作者要感谢约翰·Harbell博士的科学证据支持和时间奉献给EIT项目。作者也想感谢拜尔斯道夫公司（德国），IIVS（美国），玫琳凯公司（美国），雅芳产品公司（美国），宝洁/ Cosmital（瑞士），皮埃尔了Laboratoire法布尔（法国），哈伦实验室（英国）和LVMH Parfume（法国）参加的眼刺激试验15的多中心国际预验证和验证研究。

1.准备RHCE组织治疗 - 0天<ol><li>在收到的商业人的角膜样上皮（RHCE）试剂盒，检查材料进行企业所得税法（ 表2）所需的所有套件组件的完整性（对于包的详细信息请参见标准测定试剂盒组件（ 表1），设备及。在收据的当天，平衡组织（在其24孔运输集装箱）至RT 15分钟。 </li></ol><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 多少 </td><td> 试剂 </td><td> 条件 </td><td> 资源 </td><td> 描述 </td><td> 截止日期 </td></tr><tr><td> 1 </td><td>密封24孔板EpiOcular组织 （OCL-200） </td><td> 2-8℃， </td><td> MatTek </td><td>包含24 TISS细胞培养的UE接入，封装在琼脂糖</td><td> 72小时</td></tr><tr><td> 1瓶， 200毫升</td><td> EpiOcular分析培养基 （OCL-200-ASY） </td><td> 2-8℃， </td><td> MatTek </td><td> DMEM基础培养基</td><td> 21天</td></tr><tr><td> 1瓶， 百毫升</td><td> CA ++ Mg ++的-免费Dulbecco&#39;s-PBS（DPBS） </td><td> RT </td><td> Sigma-Aldrich公司，D5652，或当量。 </td><td>用于漂洗刀片</td><td> 1年</td></tr><tr><td> 4 </td><td> 6孔板</td><td> RT </td><td>鹘</td><td>用于试验的协议过程中保持组织</td><td> NA </td></tr><tr><td> 2 </td><td> 12孔板</td><td> RT </td><td>鹘</td><td>分析方案期间使用</td><td> NA </td></tr><tr><td> 2 </td><td> 24孔板</td><td> RT </td><td>鹘</td><td>用于执行MTT法</td><td> NA </td></tr><tr><td> 1瓶， 0.5毫升</td><td>醋酸甲酯 （CAS＃79-20-9） </td><td> RT </td><td> Sigma-Aldrich公司， 猫＃186325 </td><td>在测定中用作个人电脑</td><td> 1个月</td></tr></tbody></table> 表1：标准检测试剂盒组件。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 设备/材料 </td><td> 需要： </td></tr><tr><td>加湿培养箱（37±1℃，5±1％的CO 2，90±10％湿度） </td><td>之前和期间实验孵化组织</td></tr><tr><td>层流罩</td><td>在无菌条件下安全工作</td></tr><tr><td>真空泵（可选） </td><td>吸取媒体及解决方案</td></tr><tr><td>板阅读器光度计（对于96孔板） </td><td>读OD </td></tr><tr><td>平板振动器</td><td>精华甲的离子</td></tr><tr><td>无菌，钝边钳</td><td>处理组织刀片</td></tr><tr><td>秒表</td><td>在该协议的应用程序测试的材料和其他定时的步骤的定时</td></tr><tr><td>水浴（37±1℃） </td><td>温补媒体和MTT溶液</td></tr><tr><td>研钵和研杵</td><td>研磨粒状固体</td></tr><tr><td>容积式移液器（50微升） </td><td>的粘性和半固体材料和悬浮液中的应用</td></tr><tr><td>可调移液器（200微升-2毫升） </td><td>液体材料，测定培养基和MTT的应用</td></tr><tr><td>预消毒吸头（200微升和20微升），猫的Rainin＃HR-200F和HR-20F（或同等学历） </td><td>液体材料，测定培养基和MTT的应用</td></tr><tr><td>广口预消毒吸头（250微升），猫的Rainin＃HR-250WS（或当量alent） </td><td>的粘性和半固体材料和悬浮液中的应用</td></tr><tr><td> 8盎司/220毫升标本容器，猎鹰猫＃3540200（或同等学历） </td><td>冲洗组织</td></tr><tr><td>无菌一次性使用注射器（ 如 1毫升tuberkulin注射器Omnifix-F，贝朗Melsungen的公司，目录号9161406V） </td><td>交货〜50毫克的固体材料（可选） </td></tr><tr><td>特德佩拉微锅铲/汤匙，特德佩拉公司，猫＃13504（或同等学历，锐匙或骨刮匙， 如蛇牌，编号：FK 623） </td><td>交货〜50毫克的固体材料</td></tr><tr><td> Ca ++和Mg ++的Dulbecco磷酸盐缓冲盐水（钙++ Mg ++的自由DPBS）：Sigma-Aldrich公司，目录＃D5652（或相当的） </td><td>试验过程中冲洗组织</td></tr><tr><td>无菌去离子水，组织培养级（质量生物或同等学历） </td> &lt;TD&gt;使用数控</td></tr><tr><td> 96孔平底板，猎鹰（或同等学历） </td><td>对于读OD </td></tr><tr><td>棉签掉期（无菌） </td><td>对于干燥的组织表面（可选） </td></tr><tr><td>胶带或封口膜</td><td>甲臜提取过程中盖板</td></tr><tr><td> MTT-100测定试剂盒</td><td>含有MTT-噻唑蓝四甲基偶氮唑试剂（西格玛＃M-5655）和异丙醇萃取物。 </td></tr></tbody></table> 表2：设备和执行的EIT所需材料。 <ol start="2"><li>在无菌条件下，打开包含24孔板与RHCE组织的塑料袋，取出的无菌纱布。检查所有组织中的气泡琼脂糖凝胶和插入。下所述插入覆盖物不使用培养物的气泡的插入区域，有缺陷的组织，或组织WHI&gt; 50％<html> CH完全覆盖液体。 </li><li>标记6孔板与测试物品或控制代码和曝光时间。等分1.0毫升测定培养基（随试剂盒提供），预热至约37℃，到预标记的6孔板的孔中。 </li><li>使用无菌镊子除去含有RHCE组织每个插入并放置在插入在标记6孔板。在此步骤期间，除去任何残留的航运琼脂糖粘附到该插入物的外侧上由在无菌滤纸上印迹平缓。释放被困插入下方的气泡。 </li><li>预孵育RHCE组织在6孔板在标准培养条件下（SCC，湿润气氛用5±1％CO 2的在37±1℃）1小时。 </li><li> 1小时后，更换试验培养基用1.0毫升新鲜试验培养基预热至37℃，并培育了RHCE组织在SCC条件（隔夜= O / N）（16-24小时）。 </li></ol>。ove_title“&gt; 2预处理 - 第1天<ol><li>在O / N培养后，使用适当的移液装置适用20微升的 Ca 2+镁2+的 -贝科的磷酸盐缓冲液（DPBS，提供）。如果DPBS不会在组织蔓延，轻轻拍打上盘插入以保证DPBS弄湿整个组织表面。 </li><li>孵育RHCE组织在SCC 30±2分钟。 注意：此步骤是必要的组织水合和体内条件来模拟。 </li></ol> 3.测试材料曝光程序<ol><li>适用于每一个测试文章，并控制复制RHCE组织（N = 2）。测试品定量给料步骤为液体和固体的不同。局部应用50微升用吸管液体测试文章。的曝光时间的液体是30分钟。适用于50毫克的用一勺夷为平地（校准持有50毫克氯化钠）固体的测试文章。这位前曝光时间为固体为6小时。 注意：液体被定义为流体物质 （如，液体，凝胶，膏霜），可以利用一个移液设备施加。固体定义为非流体物质 （如，粉末，树脂状或蜡状物质），可以不使用移液管施加。 <ol><li>如果试验制品的物理状态是不容易确定，放置与试验制品的小瓶在水浴中进行15分钟（37℃）。遵循EIT协议用于液体为那些液化，在37℃试验制品。 </li><li>使用正位移移液器尤其是粘性物料。 </li></ol></li><li>第一剂量的阴性对照和阳性对照，然后剂量的试验制品。 <ol><li>应用50微升阴性对照组（NC）和阳性对照（PC）使用一个标准的吸管RHCE组织。 