于1.05的下四分位数， 即 ，如果箱线图是完全高于1.05或低于全部0.95。 - RLE情节：一个阵列被认为是离群如果RLE分布的中值大于0.1或小于-0.1以下。 </li><li>如果阵列被确定为异常值，则丢弃，并且从步骤8.1.2重新开始。 </li><li>适合于数据的总的线性模型对所关注的具体对比（ 即 ，的“处理”和“控制”情况的比较）产生用于每个探针上的微阵列设置，它可以是furt原始P值她用LIMMA包的Benjamini-霍赫贝格程序进行调整。选择每个基因的探针在20叙述保持为代表的基因进行进一步的分析。 注：从实验设计中的阻塞因子（曝光板）占模型进行数据处理。 </li></ol></li><li>网络为基础的分析。 注意：如这里实施的方法中​​详细Martin等人所述。 （在修订BMC生物信息学）。 <ol><li>对于每个感兴趣的两两比较，从所计算的（log2-）启动网络（步骤8.2）下折的变化（处理与对照）对于每个基因。 </li><li>计算用L定义的网络中的有符号拉普拉斯矩阵L（I，J）= - 符号（I〜j）条瓦特（I，J），如果有重量的瓦特节点i和j之间的（I，J）的边缘， L（I，J）=度（i）是第i和L（I，J）= 0否则加权程度。权重w（I，J）等于1，如果i和j是在主链和瓦特（I，J）= 1 / N，如果i是一个骨干节点和j是其N neighb之一ORS的成绩单层。 </li><li>为f = L3-1L2Tx计算得分的骨干，其中L 3为L的子矩阵的骨干节点和L2为L的子矩阵的行对应于转录层节点和列到骨干节点</li><li>计算由其中所有边缘标志被反转骨干网定义的网络的拉普拉斯签署，Q。 </li><li>计算NPA得分的NPA = fTQf。 </li><li>计算的置信区间，骨干边缘置换p值和转录层置换p值。 注：（ 例如 ，样品之间的生物学变异在一个实验组）除了NPA分数的置信区间，其占了实验误差，同伴统计推导来描述NPA得分的特异性所描述的生物学在网络上。因为NPA是的倍数变化的二次形式，它的方差可以基于倍数变化估计V计算ariances。置信区间是用中心极限定理随后派生。两个置换试验实施21从而第一，评估，如果结果是特定于模型的底层证据（ 即 ，基因倍数变化），从而导致一个置换P值（表示为*○当P在图中 - 值&lt;0.05）。其次，评估网络“原因和结果”图层是否显著促进了网络扰动的幅度（图中记为K *当P值&lt;0.05）。 </li><li>考虑网络的影响通过处理，如果置信区间不包含零和两个排列的p值&lt;0.05。 </li><li>计算其被定义为在骨架贡献达80％的NPA得分的关键节点的主要节点。 </li></ol></li></ol>

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M., 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=Quantitative+assessment+of+biological+impact+using+transcriptomic+data+and+mechanistic+network+models.">Quantitative assessment of biological impact using transcriptomic data and mechanistic network models.</a> Toxicology and applied pharmacolog. 272, 863-878 (2013).</li><li>Teff, Z., Priel, Z., Gheber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+forces+applied+by+cilia+depend+linearly+on+their+frequency+due+to+constant+geometry+of+the+effective+stroke.">The forces applied by cilia depend linearly on their frequency due to constant geometry of the effective stroke.</a> Biophysical journa. 94, 298-305 (2008).</li><li>Hilgendorf, C., 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=Caco-2+versus+Caco-2/HT29-MTX+co-cultured+cell+lines:+permeabilities+via+diffusion,+inside-+and+outside-directed+carrier-mediated+transport.">Caco-2 versus Caco-2/HT29-MTX co-cultured cell lines: permeabilities via diffusion, inside- and outside-directed carrier-mediated transport.</a> J Pharm Sc. 89, 63-75 (2000).</li><li>Sutto, Z., Conner, G. E., Salathe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+human+airway+ciliary+beat+frequency+by+intracellular+pH.">Regulation of human airway ciliary beat frequency by intracellular pH.</a> The Journal of physiolog. 560, 519-532 (2004).</li><li>Zhang, X., 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=Similarities+and+differences+between+smoking-related+gene+expression+in+nasal+and+bronchial+epithelium.">Similarities and differences between smoking-related gene expression in nasal and bronchial epithelium.</a> Physiological genomic. 41, 1-8 (2010).</li></ol>

