我们衷心感谢辛迪N.赵医生的帮助，凝胶电泳，安迪周和迈克尔Grodick的DNA和限制性内切酶的慷慨捐赠。我们非常感谢J.希思教授和D.测机教授慷慨的访问设备和材料。我们感谢卡恩Sorasaenee博士有益的建议。我们感谢玛丽H.唐创建在视频的示意图中使用的插图。资金由强生公司和USC Y86786提供。

注：当与RNA的工作保持一个清洁的工作环境，避免污染DNA酶和RNA酶的酶降解DNA和RNA。确保枪头和管是DNA酶和RNA酶。这也有利于向下擦拭表面的实验室和设备，如移液管，管人等具有净化解决方案。 1. RNA转录与Corrole治疗<ol><li>制备corrole和抑制剂化合物在0.01：[DNA]：的[复杂] 1的摩尔比。 注：在本例中，该比例为4.3 fmol的复杂：0.43皮摩尔DNA，其中所用的DNA模板是从E.西甲基因APET-28C矢量下在T7启动子控制的大肠杆菌 。其它DNA与T7启动子也是有效候选运行转录测定。 <ol><li>溶解放线菌素D，雷公藤，TPFC，和Ga（TPFC）的二甲亚砜（DMSO）中清洁，独立的容器中。获得的一个0.01的最终浓度：[C 1摩尔比omplex]：[DNA]通过使用连续稀释用无核酸酶的水。 <ol><li>溶解1毫克放线菌素D 1.8毫升DMSO。等份加入10μl到单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1/100稀释。等份1微升稀释到一个单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1 / 1,000稀释。 注：放线菌素D的最终溶液的浓度为4.3飞摩尔。 </li><li>溶解1毫克雷公藤中0.64毫升DMSO。等份1微升至一个单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1 / 1,000稀释。等份1微升稀释到一个单独的容器中，并稀释至1毫升无核酸酶水，以产生第二1 / 1,000稀释。 注：雷公藤甲素的最终溶液的浓度为4.3飞摩尔。 </li><li>溶解1毫克TPFC 2.9毫升DMSO。等份加入10μl到单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1/100ðilution。等份1微升稀释到一个单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1 / 1,000稀释。 注：TPFC的最终溶液的浓度为4.3飞摩尔。 </li><li>溶解1毫克嘎（TPFC）2.6毫升DMSO。等份加入10μl到单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1/100稀释。等份1微升稀释到一个单独的容器中，并稀释至1毫升无核酸酶水，以创建一个1 / 1,000稀释。 注：嘎（TPFC）的最终溶液的浓度为4.3飞摩尔。 注意：这些corroles和抑制剂是不溶于水的，必须预先溶解在DMSO中。少量的DMSO（1％以下）的不诱导细胞毒性的细胞（未发表数据）。 </li></ol></li></ol></li><li>开始前的转录反应，单独地准备必要的试剂或从商业供应商购买。 <ol><li>融冰冻结所有Ñ ​​试剂（参照表1冷冻试剂的列表）。 </li><li>允许的10X反应缓冲液和4核糖核苷酸（ATP，CTP，GTP，UTP和）解决方案的时间来混合直到它们完全溶解在溶液中。 </li><li>短暂离心所有试剂，以防止材料的损失在管的边缘。一旦解冻，存储核糖核苷酸在冰上，同时保持10X反应缓冲液在室温下。 </li><li>结合试剂在室温转录反应（参照表2试剂的列表）。 注：在10X反应缓冲的亚精胺可共沉淀模板DNA如果反应组装冰而不是RT。 </li><li>通过轻弹管或移液混合向上和向下轻轻调匀。混合后，短暂离心以在管的底部收集反应混合物中。 </li><li>孵育在37℃下将反应混合物总共2-4小时的。 注：必需的反应时间会有所不同与DNA模板大小和序列。这一步可能需要优化的特定序列。较短的模板，可能需​​要较长的反应时间为高产率的总RNA。 </li><li>从每一个小时，并储存在-20℃下经过每个反应除去器4微升等分试样。根据需要所需的时间点修改。 </li></ol></li></ol> 2. RNA离心柱<ol><li>以下的转录反应，净化用微量兼容层析柱的RNA。 注意：使用的RNA离心柱的纯化的RNA用大于20个碱基的结果在大于80％的回收率44 注：柱色谱法，是纯化的RNA一个通用和方便的方法。其它纯化方法也存在如氯化锂沉淀，苯酚：氯仿萃取，异丙醇沉淀，以及其它可商购的试剂盒。 <ol><li>均匀大力反相悬浮交联葡聚糖基质中的柱缓冲液3列或更多次。通过大幅弹柱做到这一点。 </li><li>除去从柱的顶部上限。会有一些残存的Sephadex矩阵在帽;如果帽填充有基质，更换盖到柱上并重复前面的步骤，直到大部分基质是在列。 </li><li>折断所述塔的底部前端。 注：一些液体可能逃离列。 </li><li>打包的列中。 <ol><li>将列在无菌（RNA酶和DNA酶免费）1.5 ml离心管。 </li><li>离心反应管在台式离心机在1000×g离心1分钟。 </li></ol></li><li>丢弃1.5 ml离心管以从柱中洗脱缓冲液中。 </li><li>立即放置在一个新的无菌的1.5 ml离心管的柱和样品吸取到柱床的中心。用20-50微升的样品，以避免过载的列。 </li><li>离心管的台式离心机在1000 XG1分钟。 注：洗脱液纯化RNA样本。 </li></ol></li></ol> 3.琼脂糖凝胶电泳（1％琼脂糖凝胶）42 注：溴化乙锭荧光在结合DNA和RNA，因此这些生物分子可以通过用0.5微克/毫升溴化乙锭溶液孵育他们用UV光。 <ol><li>通过将5毫克溴化乙锭在10ml水中制备的溴化乙锭（0.5毫克/毫升）的1000倍原液。 </li><li>制备的Tris乙酸EDTA（TAE）运行缓冲液用于凝胶电泳。为了使50×TAE缓冲液中结合的2.0M Tris-乙酸用0.05M乙二胺四乙酸（EDTA）和pH调节至8.5。 </li><li>将10g超纯琼脂糖​​在1L 1X TAE缓冲液和熔化与常规微波炉的琼脂糖制备1％（重量/体积）的琼脂糖凝胶。一旦冷却到50℃，加入1毫升1000倍的溴化乙锭的1％琼脂糖凝胶SOLU灰，通过轻轻摇动或者通过反转在密闭容器中混合，再倒入琼脂糖溶液成凝胶铸造平台，并允许该凝胶凝固在RT。 注：溴化乙锭是一种诱变剂和潜在的致癌物质。戴上手套，小心处理解决方案。对于溴化乙锭的正确的处理程序，请参见： <a href="http://www.sciencelab.com/msds.php?msdsId=9927667">http://www.sciencelab.com/msds.php?msdsId=9927667</a> 。 </li><li>后的凝胶凝固，放置在电泳槽的集凝胶。添加足够的TAE缓冲液，以覆盖凝胶直到井被淹没。检查TAE缓冲液的水平大约为1毫米以上的凝胶的水平。取出梳子。 注意：删除梳井被淹没后，可防止气泡被截留在井。 </li><li>制备RNA样品。结合1微升每种纯化的样品等分试样与1微升凝胶加载缓冲液（或具有1微升10×加样缓冲液和dilut移液器向上和向下微量编到10微升无核酸酶的水）调匀。 