NC是无菌的去离子水;在PC是乙酸甲酯（CAS＃79-20-9）。应用NC和PC 30分钟时测试荷兰国际集团的液体测试的文章和6小时测试时，测试固体物品。 </li></ol></li></ol> 4.测试曝光条 - 第1天<ol><li>用于液体检验物品的处理，按照在表3中给出的定时的时间表。离开各试验品的应用程序之间的1分钟的间隔，以确保相等的曝光所有组织中。 <ol><li>后30±2分钟DPBS前处理，局部应用50μl的NC和个人电脑，并且每个液体试验制品局部上使用适当的移液设备的RHCE组织。 </li><li>申请50微升液体测试品的直接在组织以覆盖上表面上。切断窄点的枪头扩大孔的粘性材料。为非常粘稠的材料，应用测试品，以一计量装置（直径大于组织插入物或塑料图钉的内径稍小的平头筒），颠倒给药ðevice并将其放置到组织上，以使受试制品均匀地接触组织表面。 </li><li>如果测试的文章不会在组织蔓延，轻轻拍打插入，以确保它涂在整个组织表面。机械散布试验制品（例如，用移液管尖端）不推荐，因为它可能会损坏组织。 </li><li>孵育组织在SCC 30±2分钟。 </li></ol></li><li>为固体试验制品治疗-第1天，按照表 4中给出的定时安排离开各试验品的应用程序之间的2分钟的间隔，以确保相等的曝光所有组织中。 <ol><li>后30±2分钟DPBS前处理，应用局部上使用适当的移液设备的RHCE组织50微升的NC和PC。 </li><li>为固体试品应用，取出插入物（2例）从井，并将其放置到一个无菌的表面（例如，上午的盖ultiwell板），以避免一个测试品溢出到培养基中。 </li><li>使用的水平校准勺子，局部应用约50毫克试验制品到组织表面的;确保该组织的表面被完全覆盖试验制品。如果试验制品不横跨组织扩散，轻轻摇动插入件从一侧到另一侧，以确保组织被完全覆盖试验制品。机械散布试验制品（例如，用移液管尖端）不推荐，因为它可能会损坏组织。 <ol><li>如果需要的话，研磨的结晶粉末用研钵和研杵来保证测试品和组织之间更好的接触。 </li><li>或者，将粉末直接到插入内部的组织培养用1毫升注射器头切断。东西粉末进入注射器当柱塞被拉回，然后通过按压柱塞向下适用。 </li><li>如果插入的外壁为contaminated 例如 ，通过粉末，用无菌纱布擦拭掉的颗粒。 </li></ol></li><li>给药后，返回到组织含有培养基的6孔板和孵化在SCC 6小时±15分钟。 </li></ol></li></ol> 5.漂洗<ol><li>准备一组每次测试第三条干净的烧杯（150毫升容量），并填写他们每个人用100毫升的DPBS。对于每个测试条，利用一组不同的3烧杯中。 </li><li>在30±2分钟曝光用于液体材料或6小时±15分钟的曝光固体材料的端部，除去并丢弃计量装置，如果它被使用。 </li><li>通过抓住塑料“衣领”用细镊子的上边缘抬起含有RHCE组织出来的介质的插入。用弯钳，以方便处理和滗析。通过利用镊子的套环保持重复的刀片一起冲洗一次组织2。要小心ñ催产素损伤组织的钳子。 </li><li>滗从组织表面的测试物品或对照到一个干净的吸收材料（纸毛巾，纱布等 ） </li><li>浸镶入DPBS，漩涡的圆周运动在DPBS约2秒的第一烧杯，提起插入物，使得它们大多填充用DPBS，倾倒出液体回烧杯中。在第一烧杯中重复这一过程三次。 </li><li>冲洗中的每个DPBS三次，第二和第三烧杯刀片以相同的方式。 </li><li>滗在插入其余到吸收材料的任何液体。旋转刀片以近似45°角（开口端向下）和触摸上唇到吸收材料。 注意：如果这是不可能去除所有可见测试材料制成的，没有进一步的漂洗应做，以避免组织损伤，由于过度处理。 </li></ol> 6.后浸泡<ol><li>冲洗后，立即浸入组织在5毫升试验培养基预先加热至RT在预标记的12孔板中。 </li><li>孵育组织为12±2分钟为液体材料或25±2分钟为浸渍在RT以有利于去除任何残留的测试品的固体材料。 </li></ol> 7.后孵化<ol><li>在结束时后浸泡浸泡期间，从组织倒出测定培养基并吸干插入到吸收材料。 </li><li>转移镶入含1毫升温测定培养基的预标记的6孔板中。 <ol><li>孵育组织为120±15分钟，在SCC用于液体检验材料。 </li><li>孵育组织18±0.25小时，在SCC固体试验材料。 </li></ol></li></ol> 8. MTT活性测定 - 第1天（协议液体）和第2天（协议固体） <ol><li> 120的后培养后进行MTT法77; 15分钟的液体和，分别为18±0.25小时为固体。 </li><li>制备1.0毫克/毫升的MTT溶液，将溶液的等分试样0.3毫升到预标记的24孔板的每个孔中。 <ol><li>使用商业试剂盒的MTT（表5）： </li><li>使用前2小时，解冻的MTT集中在室温。结合2毫升的MTT浓缩物和8毫升的MTT稀释剂，以产生1.0毫克/毫升的MTT溶液。 </li><li>储存在4℃的MTT溶液在暗处直到使用。不要存放超过1天MTT溶液。 </li></ol></li><li>在邮政孵育结束时，从6孔培养板除去每个插入并轻轻吸干上的吸收性材料。 </li><li>将镶入24孔板含0.3ml的MTT溶液。释放被困插入下方的气泡。将培养板180±10在SCC分钟。 </li><li> MTT提取<ol><li>经过180±10分钟孵化的MTT溶液，删除每个插入从24孔板并吸干插入件的底上的吸收性材料。 </li><li>对于非着色剂液体试验制品（浸没萃取）：转移插入到预标记的24孔平板含有2.0毫升萃取剂溶液（异丙醇）以便它浸没插入。 </li><li>固体和液体着色剂（非浸没萃取，以避免萃取剂溶液的污染）：转移插入到预标记的6孔平板含有1.0毫升萃取液（异丙醇），以便它不会淹没插入。 注意：执行相同的非浸没提取相应的阴性和阳性对照。 </li></ol></li><li>密封该板（例如，与板盖和孔的上边缘之间或带有标准板密封封口膜）。将平板在轨道板振荡器振摇2至3小时，在室温下，以提取的MTT。 <ol><li>另外，执行提取O / N在2-876; C黑暗无晃动。 </li></ol></li><li>对于非着色剂液体试验制品（浸没萃取）：在萃取期间结束，倒出从每个液体插入回入井和丢弃含有RHCE组织中的插入。 <ol><li>混合萃取溶液和根据该板配置 （图2）传输2 200微升等份到预标记的96孔板的适当孔中。 </li></ol></li><li>固体和液体着色剂（非浸没萃取）：在萃取期间结束，丢弃组织（确保不刺破组织）。 <ol><li>加将1.0ml提取剂溶液的进24孔板含有从组织中提取的溶液的各孔中。混合萃取溶液和根据该板配置 （图2）传输2 200微升等份到预标记的96孔板的适当孔中。 </li></ol></li><li>决定T他所提取的样品中的在550和590nm的单一波长的光密度（OD）（应该是一个实验室内一致）上读板器或分光光度计。 <ol><li>的情况下所造成的不溶性固体浑浊提取液，离心之前测量OD（冷却离心机至4℃，以避免蒸发）的解决方案。如果漂洗不删除测试制品（T​​A）和TA干扰MTT减少，额外的控制，必须使用。请参阅详细的SOP以校正MTT还原16。 </li><li>万一的TA被示出具有，或使之显色可与MTT测量交互，附加的测试必须被执行以确定颜色的结合到量，并随后从组织中提取。请参阅详细的SOP以校正彩色试验制品16。 </li></ol></li></ol> 9.计算为组织的生存力试验（表6和图3）＆＃160; <ol><li>通用计算<ol><li>计算空白对照孔（OD座）每个实验的平均OD值。 </li><li>来自相同实验（座校正数据）的每个OD值减去OD座。 </li><li>计算这两个等分试样的每个组织（=校正OD）的平均值。 </li></ol></li><li>计算出每对每个对照和测试条相对于平均阴性对照（100％对照）两个复制组织的百分存活率。 存活率（％）= [校正的OD治疗组织/校正OD阴性对照]×100％ </li><li>计算存活率（两次重复的组织之间的存活率差别）的差别。 </li><li>计算平均值测试制品存活率（TA生存力），并根据该预测模型分类测试制品。 </li></ol> 10.预测模型（图3） <ol><li>如果TA处理的组织生存能力&gt; 60.0相对于NC处理的组织viability，标记检品为无刺激性（NI）（GHS无类别）。 </li><li>如果TA处理的组织的生存力是≤60.0相对于数控处理的组织活力，标号测试品为刺激物（Ⅰ）（GHS类别1和2）。 注：EIT测试结果都算合格，如果： <ul><li>企业所得税NC OD&gt; 0.8和&lt;2.5; <nc li=""></nc></li><li>的EIT的PC组织活力（％，相对于北卡罗莱纳州）是≤50.0％; </li><li>两个复制组织（北卡罗莱纳州，PC和测试物品）之间的差&lt;20.0％。 </li></ul></li></ol>