这项研究是由美国菲利普莫里斯国际公司。

这里，我们已经证明人类器官支气管和鼻腔组织模型的适用性评估重复CS曝光的影响。作为替代动物试验，一些曝光系统的气溶胶暴露在体外毒理学评估被开发（ 例如 ，Vitrocell，Cultex，艾丽斯等）。这些曝光模块也可以用于空气中的污染物，空气中的颗粒，纳米颗粒等的毒理评估在这项研究中，我们使用了Vitrocell系统，可以容纳多达48个不同的样品同时，允许对较大规模的实验和下部之间的变异治疗。对于每一个气溶胶暴露在体外组织培养污染的风险仍然是一个重大的风险，其缓解需要一个小心处理的组织培养在整个实验。 电子期间微量天平测量颗粒沉积在实时xposure实验允许监视来自曝光系统中产生的CS剂量符合期望。以确保所测量的颗粒沉积的精度，设定安装在线测量正确的曝光之前是临界指数， 例如 ，规模设置为0。此外，由于微天平（厘米2）的区域之间的差的和组织培养插入物（0.33 平方厘米），我们调整最后计算在培养插入物的区域：只有33％在微量天平沉积的反映在培养插入物的实际沉积。 测量TEER的确定紧结屏障功能，并评估上皮层的破坏是一个相对容易的过程来实现，这是我们这里报告。然而，因为支气管和鼻腔组织模型包含黏液产生穿插杯状细胞，根尖洗涤需要TEER测定值之前必须执行urement。心尖​​洗涤是至关重要的，因为该粘液层的存在和厚度可偏压TEER的测量的可变性，在CS曝光的影响interferring。这个概念是在与什么是报告的Hilgendorf和同事，其中Caco-2细胞的渗透性受共培养与粘液产生杯状细胞系HT29 23的协议。粘液需要之前TEER测量被洗掉，因为在曝光前右心尖洗涤可以与组织反应的CS干扰。因此，进行测量曝光前三天，之前曝光不正确。 我们发现，CYP1A1 / CYP1B1活动可以从以下CS暴露的器官培养模型测量虽然活性仅略有增加CS。这个弱信号可由CYP1A1 / 1B1基板的较长的温育扩增（ 即 ，萤光素-CEE），例如用于24小时（数据未显示）。一个在目前的工作中的CYP活性测量的局限性是缺乏标准化到CYP蛋白质水平或向细胞计数，其可以被考虑为将来的研究，以确保对酶活性的改变不影响由任一蛋白水平或细胞计数的改变。 我们报道了CS暴露抑制纤毛击败两个鼻腔和支气管组织模型。类似的观察结果在不同的哺乳动物和非哺乳动物模型12完成。为睫状跳动测量，从而确保组织被处理，并且处理过的相似的方式是非常关键的，例如，如果介质变更实施，应适用于所有的样品。许特特和他的同事报道，pH值影响哺乳动物的纤毛跳动频率24。因此，比较以不同朱侃，陈万平/混合物，pH值处理的细胞之间跳动频率时调整，应考虑到最小化睫状打浆测量的可变性。此外，在该测量进行的温度也是关键的，因为睫状跳动的频率下降随着温度的降低。为了最大限度地减少由于这些变化，一个阶段顶培养箱，配备有温度，CO 2，湿度控制在这项研究中，使用的可变性。尽管这样，我们观察到，在睫状肌的CS曝光后的支气管组织跳动频率分别为高度可变（ 即 ，增加在一个插入件和减小另一个插入件），这表明睫状打浆是曝光后高度干扰的权利。与此相反，我们观察到了不存在可测量的睫状打浆频率中的CS曝光后的鼻组织，这表明鼻组织的反应更加敏感和一致。这是在协议与先前的出版物表明该鼻组织具有较低的容量来解毒作为与支气管25相比。 最后，我们发现，基因表达的器官支气管及鼻部组织模式受到CS分析可以证明CS对异生代谢产生影响。有趣的是，在异生素代谢所观察到的改变的器官支气管和鼻腔的体外模型暴露的CS表现得像是在体内的情况在吸烟者中更详细地在先前的出版物15中讨论。用于基因表达分析，使用机器人的仪器使高通量分析成为可能。此外，自动机器人装卸进一步增加的基因表达的结果的一致性和准确性。然而，快速采集的组织样品是至关重要的，以避免RNA提取过程中的RNA降解。的RNA加工和这里描述转录方法，也可以应用到体内的组织样本。