注：10×加样缓冲液中含有20％的Ficoll 400，0.1M的Na 2 EDTA，pH值8.0，1.0％SDS，0.25％溴酚蓝。 0.25％二甲苯蓝，也可以加入到加样缓冲液，因为它运行〜50％速度是溴酚蓝，并且可以用于监测非常长的运行是有用的。42 </li><li>通过仔细吹打溶液到每个孔的底部装入每个样品的凝胶。小心不要留下任何气泡或混合井之间的样本。 注：核糖核酸梯也可以用于确定转录物的大小。 </li><li>附加的引线，使得DNA会迁移到朝向正引线的凝胶。设置电压到需要的水平，通常为1至10伏/凝胶厘米。运行凝胶然而长是足够的RNA片段之间显著分离，使用染料的迁移来监测分离的进度。 ñOTE：250 V A23厘米×25厘米的凝胶大约需要1小时，而一个8厘米×8厘米凝胶在150 V大约需要20分钟。小RNA片段具有更好的分辨率，更高的电压运行时。 </li><li>关掉电源时从装载缓冲染料已迁移判断足以使RNA片段之间的分离距离。 </li><li>图像在UV光下的溴化乙锭的RNA会发出荧光。 </li><li>取的图像的图像，并比较来自每个条件的纯化的RNA的荧光强度。 </li></ol>通过紫外 - 可见光谱4. RNA定量<ol><li>放在一个的NanoDrop 2000或类似的机器2微升H 2 O，并测量空白。 </li><li>下一步，放置2微升每种RNA样品的，下面的净化，在分光光度计上并测量紫外 - 可见从200纳米至800纳米的波长范围。 注：一个A 260读数为1.0，相当于核糖核酸约40微克/毫升，和纯RNA具有A 260 / A 280比值为2.1。45 </li><li>在的情况下的吸光度大于1的OD，稀释于无核酸酶水样品，得到的OD小于1。 </li><li>用绘图软件绘制各种样品，并在260 nm的OD比较。 </li></ol>

The authors have nothing to disclose.

该测定表明，添加Ga（TPFC）的抑制RNA的转录可比已知的DNA结合抗癌络合物放线菌素D和雷公藤。嘎（TPFC）（GI 50 = 58.1-154.7μM）的细胞毒性行为可能由于其抑制性能。因为没有转录抑制中观察到TPFC，TPFC的细胞毒性是不是由于RNA的转录抑制，但是由于尚未研究其他手段。 在执行了4转录反应，DNA用放线菌素D，雷公藤，TPFC或Ga（TPFC）处理，在[复]：[模板DNA碱基]为0.01，分别...

化疗通常涉及广谱细胞毒性剂与不期望的副作用和有限的定位，但与癌症生物学的更多的理解，存在用于抗癌剂不断增加的需求，以更高的癌症靶向功效和副作用较少。1人类癌细胞经常成为依赖单一活化或过表达癌基因的生存。2因此，许多抗癌药物是通过它们抑制RNA的转录的能力进行评估。治疗，阻止这些转化基因的表达是有效的消除癌细胞，并导致细胞死亡。3转化的细胞是在RNA的转录干扰更敏感比是相应的正常细胞。4抗癌药物抑制转录预期选择性地抑制这是必要的癌症细胞生存的癌基因的5因此表达，RNA的转录我nhibition是要找出潜在的抗癌候选药物，并进一步了解其作用机制的有效途径。此协议表明镓（TPFC）抑制RNA的转录顺序相同的化疗药物放线菌素D和雷公藤上;类似的比较，可以使用此协议与其他抗癌候选药物进行。放线菌素D是一种RNA的转录抑制剂通常用于治疗妊娠滋养细胞癌，睾丸癌，肾母细胞瘤，rhabdomyosacoma和尤因氏肉瘤6。放线菌素D被用于癌症治疗近五十年，因为它是第一个被FDA批准在1964.Triptolide是已经在体外和各种肿瘤携带动物模型30年已经研究选择性转录抑制剂。