<ol>
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我们已经提出了眼睛刺激试验（图1），这是为EpiOcular组织模型开发的。的EIT能够从材料不需要标记（GHS没有类别）与敏感性和特异性17. EIT的高度分离眼刺激物和腐蚀（GHS类别1和2的组合），为这里提出GHS类别之间不区分1，从第2类化学品。的EIT被验证为分类和大范围的化学品，包括化妆品和​​药物成分的眼刺激潜在标记。与其他体外试验相结合，企业所得税将作为替代体内兔眼刺激试验。 的EIT使用两个类似的，但不同的协议用 ​​于液体和固体材料，其变化在曝光和后曝光潜伏期（图1）的长度。在EIT中使用的端点是组织活力，通过MTT测定法，已在验证人上皮组织模型20,21以前使用过确定。为了进行该试验中，除了标准的细胞培养设备没有特殊的设备是必要的。由于高层次组织到组织再现性，n = 2的组织，而不是通常建议n = 3的被使用。的能力以使用n = 2的组织是该协议的一个重要方面，因为它允许有经验的操作，以同时处理两个组织，从而最大限度地减少可能出现的测定法的变异性是由于不同的处理从个人组织14。此外，通过使用每个试验制品n = 2的组织中，对相同的物理状态（液体或固体）10测试物质刺激性，随着阳性和阴性对照，可以使用一种试剂盒（24组织）来评价。 即确保物料可靠分类其他要点是规格为阳性对照スbstance（组织活力≤50.0％），重复的组织（差值&lt;20.0％），和阴性对照的OD读数（&gt; 0.8和&lt;2.5）之间的再现性。 当执行EIT测试，它附着在验证协议和所建议的剂量和漂洗的时间表（ 表3和4）是重要的，因为从协议或变化潜伏期偏差可以导致改变的结果。同样地，从3小时的时间，MTT温育偏差将导致不同的MTT读数并可能影响测定结果。 有时，测试化学品可能具有可以与MTT组织活力测定或导致MTT减少干扰的光学或其他特性。例如，一个测试化学品可以直接减少的MTT成蓝紫色的反应产物，或者可以是有色物质吸收光在同一范围内MTT甲（〜570纳米）。然而，这些测试化学品将PR仅ESENT一个问题，如果在MTT测定的时间，材料的足够量仍然是存在于（或通过吸收）该组织。为了避免这种干扰，广泛漂洗程序被纳入EIT协议。如果漂洗不删除的TA和TA干扰MTT减少，额外的控制必须用于检测并纠正它。简单地说，如果直接MTT还原测定化学物质被怀疑，50μl的问题的化学品（或50毫克固体）温育3小时以在SCC（北卡罗莱纳州，加入50μl无菌去离子水工作MTT溶液，应同时运行）。如果MTT溶液变为蓝紫色，测试品被推定为已经降低了MTT。在这种情况下使用冷冻杀死组织控制功能检查应进行评估测试材料是否被结合到组织并导致假MTT还原信号。如果在TA-露出，杀死组织控制可察觉MTT还原（相对于在未处理的活组织的量），试验制品的平均值组织活力必须减去杀死对照的平均存活校正。 在EIT犯错上安全的侧面，这表现在假阴性分类14,15,18的低发病率。重要的是，没有一个GHS 1类化学物质，这是腐蚀性的眼睛和代表最严重的眼部危险，被归类为无刺激性在该测定14,15,18,19。最后，对RHCE的体外试验方法的主要优点之一是测试纯液体和固体材料（这是无法实现的二维的，浸没的细胞培养）的可能性。 企业所得税将在确定根据联合国GHS分类和标签制度范围广泛的材料眼部刺激潜在显著贡献。动物的替代，以确定Ø丘拉尔毒性已毒理学研究的一个目标多年。该EIT的测试方法已经完成了EURL ECVAM支持的正式验证研究在2014年和EpiOcular企业所得税实施到经合组织测试准则OECD TG 492在2015年。