Lungs are directly and constantly exposed to inhaled air that may contain diverse toxicants such as pollutants and constituents of cigarette smoke (CS). Studying the impact of exposure to those toxicants on respiratory tissues is most informative when done in a manner that resembles in vivo exposure. Compared with the classical 2D immersed cell cultures (e.g., normal human bronchial epithelial cells (NHBE)), 3D organotypic tissue models better recapitulate the morphological, physiological, and molecular aspects of the human airway epithelium in vivo1,2: the 3D tissue models contain the diversity of the cell types observed in vivo, including differentiated epithelial cells, ciliated and non-ciliated cells, goblet cells, and basal cells. They have functional tight junctions and exhibit a mucociliary phenotype 1-3. Moreover, the cultures can be grown on a permeable porous membrane, in an air-liquid interface, allowing a direct exposure to aerosol at the apical side (whereas the basolateral side is immersed in culture medium) 3-5. Dvorak and colleagues reported that gene expression profiles of bronchial tissue models were similar to those obtained from human bronchial brushings 3. In addition, Mathis and colleagues showed that the responses of these tissue models to CS were similar to the differences observed between bronchial epithelial cells obtained from smokers and cells obtained from non-smokers 6. Finally, because the bronchial tissue models could be cultured for up to several months 4,5, they could potentially be used to examine the effects of long-term exposure of test items. 
Cytotoxicity assessments are common parameters measured following chemical insults or to assess the toxicity of specific compounds or mixtures. For instance, membrane integrity can be measured by a luminescent assay and allows the measurement of a dose-dependent cytotoxic effect on the cell culture 7. However, to assess pathophysiological effects of compounds at subtoxic concentrations, other parameters should be measured. For example, tissue integrity determined using the transepithelial electrical resistance (TEER) assay ensures the functionality of tight junctions and monitors the disruption of the epithelial layer 8,9. Ciliary beating frequency also allows the measurement of CS-related effects on respiratory tissues. A normal beating frequency for the cilia lining bordering the upper and lower respiratory tract is important to protect against airway infections 10. Each of the ciliated columnar epithelial cells of the respiratory epithelium has 200-300 cilia beating at a particular frequency to eliminate infectious agents or inhaled particulate matter trapped in the mucus released by interspersed goblet cells 11. CS contains chemicals that may inhibit ciliary beating 12, leading to a reduced protection of the respiratory tract. This work shows that ciliary beating can be measured in organotypic tissue models. This approach allows assessment of whether epithelial cells exhibit their normal function in the organotypic tissue culture. CS also activates xenobiotic metabolism responses in the respiratory tract to metabolize tobacco smoke constituents 13. The activity of the phase I xenobiotic metabolism enzymes, CYP1A1 and CYP1B1, of the tissue models can be measured. Additionally, as previously reported, global gene expression can be measured in the organotypic bronchial tissue models 6,14,15. A transcriptomic data and network-based systems biology approach is leveraged to assess the impact of CS on xenobiotic metabolism 15. 
The methodologies used to expose organotypic 3D bronchial and nasal tissue models to mainstream CS using an in vitro exposure system and to measure the tissue responses to this exposure compared to fresh air exposure (control) are detailed here.