消费产品，如化妆品，洗涤剂，和家用清洁剂包括多种化学物质，他们可能发生严重的损坏，如果它们接触眼睛。因此，这些药物对眼睛的刺激的测试要求美国和欧盟的监管机构，以确保消费者的安全1。混合物和配方的眼睛刺激潜力的评估也与REACH（注册，评估，授权和化学产品的限制）符合立法化妆品成分根据欧盟化妆品指令对化学品的运输标签的要求，并为农药和家用产品2的标签。目前，监管机构需要用化学品分类和标签（GHS）3的全球协调制度眼部危害评估。全球统一制度主要基于的Draize眼刺激试验，应用最广泛的眼刺激试验，其中外来物质和MIxtures被直接引入到兔眼4的结膜囊。据GHS分类，GHS第1类（眼腐蚀性）是指测试的化学物质，造成严重的初始伤害眼睛组织或眼睛和视力这是不是在21天内曝光3,5完全可逆的严重损害。 GHS分类2是指测试产生的眼睛是在21天的接触完全可逆显著变化的化学物质。测试的化学品是不腐蚀或刺激物被称为GHS没有类别。 对于超过40年，在的Draize兔眼测试被批评为缺乏重现性，对人类的反应高估，并利用活体动物5-8。这些问题已经鼓励许多建议进行细化，还原，并更换新的体内试验 9。对于需要验证的非动物替代品，进一步加强通过采用第七修正案化妆品指令，它禁止使用动物的化妆品的安全性评价（2005年）和配料（2009年）2。 自1996年以来，该重建角膜样组织模型已被广泛用于化妆品行业来评估原料，表面活性剂为基础的配方的刺激性，并且加剧被用于设计的混合物，或在，眼睛附近10-13。使用的RHCE组织模型的允许测试材料的直接局部施用到在其天然，未稀释的形式组织表面。以这种方式，非水溶性制剂可以不与溶剂稀释它们进行测试。在回答EURL ECVAM的（欧盟参考实验室欧洲中心的替代方法的验证）要求提供广泛适用，简单，经济的方法眼刺激试验（EIT），它采用了SINGLË曝光时间，并能够从材料不需要标记分离眼刺激和腐蚀被开发（图1）14。基于该结果83的化学品（44液体和39固体），在EIT取得95.5 / 68.2 / 81.8％的灵敏度/特异性和准确度（SS＆A）为液体，100.0 / 68.4 / 84.6％SS＆一种用于固体和97.6 / 68.3 /和整体SS＆A 83.1％。 2007年，由欧洲化妆品（原COLIPA）的EURL ECVAM的主持下，举办了多个实验室预验证研究评估了企业所得税的相关性和可靠性，把它正式确认15的目标。在这项研究中，298独立审判七个独立的实验室进行。研究结果表明在预测99.7％协议，在所有参与的实验室15变异系数低。其结果是，在2010年企业所得税协议已进入正式的EURL ECVAM验证计划。验证研究使用104编码测试的化学物质，包括个人的物质和化学混合物，其在体内的参考数据（的Draize眼刺激数据）可用。在此基础上的成功，经合组织测试准则草案已提交于2014年。预计企业所得税将显著到的根据联合国GHS分类和标签制度范围广泛的材料眼部刺激潜在的分类作出贡献。

eye irritation test (eit) for hazard identification of eye irritating chemicals using reconstructed human cornea-like epithelial (rhce) tissue model

为了符合第七修正案欧盟化妆品指令和欧盟REACH法规，需要对眼部的毒性在人可靠和准确的评估，验证非动物替代方法。为了满足这一需要，我们开发了一种眼刺激试验（EIT），其利用三维重构的人角膜状上皮（RHCE）组织模型，该模型是基于正常的人类细胞。的EIT是能够分离眼刺激物和腐蚀（GHS类别1和2的组合）和那些不要求标记（GHS没有类别）。测试利用两个不同的协议，是设计有液体化学品和第二次类似的协议为固体试验制品。的EIT预测模型采用了单曝光周期（30分钟为液体，6小时为固体）和一个单一组织的生存力的截止（如通过MTT试验测定60.0％）。基于该结果83的化学品（44液体和39固体）的EIT取得95.5 / 68.2 / 81.8％SENSitivity /特异性和准确性（SS＆A）的液体，100.0 / 68.4 / 84.6％，SS＆A的固体和97.6 / 68.3 /和整体SS＆A 83.1％。的EIT将显著在不使用动物以满足监管测试要求进行分类的一系列的液体和固体的化学物质的眼部刺激潜在贡献。该EpiOcular企业所得税法于2015年实施到作为TG 492经合组织试验准则。

用10试验制品（TA）和阴性和阳性对照进行代表EIT结果呈现在表6和图3中的平均OD = 1.31用于NC对应于100％组织活力，因此在PC（平均OD = 0.41）有31.2％的相对组织活力。如果是在15个有效独立的实验使用液体暴露协议和8个独立有效的实验，4个实验室，使用固体曝光协议执行了7实验室所得税协议，一般的组织活力为使用液体协议的PC为36.4±4.0 ％和固体协议32.3±6.4％。在所有情况下，阳性对照的结果都低于60.0％15截止值。 如图3，TA1，TA2，TA4，TA7，TA8和过组织生存力&gt; 60.0％，因此被归类为“NI”。 TA3，TA5，TA6，TA9，TA10和过组织生活力X04; 60.0％，因此被归类为“I”。重复的组织之间的组织活力的差异与异常TA2所有助教&lt;20.0％。因此，结果所有的测试文章，有一个例外TA2的，被认为是“合格”的，因为他们满足了所有的EIT验收标准（10.2节）。因为为TA2在初始实验重复组织之间的高变异性，第二个实验是必要的，以获得合格的EIT的结果。 该EIT的测试方法这里所描述的利用红帽认证工程师组织模型用于眼部刺激的评估几个multilaboratory验证研究，包括正式审定通过EURL ECVAM /化妆品欧洲15,17-19。在所有的研究中，所得税被证明是可重复的，并能够正确地识别化学品（包括物质和混合物），不要求分类和标签的眼刺激或严重眼损伤交流盘带联合国GHS 15,17-19。该EIT的测试方法完成了验证管理集团（VMG）的验收标准眼刺激的敏感性，特异性和总精度，目前它正在等待正式实施，作为活体兔的Draize测试19的部分替代。 <img alt="图1" src="/files/ftp_upload/52979/52979fig1.jpg" /> 图1：企业所得税协议液体和固体的测试文章概要所用的缩写：AM，检测中。 SCC，标准培养条件; PBS，Dulbecco氏磷酸盐缓冲盐水;室温，室温。 <img alt="图2" src="/files/ftp_upload/52979/52979fig2.jpg" /> 图2：用于MTT组织的生存力试验的标准化96孔板的配置的两个200微升等份是TRAnsferred到预标记的96孔板的适当孔中。所用的缩写：NC，阴性对照; PC，阳性对照; TA1-TA20，测试文章1-20;空白，萃取液。 96 MTT板配置使用Excel电子表格设计计算RHCE组织活力和经济转型的结果。 <img alt="图3" src="/files/ftp_upload/52979/52979fig3.jpg" /> 获得了10个测试文章，NC和使用RHCE组织模型PC控制EIT结果的图表从Excel电子表格设计提出经济转型的结果产生：图3。试验的化学物质减少组织的生存力≤相60.0％为NC被分类为刺激物（“I”，TA3，TA5，TA6，TA9，和TA10）和测试化学品有组织的生存力&gt; 60.0％被分类为无刺激物（“ NI“，TA1，TA2，TA4，TA7，TA8和）。 <table border="0" cellpadding="0" cellspaci纳克=“0”&gt; <tbody><tr><td colspan="7">开始时间为EIT协议的工作步骤（一操作员一天的工作） </td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">每一行对应于一对组织</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 订购步骤： </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 五 </td><td> 6 </td><td> 7 </td></tr><tr><td> 液体： </td><td> PBS </td><td> TA 暴光 </td><td> 后 浸透 </td><td> 后 <td> MTT 反应 </td><td> MTT 抽 </td><td> 测量 </td></tr><tr><td></td><td> 30分钟 </td><td> 30分钟 </td><td> 12分钟 </td><td> 120分钟 </td><td> 180分钟 </td><td> 120分钟 </td><td> OD </td></tr><tr><td> NC </td><td> 9:00 </td><td> 9:30 </td><td> 10:00 </td><td> 10:12 </td><td> 12:12 </td><td> 15:12 </td><td>后</td></tr><tr><td> 个人计算机 </td><td> 9:01 </td><td> 9:31 </td><td> 10:01 </td><td> 10:13 </td><td> 12:13 </td><td> 15:13 </td><td> 17点30 </td></tr><tr><td> TA-1 </td><td> 9:02 </td><td> 9:32 </td><td> 10:02 </td><td> 10:14 </td><td> 12:14 </td><td> 15:14 </td><td></td></tr><tr><td> TA-2 &lt;/ STRONG&gt; </td><td> 9点03分</td><td> 9:33 </td><td> 10:03 </td><td> 10:15 </td><td> 12:15 </td><td> 15:15 </td><td></td></tr><tr><td> TA-3 </td><td> 9:04 </td><td> 9点34分</td><td> 10:04 </td><td> 10:16 </td><td> 12:16 </td><td> 15:16 </td><td></td></tr><tr><td> TA-4 </td><td> 9:05 </td><td> 9:35 </td><td> 10:05 </td><td> 10:17 </td><td> 12:17 </td><td> 15:17 </td><td></td></tr><tr><td> TA-5 </td><td> 9时06分</td><td> 9:36 </td><td> 10时06 </td><td> 10:18 </td><td> 12:18 </td><td> 15:18 </td><td></td></tr><tr><td> TA-6 </td><td> 9:07 </td><td> 9:37 </td><td> 10:07 </td><td> 10:19 </td><td> 12:19 </td><td> 15:19 </td><td></td></tr><tr><td> TA-7 </td><td> 9:08 </td><td> 9:38 </td><td> 10时08分</td><td> 10:20 </td><td> 12:20 </td><td> 15:20 </td><td></td></tr><tr><td><stro<html> NG&gt; TA-8 </td><td> 9:09 </td><td> 9:39 </td><td> 10:09 </td><td> 10:21 </td><td> 12:21 </td><td> 15:21 </td><td></td></tr><tr><td> TA-9 </td><td> 9:1​​0 </td><td> 9:40 </td><td> 10:10 </td><td> 10:22 </td><td> 12:22 </td><td> 15:22 </td><td></td></tr><tr><td> TA-10 </td><td> 9:1​​1 </td><td> 9:41 </td><td> 10:11 </td><td> 10:23 </td><td> 12:23 </td><td> 15:23 </td><td></td></tr></tbody></table> 表3：用于液体检验物品的测试样本时间调度协议步骤，包括预润湿用DPBS，测试条中的应用（TAS），冲洗和组织后浸泡，后潜伏期，MTT法，MTT法提取，和MTT外径的测量列于列。时报重复的组织被组织成行。整个实验测试10助教和控制可以在一天内完成。 e_content“&gt; <table border="0" cellpadding="0" cellspacing="0" height="439" width="582"><tbody><tr><td colspan="6">开始时间为EIT协议的步骤（2天工作的一个操作员） </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td colspan="4">每一行对应于一对组织</td><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>第1天</td><td></td><td></td><td></td><td></td><td colspan="2">第2天（第二天） </td><td></td></tr><tr><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td> 订购步骤： </td><td> 1 </strong&gt;的</td><td> 2 </td><td> 3 </td><td> 4 </td><td> 五 </td><td> 6 </td><td> 7 </td></tr><tr><td> 固体： </td><td> PBS </td><td> TA 暴光 </td><td> 后 浸透 </td><td> 后 Incubaction </td><td> MTT 反应 </td><td> MTT 抽 </td><td> 测量 </td></tr><tr><td></td><td> 30分钟 </td><td> 6小时 </td><td> 26分钟 </td><td> 18小时 </td><td> 180分钟 </td><td> 120分钟 </td><td> OD </td></tr><tr><td> NC </td><td> 9:00 </td><td> 9:30 </td><td> 15:30 </td><td> 15:56 </td><td> 9:56 </td><td> 12:56 </td&gt; <td>后</td></tr><tr><td> 个人计算机 </td><td> 9:02 </td><td> 9:32 </td><td> 15:32 </td><td> 15:58 </td><td> 9:58 </td><td> 12点58分</td><td> 15:30 </td></tr><tr><td> TA-1 </td><td> 9:04 </td><td> 9点34分</td><td> 15:34 </td><td> 16:00 </td><td> 10:00 </td><td> 13:00 </td><td></td></tr><tr><td> TA-2 </td><td> 9时06分</td><td> 9:36 </td><td> 15:36 </td><td> 16:02 </td><td> 10:02 </td><td> 13:02 </td><td></td></tr><tr><td> TA-3 </td><td> 9:08 </td><td> 9:38 </td><td> 15点38分</td><td> 16:04 </td><td> 10:04 </td><td> 13:04 </td><td></td></tr><tr><td> TA-4 </td><td> 9:1​​0 </td><td> 9:40 </td><td> 15:40 </td><td>十六点06分</td><td> 10时06 </td><td>十三时06分</td><td></td></tr><tr><td> TA-5 </td><td> 9:1​​2 </td><td> 9:42 </td><td> 15:42 </td><td> 16点08 </td><td> 10时08分&lt;/ TD&gt; <td> 13时08 </td><td></td></tr><tr><td> TA-6 </td><td> 9:1​​4 </td><td> 9:44 </td><td> 15:44 </td><td> 16:10 </td><td> 10:10 </td><td> 13:10 </td><td></td></tr><tr><td> TA-7 </td><td> 9:1​​6 </td><td> 9:46 </td><td> 15:46 </td><td> 16:12 </td><td> 10:12 </td><td> 13:12 </td><td></td></tr><tr><td> TA-8 </td><td> 9点18分</td><td> 9:48 </td><td> 15:48 </td><td> 16:14 </td><td> 10:14 </td><td> 13:14 </td><td></td></tr><tr><td> TA-9 </td><td> 9:20 </td><td> 9:50 </td><td> 15:50 </td><td> 16:16 </td><td> 10:16 </td><td> 13:16 </td><td></td></tr><tr><td> TA-10 </td><td> 9:22 </td><td> 9:52 </td><td> 15:52 </td><td> 16:18 </td><td> 10:18 </td><td> 13:18 </td><td></td></tr></tbody></table> 表4：固体的测试文章测试样品时间表。