<table border="0" cellpadding="0" cellspacing="0" width="1545">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:356px;">Name of Material/ Equipment</td>
			<td style="width:367px;">Company</td>
			<td style="width:655px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MucilAir-human fibroblasts-bronchia</td>
			<td>Epithelix S&#225;rl, Geneva, Switzerland</td>
			<td>http://www.epithelix.com/content/view/122/19/lang,en/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MucilAir Culture Medium</td>
			<td>Epithelix S&#225;rl, Geneva, Switzerland</td>
			<td>http://www.epithelix.com/content/view/84/16/lang,en/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">VITROCELL</td>
			<td>VITROCELL systems GmbH, Waldkirch, Germany</td>
			<td>http://www.vitrocell.com/index.php?Nav_Nummer=2&#38;R=</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3R4F reference cigarette</td>
			<td>University of Kentucky</td>
			<td>http://www2.ca.uky.edu/refcig/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">30-port carousel smoking machine SM2000</td>
			<td>Philip Morris, Int.</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">CiliaMetrix camera and software</td>
			<td>La Haute &#201;cole de Gestion (HESGE), Geneva, Switzlerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Leica DMIL microscope&#160;</td>
			<td>Leica, Heerbrugg, Switzerland</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LED light source</td>
			<td>Titan Tool Supplies, Buffalo, NY</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chopstick Electrode STX-2</td>
			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/stx2-chopstick-electrode-set-for-evom2/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EVOMXTM Epithelial Voltohmmeter&#160;</td>
			<td>World Precision Instruments</td>
			<td>http://www.wpiinc.com/products/physiology/evom2-evom2-epithelial-voltohmmeter-for-teer/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferin Detection Reagent&#160;</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MagNA Lyser Instrument&#160;</td>
			<td>Roche</td>
			<td>http://www.roche.com/products/product-details.htm?region=us&#38;type=product&#38;id=66</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chloroform&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>http://www.sigmaaldrich.com/catalog/product/sial/288306?lang=en&#38;region=</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAcube&#160;</td>
			<td>Qiagen</td>
			<td align="right">9001882</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>http://www.nanodrop.com/</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agilent 2100 Bioanalyzer&#160;</td>
			<td>Agilent</td>
			<td>http://www.genomics.agilent.com/en/Bioanalyzer-System/2100-Bioanalyzer-Instruments/?cid=AG-PT-106</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Affymetrix GeneChip High throughput 3&#8217;IVT Express Kit</td>
			<td>Affymetrix</td>
			<td>http://www.affymetrix.com/catalog/prod370001/AFFY/High-Throughput-(HT)-Whole-Transcript-(WT)-Kit</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scanner 3000 7G&#160;</td>
			<td>Affymetrix</td>
			<td>http://www.affymetrix.com/catalog/131503/AFFY/Scanner-3000-7G</td>
			<td></td>
		</tr>
	</tbody>
</table>

impact assessment of repeated exposure of organotypic 3d bronchial and nasal tissue culture models to whole cigarette smoke

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease or lung cancer. To understand the underlying mechanisms of disease development, it would be important to examine the impact of CS exposure directly on lung tissues. However, this approach is difficult to implement in epidemiological studies because lung tissue sampling is complex and invasive. Alternatively, tissue culture models can facilitate the assessment of exposure impacts on the lung tissue. Submerged 2D cell cultures, such as normal human bronchial epithelial (NHBE) cell cultures, have traditionally been used for this purpose. However, they cannot be exposed directly to smoke in a similar manner to the in vivo exposure situation. Recently developed 3D tissue culture models better reflect the in vivo situation because they can be cultured at the air-liquid interface (ALI). Their basal sides are immersed in the culture medium; whereas, their apical sides are exposed to air. Moreover, organotypic tissue cultures that contain different type of cells, better represent the physiology of the tissue in vivo. In this work, the utilization of an in vitro exposure system to expose human organotypic bronchial and nasal tissue models to mainstream CS is demonstrated. Ciliary beating frequency and the activity of cytochrome P450s (CYP) 1A1/1B1 were measured to assess functional impacts of CS on the tissues. Furthermore, to examine CS-induced alterations at the molecular level, gene expression profiles were generated from the tissues following exposure. A slight increase in CYP1A1/1B1 activity was observed in CS-exposed tissues compared with air-exposed tissues. A network-and transcriptomics-based systems biology approach was sufficiently robust to demonstrate CS-induced alterations of xenobiotic metabolism that were similar to those observed in the bronchial and nasal epithelial cells obtained from smokers.

在下面所描述的效果，器官组织被初级人体上皮细胞的健康，不吸烟的隔离，高加索捐助者和成纤维细胞使用了15重组。 3D支气管和鼻腔组织模型 在体外的支气管和鼻腔器官模型在细胞水平上的人的呼吸道上皮（ 图2）类似。 CS曝光 器官支气管和鼻腔组织模型可反复暴露于主流的CS在空气 - 液体界面。用于这里示出的结果的烟雾暴露实验步骤示于图1。 纤毛跳动记录 纤毛打浆被记录在如图中视频空气暴露组织。 CS暴露与减少纤毛跳动频率有关：强峰周围观察在露出的假和前曝光4赫兹观察不到的CS曝光（ 图3）之后的任何时间。这降低频率会使纤毛跳动去除粘液和传染性病原体22效率较低。 TEER和CYP1A1 / 1B1活性测定 TEER测量48小时的最后曝光后，确保组织仍然正常工作。 CYP1A1 / 1B1活性也测定曝光的结束后48小时， 如图1中的CS-暴露组织中的TEER值没有显著不同，因为相比于空气暴露的组织，确认有烟雾没有重大的细胞毒作用在该浓度。在48小时后曝光，将组织的CYP1A1 / 1B1活性略有增加，这表明了组织防御机制以下的曝光（ 图4）的适度增加。 基因表达谱和基于网络的系统，双向易学的方法来学习CS暴露对异生代谢改变的影响 组织收集在48小时暴露后的时间点。接着，微阵列分析进行，以产生CS-基因表达谱和空气暴露组织。使用来自基因表达图谱杠杆基于网络的系统生物学方法对CS曝光上的生物外源性代谢的影响进行了研究，并如先前报道15。使用一个基于网络的系统生物学方法表明，在骨干节点的异型生物代谢网络模型的级别的CS曝光的影响（ 图5）之间， 在体外和体内数据集被很好地相关。与此相反，在每个单独的基因表达的改变程度，差的相关性， 在体外 （CS-暴露与空气接触），并在体内 （吸烟者与不吸烟者）之间观察到。 CS的重复暴露于器官支气管组织模型的图1示意图步骤缩写：CYP，细胞色素P450的; CS，香烟烟雾， TEER，跨上皮电阻。 <img alt="图2" src="/files/ftp_upload/52325/52325fig2.jpg" /> 图2.器官支气管和鼻腔上皮组织培养模型。卡通表示在空气-液体界面的支气管（A）和鼻（B）的组织培养插入物。 在体外模型中包含的苏木精-伊红染色的细胞的顶层所示纤毛细胞（ 下支气管，D为经鼻）如先前报道15。模型共培养与成纤维细胞是重要<html>用于生长和上皮细胞分化（用箭头表示）。染色气道系标记：P63（E支气管，女鼻）和MUC5AC（G支气管，H鼻）显示为此前15日报道， <a href="https://www.jove.com/files/ftp_upload/52325/52325fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/52325/52325fig3.jpg" /> 图3.纤毛跳动频率。纤毛跳动频率（CBF），测定在空气中暴露并在培养之前和曝光之后。下降的CBF的CS暴露后，看到（从两个刀片代表性结果显示为复制1（R1）和复制2（R 2））。 广告/ 52325 / 52325fig4.jpg“/&gt; 图4.测量CYP1A1 / CYP1B1活性和TEER的。左pannels表明在支气管（A）和鼻（B）的组织模型的CYP1A1 / CYP1B1活性。右侧面板指示在支气管（C）和鼻（D）的组织模型的TEER测定。装置±SD被示出。缩写：CS，香烟烟雾， CYP，细胞色素P450; TEER，跨上皮电阻; RLU，相对luminescense单位。 <img alt="图5" src="/files/ftp_upload/52325/52325fig5.jpg" /> 图5.基于网络的系统生物学方法对CS暴露在异生代谢的上下文中的影响的评估。的基因表达水平的变化被用来计算异型生物代谢网络模型的骨干节点的激活（A）的 </<html> STRONG&gt;报道在以前的出版物15。右边的图显示了骨干节点，也被称为量化的概念，“微分骨干网的价值，”采用NPA方法。蓝色椭圆表示的骨干节点的活性（ 即 ，功能层）和绿色球代表的基因的表达（ 即 ，转录层）。 CS暴露的器官组织支气管的影响（ 体外，CS-暴露与空气接触），在暴露后的48小时相比，这些吸烟对经支气管镜和鼻上皮收集burshing下鼻甲人支气管上皮（GSE16008 ）（ 在体内，吸烟者与非吸烟者）（B）中 。插图中，基因表达的相关性之间的体外和体内数据集变化。缩写：CS，香烟烟雾， NPA，网络扰动幅度。ove.com/files/ftp_upload/52325/52325fig5large.jpg“目标=”_空白“&gt;点击此处查看该图的放大版本。