协议的步骤，包括预湿用DPBS，测试条的应用组织，冲洗和后浸泡，后潜伏期，MTT法，MTT提取和MTT OD测量在列介绍。时报重复的组织被组织成行。整个实验测试10助教和控制进行了为期两天的时间。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 多少 </td><td> 试剂 </td><td> 储存条件 </td><td> 资源 </td><td> 描述 </td><td> 截止日期 </td></tr><tr><td> 1瓶， 2毫升</td><td> MTT精矿（MTT-100-CON） </td><td>避光（-20℃） </td><td> MatTek </td><td>冷冻浓缩MTT </td><td> 2个月</td></tr><tr><td> 1瓶， 8毫升</td><td> MTT稀释剂</td><td> 2-8＃186的温度</td><td> MatTek </td><td>对于MTT稀释浓缩在之前MTT法使用</td><td> 2个月</td></tr><tr><td> 1瓶， 60毫升</td><td>异丙醇 （CAS＃67-63-0） </td><td> RT </td><td> Sigma-Aldrich公司</td><td>萃取液</td><td> NA </td></tr></tbody></table> 表5：MTT-100检测试剂盒组件。 <table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td> 代号N° </td><td> 薄纸 </td><td colspan="2"> 原始数据 </td><td colspan="2"> 空白校正的数据 </td><td rowspan="2"> 平均外径 </td><td rowspan="2"> ％生存能力 </td></tr><tr><td></td><td> ñ </td><td> Aliq。 1 </td><td> Aliq。 2 </td><td> Aliq。 1 </td><td> Aliq。 2 </td></tr><tr><td> NC </td><td>1 </td><td> 1.316 </td><td> 1.352 </td><td> 1.316 </td><td> 1.352 </td><td> 1.334 </td><td> 101.6 </td></tr><tr><td></td><td> 2 </td><td> 1.277 </td><td> 1.309 </td><td> 1.277 </td><td> 1.309 </td><td> 1.293 </td><td> 98.4 </td></tr><tr><td>个人计算机</td><td> 1 </td><td> 0.379 </td><td> 0.397 </td><td> 0.379 </td><td> 0.397 </td><td> 0.388 </td><td> 29.6 </td></tr><tr><td></td><td> 2 </td><td> 0.419 </td><td> 0.442 </td><td> 0.419 </td><td> 0.442 </td><td> 0.431 </td><td> 32.8 </td></tr><tr><td> TA1 </td><td> 1 </td><td> 1.213 </td><td> 1.244 </td><td> 1.213 </td><td> 1.244 </td><td> 1.229 </td><td> 93.5 </td></tr><tr><td></td><td> 2 </td><td> 1.355 </td><td> 1.355 </td><td> 1.355 </td><td> 1.355 </td><td> 1.355 </td><td> 103.2 </td></tr><tr><td> TA2 </td><td> 1 </td><td> 1.210 </td><td> 1.122 </td><td> 1.210 </td><td> 1.122 </td><td> 1.166 </td><td> 88.7 </td></tr><tr><td></td><td> 2 </td><td> 0.828 </td><td> 0.837 </td><td> 0.828 </td><td> 0.837 </td><td> 0.833 </td><td> 63.4 </td></tr><tr><td> TA3 </td><td> 1 </td><td> 0.167 </td><td> 0.168 </td><td> 0.167 </td><td> 0.168 </td><td> 0.167 </td><td> 12.7 </td></tr><tr><td></td><td> 2 </td><td> 0.138 </td><td> 0.136 </td><td> 0.138 </td><td> 0.136 </td><td> 0.137 </td><td> 10.4 </td></tr><tr><td> TA4 </td><td> 1 </td><td> 1.137 </td><td> 1.160 </td><td> 1.137 </td><td> 1.160 </td><td> 1.149 </td><td> 87.4 </td></tr><tr><td></td><td> 2 </td><td> 1.262 </td><td> 1.191 </td><td> 1.262 </td><td> 1.191 </td><td> 1.227 </td><td> 93.4 </td></tr><tr><td> TA5 </td><td> 1 </td><td> 0.610 </td><td> 0.621 </td><td> 0.610 </td><td> 0.621 </td><td> 0.616 </td><td> 46.​​9 </td></tr><tr><td></td><td> 2 </td><td> 0.480 </td><td> 0.484 </td><td> 0.480 </td><td> 0.484 </td><td> 0.482 </td><td> 36.7 </td></tr><tr><td> TA6 </td><td> 1 </td><td> 0.502 </td><td> 0.513 </td><td> 0.502 </td><td> 0.513 </td><td> 0.508 </td><td> 38.7 </td></tr><tr><td></td><td> 2 </td><td> 0.396 </td><td> 0.407 </td><td> 0.396 </td><td> 0.407 </td><td> 0.402 </td><td> 30.6 </td></tr><tr><td> TA7 </td><td> 1 </td><td> 1.048 </td><td> 1.050 </td><td> 1.048 </td><td> 1.050 </td><td> 1.049 </td><td> 79.9 </td></tr><tr><td></td><td> 2 </td><td> 1.149 </td><td> 1.150 </td><td> 1.149 </td><td> 1.150 </td><td> 1.150 </td><td> 87.5 </td></tr><tr><td> TA8 </td><td> 1 </td><td> 1.032 </td><td> 1.034 </td><td> 1.032 </td><td> 1.034 </td><td> 1.033 </td><td> 78.7 </td></tr><tr><td></td><td> 2 </td><td> 0.941 </td><td> 0.935 </td><td> 0.941 </td><td> 0.935 </td><td> 0.938 </td><td> 71.4 </td></tr><tr><td> TA9 </TD&gt; <td> 1 </td><td> 0.022 </td><td> 0.022 </td><td> 0.022 </td><td> 0.022 </td><td> 0.022 </td><td> 1.7 </td></tr><tr><td></td><td> 2 </td><td> 0.144 </td><td> 0.149 </td><td> 0.144 </td><td> 0.149 </td><td> 0.147 </td><td> 11.2 </td></tr><tr><td> TA10 </td><td> 1 </td><td> 0.150 </td><td> 0.150 </td><td> 0.150 </td><td> 0.150 </td><td> 0.150 </td><td> 11.4 </td></tr><tr><td></td><td> 2 </td><td> 0.254 </td><td> 0.255 </td><td> 0.254 </td><td> 0.255 </td><td> 0.255 </td><td> 19.4 </td></tr></tbody></table><table border="0" cellpadding="0" cellspacing="0"><tbody><tr><td></td><td>意味着</td><td> DIF。 </td><td>平均</td><td> DIF。 </td><td> DIF。/ 2 </td><td colspan="2">分类</td></tr><tr><td></td><td>外径</td><td>外径</td><td>存活率[％] </td><td>存活率的</td><td></td><td></td><td></td></tr><tr><td> NC </td><td> 1.314 </td><td> 0.041 </td><td> 100.0 </td><td> 3.12 </td><td> 1.56 </td><td> NI </td><td>合格</td></tr><tr><td>个人计算机</td><td> 0.410 </td><td> 0.043 </td><td> 31.2 </td><td> 3.23 </td><td> 1.62 </td><td>一世</td><td>合格</td></tr><tr><td> TA1 </td><td> 1.292 </td><td> 0.127 </td><td> 98.3 </td><td> 9.63 </td><td> 4.81 </td><td> NI </td><td>合格</td></tr><tr><td> TA2 </td><td> 0.999 </td><td> 0.333 </td><td> 76.1 </td><td> 25.36 </td><td> 12.68 </td><td> NI </td><td> D&gt; 20 </td></tr><tr><td> TA3 </td><td> 0.152 </td><td> 0.030 </td><td> 11.6 </td><td> 2.32 </td><td> 1.16 </td><td>一世</td><td>合格</td></tr><tr><td> TA4 </td><td> 1.188 </td><td> 0.078 </td><td> 90.4 </td><td> 5.94 </td><td> 2.97 </td><td> NI </td><td>合格</td></tr><tr><td> TA5 </td><td> 0.549 </td><td> 0.134 </td><td> 41.8 </td><td> 10.16 </td><td> 5.08 </td><td>一世</td><td>合格</td></tr><tr><td> TA6 </td><td> 0.455 </td><td> 0.106 </td><td> 34.6 </td><td> 8.07 </td><td> 4.03 </td><td>一世</td><td>合格</td></tr><tr><td> TA7 </td><td> 1.100 </td><td> 0.101 </td><td> 83.7 </td><td> 7.65 </td><td> 3.82 </td><td> NI </td><td>合格</td></tr><tr><td> TA8 </td><td> 0.986 </td><td> 0.095 </td><td> 75.0 </td><td> 7.23 </td><td> 3.62 </td><td> NI </td><td>合格</td></tr><tr><td> TA9 </td><td> 0.085 </td><td> 0.125 </td><td> 6.4 </td><td> 9.48 </td><td> 4.74 </td><td>一世</td><td>合格</td></tr><tr><td> TA10 </td><td> 0.203 </td><td> 0.105 </td><td> 15.4 </td><td> 7.95 </td><td> 3.98 </td><td>一世</td><td>合格</td></tr></tbody></table> 表6：10测试的文章获得EIT的结果，NC和PC控制的表是督促通过Excel电子表格设计，计算组织活力和EIT结果uced。试验的化学物质减少组织的生存力≤相60.0％为NC被分类为刺激物（“I”，TA3，TA5，TA6，TA9，和TA10）和测试的化学品有组织的生存力&gt; 60.0％被分类为无刺激物（“ NI“，TA1，TA2，TA4，TA7，TA8和）。

Watch this Scientific Journal Video about Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model at JoVE.com

Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

我们已经制定了一个眼刺激试验一种利用三维重建人类角膜样上皮（RHCE）组织模型。该测试能够眼部刺激性和腐蚀性的材料（GHS分类1和2的组合），以及那些不要求标注（GHS无类别）来区分。

眼刺激试验（EIT）使用重构型人角膜上皮样（RHCE）组织模型眼刺激性化学品危险性鉴定

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and ...

eye-irritation-test-eit-for-hazard-identification-eye-irritating

Research

JoVE Journal

Bioengineering

Eye Irritation Test (EIT) for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial (RhCE) Tissue Model

37.0K 次观看.  MatTek Corporation. 我们已经制定了一个眼刺激试验一种利用三维重建人类角膜样上皮（RHCE）组织模型。该测试能够眼部刺激性和腐蚀性的材料（GHS分类1和2的组合），以及那些不要求标注（GHS无类别）来区分。 

视频: Eye Irritation Test EIT for Hazard Identification of Eye Irritating Chemicals using Reconstructed Human Cornea-like Epithelial RhCE Tissue Model

Evaluation of Polymeric Gene Delivery Nanoparticles by Nanoparticle Tracking Analysis and High-throughput Flow Cytometry

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a technology for regenerative medicine2. Unlike viruses, which have significant safety issues, polymeric nanoparticles can be designed to be non-toxic, non-immunogenic, non-mutagenic, easier to synthesize, chemically versatile, capable of carrying larger nucleic acid cargo and biodegradable and/or environmentally responsive. Cationic polymers self-assemble with negatively charged DNA via electrostatic interaction to form complexes on the order of 100 nm that are commonly termed polymeric nanoparticles. Examples of biomaterials used to form nanoscale polycationic gene delivery nanoparticles include polylysine, polyphosphoesters, poly(amidoamines)s and polyethylenimine (PEI), which is a non-degradable off-the-shelf cationic polymer commonly used for nucleic acid delivery1,3 . Poly(beta-amino ester)s (PBAEs) are a newer class of cationic polymers4 that are hydrolytically degradable5,6 and have been shown to be effective at gene delivery to hard-to-transfect cell types such as human retinal endothelial cells (HRECs)7, mouse mammary epithelial cells8, human brain cancer cells9 and macrovascular (human umbilical vein, HUVECs) endothelial cells10.
A new protocol to characterize polymeric nanoparticles utilizing nanoparticle tracking analysis (NTA) is described. In this approach, both the particle size distribution and the distribution of the number of plasmids per particle are obtained11. In addition, a high-throughput 96-well plate transfection assay for rapid screening of the transfection efficacy of polymeric nanoparticles is presented. In this protocol, poly(beta-amino ester)s (PBAEs) are used as model polymers and human retinal endothelial cells (HRECs) are used as model human cells. This protocol can be easily adapted to evaluate any polymeric nanoparticle and any cell type of interest in a multi-well plate format.

A protocol for nanoparticle tracking analysis (NTA) and high-throughput flow cytometry to evaluate polymeric gene delivery nanoparticles is described. NTA is utilized to characterize the nanoparticle particle size distribution and the plasmid per particle distribution. High-throughput flow cytometry enables quantitative transfection efficacy evaluation for a library of gene delivery biomaterials.

高分子基因传递纳米粒子的纳米粒子跟踪分析和高通量流式细胞仪评价

Non-viral gene delivery using polymeric nanoparticles has emerged as an attractive approach for gene therapy to treat genetic diseases1 and as a ...

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 ...

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

人体软骨组织制作采用三维喷墨打印技术

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury leading to permanent pathological damage. Existing conventional assays allow the analysis of leukocyte adhesion molecules only after the extraction of leukocytes from the blood. This requires the blood to undergo several steps before peripheral blood leukocytes (PBLs) can be ready for analysis, which in turn can stimulate PBLs influencing the research findings. The autoperfused micro flow chamber assay, however, allows scientists to study early leukocytes functional dysregulation using the systemic flow of a live mouse while having the freedom of manipulating a coated chamber. Through a disease model, the functional expression of leukocyte adhesion molecules can be assessed and quantified in a micro-glass chamber coated with immobilized endothelial adhesion molecules ex vivo. In this model, the blood flows between the right common carotid artery and left external jugular vein of a live mouse under anesthesia, allowing the interaction of native PBLs in the chamber. Real-time experimental analysis is achieved with the assistance of an intravital microscope as well as a Harvard Apparatus pressure device. The application of a flow regulator at the input point of the glass chamber allows comparable physiological flow conditions amongst the experiments. Leukocyte rolling velocity is the main outcome and is measured using the National Institutes of Health open-access software ImageJ. In summary, the autoperfused micro flow chamber assay provides an optimal physiological environment to study leukocytes endothelial interaction and allows researchers to draw accurate conclusions when studying inflammation.