Watch this Scientific Journal Video about Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke at JoVE.com

Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

反复接触器官的3D支气管及鼻部组织文化模型对整个卷烟烟雾的影响评估

Cigarette smoke (CS) has a major impact on lung biology and may result in the development of lung diseases such as chronic obstructive pulmonary disease ...

impact-assessment-repeated-exposure-organotypic-3d-bronchial-nasal

Research

JoVE Journal

Bioengineering

11.0K 次观看.  Philip Morris Products S.A.. The aim of this protocol is to expose human organotypic 3D bronchial and nasal tissue models to mainstream cigarette smoke (CS) at the air-liquid interface. The impact of CS on the tissues is then investigated using a cytochrome P450 activity assay, a cilia beating measurement, and a systems biology approach.

视频: Impact Assessment of Repeated Exposure of Organotypic 3D Bronchial and Nasal Tissue Culture Models to Whole Cigarette Smoke

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.  ...

科研

生物工程

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

一个人的3D组织工程<em&gt;在体外</em&gt;肿瘤测试系统

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

样品制备策略的3D细胞培养模型质谱成像

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

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

Electronic cigarettes are the most popular tobacco product among middle and high schoolers and are the most popular alternative tobacco product among adults. High quality, reproducible research on the consequences of electronic cigarette use is essential for understanding emerging public health concerns and crafting evidence based regulatory policy. While a growing number of papers discuss electronic cigarettes, there is little consistency in methods across groups and very little consensus on results. Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This protocol details instructions for the assembly and operation of said device, and demonstrates the use of the generated extract in two sample applications: an in vitro cell viability assay and gas-chromatography mass-spectrometry. This method provides a tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

Here, we describe a programmable laboratory device that can be used to create extracts of conventional cigarette smoke and electronic cigarette aerosol. This method provides a useful tool for making direct comparisons between conventional cigarettes and electronic cigarettes, and is an accessible entry point into electronic cigarette research.

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 ...

Preclinical Cardiac Electrophysiology Assessment by Dual Voltage and Calcium Optical Mapping of Human Organotypic Cardiac Slices