Assessing Leukocyte-endothelial Interactions Under Flow Conditions in an Ex Vivo Autoperfused Microflow Chamber Assay

Here, we present a protocol that allows the investigator to assess leukocyte recruitment dynamics ex vivo by connecting a chamber coated with endothelial-derived adhesion molecules to the circulatory system of a mouse. This method offers significant advantages since it allows for leukocyte assessment under relative biological conditions.

评估白细胞内皮的相互作用下水流条件的<em&gt;离体</em&gt;自动注入微流室法

Leukocyte-endothelial interactions are early and critical events in acute and chronic inflammation and can, when dysregulated, mediate tissue injury ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

三维组织工程人食管黏膜型号生产，表征和潜在用途

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Encapsulated Cell Technology for the Delivery of Biologics to the Mouse Eye

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, typically via intravitreal injections. Encapsulated cells expressing the biologic of choice are becoming a tool for local protein production and release (e.g., via long-term drug delivery). In addition, encapsulation systems utilize permeable materials that allow diffusion of nutrients, waste, and therapeutic factors into and out of cells. This occurs while masking the cells from the host immune response, avoiding the need for suppression of the host immune system. This protocol describes the use of alginate as a polymer in microencapsulation coupled with the electrospray method as a microencapsulation technique. ARPE-19 cells, a spontaneously arising human RPE cell line, has been used in long-term cell therapy experiments due to its lifetime functionality, and it is used here for encapsulation and delivery of the capsules to mouse eyes. The manuscript summarizes the steps for cell microencapsulation, quality control, and ocular delivery.

Presented here is a protocol for the use of alginate as a polymer in microencapsulation of immortalized cells for long-term delivery of biologics to rodent eyes.

用于向小鼠眼睛输送生物制剂的封装细胞技术

Many current therapeutics under development for diseases of the posterior pole of the eye are biologics. These drugs need to be administered frequently, ...

Growth of Human and Sheep Corneal Endothelial Cell Layers on Biomaterial Membranes

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is deleterious for the cells as it reduces their ability to form a mature and functional layer. Here, we present a method for establishing and subculturing human and sheep corneal endothelial cell cultures that minimizes the loss of cell-to-cell contact. Explants of corneal endothelium/Descemet&#39;s membrane are taken from donor corneas and placed into tissue culture under conditions that allow the cells to collectively migrate onto the culture surface. Once a culture has been established, the explants are transferred to fresh plates to initiate new cultures. Dispase II is used to gently lift clumps of cells off tissue culture plates for subculturing. Corneal endothelial cell cultures that have been established using this protocol are suitable for transferring to biomaterial membranes to produce tissue-engineered cell layers for transplantation in animal trials. A custom-made device for supporting biomaterial membranes during tissue culture is described and an example of a tissue-engineered graft composed of a layer of corneal endothelial cells and a layer of corneal stromal cells on either side of a collagen type I membrane is presented.

This protocol describes the critical steps required to establish and grow corneal endothelial cell cultures from explants of human or sheep tissue. A method for subculturing corneal endothelial cells on membranous biomaterials is also presented.

生物材料膜上人与羊角膜内皮细胞层的生长

Corneal endothelial cell cultures have a tendency to undergo epithelial-to-mesenchymal transition (EMT) after loss of cell-to-cell contact. EMT is ...

In Vitro Model of Human Cutaneous Hypertrophic Scarring using Macromolecular Crowding

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol applies a macromolecular crowding (MMC) technique to mimic this physiological crowding through the addition of neutral polymers (crowders) to cell cultures in vitro. Previous studies using Ficoll or dextran as crowders demonstrate that the expression of collagen I and fibronectin in WI38 and WS-1 cell lines are significantly enhanced using the MMC technique. However, this technique has not been validated in primary hypertrophic scar-derived human skin fibroblasts (hHSFs). As hypertrophic scarring arises from the excessive deposition of collagen, this protocol aims to construct a collagen-rich in vitro hypertrophic scar model by applying the MMC technique with hHSFs. This optimized MMC model has been shown to possess more similarities with in vivo scar tissue compared to traditional 2-dimensional (2-D) cell culture systems. In addition, it is cost-effective, time-efficient, and ethically desirable compared to animal models. Therefore, the optimized model reported here offers an advanced &#8220;in vivo-like&#8221; model for hypertrophic scar-related studies.

This protocol describes the use of macromolecular crowding to create an in vitro human hypertrophic scar tissue model that resembles in vivo conditions. When cultivated in a crowded macromolecular environment, human skin fibroblasts exhibit phenotypes, biochemistry, physiology, and functional characteristics resembling scar tissue.

利用大分子挤出的人类食性增大疤痕的体外模型

It has been shown that in vivo tissues are highly crowded by proteins, nucleic acids, ribonucleoproteins, polysaccharides, etc. The following protocol ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

大规模培育三维重建人类表皮

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Quantifying Fibrillar Collagen Organization with Curvelet Transform-Based Tools

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression of a wide range of diseases including breast, ovarian, kidney, and pancreatic cancers. Freely available fiber quantification software tools are mainly focused on the calculation of fiber alignment or orientation, and they are subject to limitations such as the requirement of manual steps, inaccuracy in detection of the fiber edge in noisy background, or lack of localized feature characterization. The collagen fiber quantitation tool described in this protocol is characterized by using an optimal multiscale image representation enabled by curvelet transform (CT). This algorithmic approach allows for the removal of noise from fibrillar collagen images and the enhancement of fiber edges to provide location and orientation information directly from a fiber, rather than using the indirect pixel-wise or window-wise information obtained from other tools. This CT-based framework contains two separate, but linked, packages named &#8220;CT-FIRE&#8221; and &#8220;CurveAlign&#8221; that can quantify fiber organization on a global, region of interest (ROI), or individual fiber basis. This quantification framework has been developed for more than ten years and has now evolved into a comprehensive and user-driven collagen quantification platform. Using this platform, one can measure up to about thirty fiber features including individual fiber properties such as length, angle, width, and straightness, as well as bulk measurements such as density and alignment. Additionally, the user can measure fiber angle relative to manually or automatically segmented boundaries. This platform also provides several additional modules including ones for ROI analysis, automatic boundary creation, and post-processing. Using this platform does not require prior experience of programming or image processing, and it can handle large datasets including hundreds or thousands of images, enabling efficient quantification of collagen fiber organization for biological or biomedical applications.

Here, we present a protocol to use a curvelet transform-based, open-source MATLAB software tool for quantifying fibrillar collagen organization in the extracellular matrix of both normal and diseased tissues. This tool can be applied to images with collagen fibers or other types of line-like structures.

使用基于曲线的转换工具量化纤维胶原蛋白组织

Fibrillar collagens are prominent extracellular matrix (ECM) components, and their topology changes have been shown to be associated with the progression ...