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between animal and clinical trials. Numerous animal and cell models have been used to examine the effects of drugs, yet these responses often differ in humans. Human cardiac slices offer an advantage for drug testing in that they are directly derived from viable human hearts. In addition to having preserved multicellular structures, cell-cell coupling, and extracellular matrix environments, human cardiac tissue slices can be used to directly test the effect of innumerable drugs on adult human cardiac physiology. What distinguishes this model from other heart preparations, such as whole hearts or wedges, is that slices can be subjected to longer-term culture. As such, cardiac slices allow for studying the acute as well as chronic effects of drugs. Furthermore, the ability to collect several hundred to a thousand slices from a single heart makes this a high-throughput model to test several drugs at varying concentrations and combinations with other drugs at the same time. Slices can be prepared from any given region of the heart. In this protocol, we describe the preparation of left ventricular slices by isolating tissue cubes from the left ventricular free wall and sectioning them into slices using a high precision vibrating microtome. These slices can then either be subjected to acute experiments to measure baseline cardiac electrophysiological function or cultured for chronic drug studies. This protocol also describes dual optical mapping of cardiac slices for simultaneous recordings of transmembrane potentials and intracellular calcium dynamics to determine the effects of the drugs being investigated.

This protocol describes the procedure for sectioning and culturing human cardiac slices for preclinical drug testing and details the use of optical mapping for recording transmembrane voltage and intracellular calcium signals simultaneously from these slices.

人体器官性心脏切片双电压和钙光学映射的临床前心脏电生理评估

Human cardiac slice preparations have recently been developed as a platform for human physiology studies and therapy testing to bridge the gap between ...

Advanced 3D Liver Models for In vitro Genotoxicity Testing Following Long-Term Nanomaterial Exposure

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of robust, predictive in vitro test systems is essential. Hepatic toxicology is key when considering ENM exposure, as the liver serves a vital role in metabolic homeostasis and detoxification as well as being a major site of ENM accumulation post exposure. Based upon this and the accepted understanding that 2D hepatocyte models do not accurately mimic the complexities of intricate multi-cellular interactions and metabolic activity observed in vivo, there is a greater focus on the development of physiologically relevant 3D liver models tailored for ENM hazard assessment purposes in vitro. In line with the principles of the 3Rs to replace, reduce and refine animal experimentation, a 3D HepG2 cell-line based liver model has been developed, which is a user friendly, cost effective system that can support both extended and repeated ENM exposure regimes (&#8804;14 days). These spheroid models (&#8805;500 &#181;m in diameter) retain their proliferative capacity (i.e., dividing cell models) allowing them to be coupled with the &#8216;gold standard&#8217; micronucleus assay to effectively assess genotoxicity in vitro. Their ability to report on a range of toxicological endpoints (e.g., liver function, (pro-)inflammatory response, cytotoxicity and genotoxicity) has been characterized using several ENMs across both acute (24 h) and long-term (120 h) exposure regimes. This 3D in vitro hepatic model has the capacity to be utilized for evaluating more realistic ENM exposures, thereby providing a future in vitro approach to better support ENM hazard assessment in a routine and easily accessible manner.

This procedure was established to be used for developing advanced 3D hepatic cultures in vitro, which can provide a more physiologically relevant assessment of the genotoxic hazards associated with nanomaterial exposures over both an acute or long-term, repeated dose regimes.

长期纳米材料暴露后体外基因毒性测试的高级 3D 肝脏模型

Due to the rapid development and implementation of a diverse array of engineered nanomaterials (ENM), exposure to ENM is inevitable and the development of ...

An Open Source Technology Platform to Manufacture Hydrogel-Based 3D Culture Models in an Automated and Standardized Fashion

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These issues represent drawbacks in workflows that can ultimately result in irreproducibility of research findings. Manual-based workflows are further limiting the advancement and widespread adoption of viscous materials, such as hydrogels used for biomedical applications. These challenges can be overcome by using automated workflows with standardized mixing processes to increase reproducibility. In this study, we present step-by-step instructions to use an open source protocol designer, to operate an open source workstation, and to identify reproducible mixtures. Specifically, the open source protocol designer guides the user through the experimental parameter selection and generates a ready-to-use protocol code to operate the workstation. This workstation is optimized for pipetting of viscous materials to enable automated and highly reliable handling by the integration of temperature docks for thermoresponsive materials, positive displacement pipettes for viscous materials, and an optional tip touch dock to remove excess material from the pipette tip. The validation and verification of mixtures are performed by a fast and inexpensive absorbance measurement of Orange G. This protocol presents results to obtain 80% (v/v) glycerol mixtures, a dilution series for gelatin methacryloyl (GelMA), and double network hydrogels of 5% (w/v) GelMA and 2% (w/v) alginate. A troubleshooting guide is included to support users with protocol adoption. The described workflow can be broadly applied to a number of viscous materials to generate user-defined concentrations in an automated fashion.

This protocol serves as a comprehensive tutorial for standardized and reproducible mixing of viscous materials with a novel open source automation technology. Detailed instructions are provided on the operation of a newly developed open source workstation, the usage of an open source protocol designer, and the validation and verification to identify reproducible mixtures.

以自动化和标准化方式制造基于水凝胶的3D培养模型的开源技术平台

Current mixing steps of viscous materials rely on repetitive and time-consuming tasks which are performed mainly manually in a low throughput mode. These ...

Isolation and Culture of Primary Human Gingival Epithelial Cells using Y-27632

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is an important structure of gingival tissue, especially in the repair and regeneration of periodontal tissue. Studying the functions of gingival epithelial cells has crucial scientific value, such as repairing oral defects and detecting the compatibility of biomaterials. As human gingival epithelial cells are highly differentiated keratinized cells, their lifespan is short, and they are difficult to passage. So far, there are only two ways to isolate and culture gingival epithelial cells, a direct explant method and an enzymatic method. However, the time required to obtain epithelial cells using the direct explant method is longer, and the cell survival rate of the enzymatic method is lower. Clinically, the acquisition of gingival tissue is limited, so a stable, efficient, and simple in vitro isolation and culture system is needed. We improved the traditional enzymatic method by adding Y-27632, a Rho-associated kinase (ROCK) inhibitor, which can selectively promote the growth of epithelial cells. Our modified enzymatic method simplifies the steps of the traditional enzymatic method and increases the efficiency of culturing epithelial cells, which has significant advantages over the direct explant method and the enzymatic method.

Here we present a modified method for the isolation and culture of human gingival epithelial cells by adding the Rock inhibitor, Y-27632, to the traditional method. This method is easier, less time-consuming, enhances stem cell properties, and produces larger numbers of high-potential epithelial cells both for the laboratory and for clinical applications.

使用Y-27632的原发性人类金吉瓦尔表皮细胞的隔离与培养

The gingival tissue is the first structure that protects periodontal tissues and plays meaningful roles in many oral functions. The gingival epithelium is ...

使用 3D 声学组装装置可扩展生成均匀的单元球体

Three-Dimensional Acoustic Assembly Device for Mass Manufacturing of Cell Spheroids

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method for manufacturing high-quality and high-throughput cell spheroids using a 3D acoustic assembly device through maneuverable procedures. The acoustic assembly device consists of three lead zirconate titanate (PZT) transducers, each arranged in the X/Y/Z plane of a square polymethyl methacrylate (PMMA) chamber. This configuration enables the generation of a 3D dot-array pattern of levitated acoustic nodes (LANs) when three signals are applied. As a result, cells in the gelatin methacryloyl (GelMA) solution can be driven to the LANs, forming uniform cell aggregates in three dimensions. The GelMA solution is then UV-photocured and crosslinked to serve as a scaffold that supports the growth of cell aggregates. Finally, masses of matured spheroids are obtained and retrieved by subsequently dissolving the GelMA scaffolds under mild conditions. The proposed new 3D acoustic cell assembly device will enable the scale-up fabrication of cell spheroids, and even organoids, offering great potential technology in the biological field.

作者聚焦：用于高质量 3D 细胞球状体培养的无支架声学组装方法的开发 

Cell spheroids have been considered one potential model in the field of biological applications. This article describes protocols for scalably generating cell spheroids using a 3D acoustic assembly device, which provides an efficient method for the robust and rapid fabrication of uniform cell spheroids.

用于细胞球体批量生产的三维声学装配装置

Cell spheroids are promising three-dimensional (3D) models that have gained wide applications in many biological fields. This protocol presents a method ...