7 corroles的两亲性质的大环赋予比其他类药物显著优点，如小分子或生物第8-14的大环字符允许蜂窝渗透性大于预期对于如此大的分子，它们是大到足以与大分子的表面，如那些蛋白质的相互作用。8 Corroles已知以形成紧密的非共价配合物的生物分子和药物。10除了corrole框架的固有细胞毒性，我们已经表明，磺化corrole充当化学治疗剂与载体分子，特别是DNA的嵌入蒽环类药物阿霉素。当磺化corrole物共施用阿霉素，3倍的增强中阿霉素的IC 50观察到，DU-145细胞。9 corrole框架是稳定的，并具有固有的吸光度和荧光性质是，官能化时，经过独特的吸光度的变化可用于表征。10官能支架的不固有地affec吨的corrole，9-15的光物理性质，但，因为看到带有磺化corrole，选择性地修改所述corrole框架可以基本上改变其生物学特性。16，我们先前评估6 metallocorroles与七人癌细胞系。结果表明，对人体癌细胞的毒性是依赖于特定的金属离子，以及官能团取代。例如，磺化镓corroles经历高的细胞摄取和渗透选择性进入脑转移性前列腺癌细胞（DU-145）的核;同样corrole，虽然它不渗入的其他细胞系的细胞核中，显示出更大的细胞毒性乳腺癌（MDA-MB-231），黑色素瘤（SK-MEL-28），和卵巢（OVCAR-3）的癌细胞比前列腺癌。9 初始基于细胞的试验表明，这些化合物显示出希望作为抗癌治疗剂，其优点菲尔特呃调查行动的机制。转录抑制观察到某些有机金属配合物17-27，我们试图研究这个过程中作为一个可能的机制corrole家庭的细胞毒行为。此转录测定法提供了用于评估转录的抑制，这将导致对这些分子在活细胞中的效应的更详细的信息的简单的，廉价且简便的方法。 这里，镓的转录抑制（Ⅲ）5,10,15-（三）pentafluorophenylcorrole（镓（TPFC））和它的游离碱类似物5,10,15-（三）pentafluorophenylcorrole（TPFC）（ 图1）进行测试。不像一些过渡金属配合物，镓（III）为氧化还原非活性，因此不直接参与的氧化还原类的代谢途径的氧化还原过程。28无论如何，镓（Ⅲ）并不表现出细胞毒特性，并已被研究用于治疗目的。镓是第二个最有前途的金​​属铂之后抗癌治疗，并经历了多次的研究和调查;硝酸盐和氯化物镓盐在防治肝癌，淋巴瘤，膀胱癌等多种疾病的临床试验进行了评估。29-34镓（III），因此理想的抗癌corrole研究。初始数据表明镓（TPFC）和TPFC具有低GI 50，所需的药物浓度，以抑制的最大细胞增殖的50％，与各种癌细胞株（参见图2）;这肯定了关于这两种化合物的进一步的实验的有效性，以确定它们抑制性质。我们比较这些化合物与普通抗癌药物放线菌素D和雷公藤甲素。放线菌素D插层的DNA，抑制RNA的伸长率，并诱导细胞凋亡的某些细胞系在皮摩尔浓度6,35-37雷公藤已经显示出抑制肿瘤生长。它与人XPB / ERCC3，亚基Ø˚F转录因子TFIIH，从而抑制RNA聚合酶II的活性。6-7,38-40 虽然众所周知，corroles表现出细胞毒性性质，存在关于从官能化所产生的不同的机制的信息很少。 RNA转录抑制Corrole将提供他们与生物大分子的相互作用更大的洞察力。已知结合至DNA的其它配合物，如二铑（II，II）配合物，铬（III）络合物，钌（II）多吡啶配合物，铑（Ⅲ）配合物，以及各种其他经受RNA的转录测定法，18- 27致使其与生物大分子相互作用的更深入的了解。这个浅显的和广泛使用的实验也是一个不错的初步测试，以评估特定分子的细胞毒性特性并确定它是否值得进一步的生物测试。所述RNA转录测定法也可进行多种修饰，如varyin克使用的化合物或酶的数量;变的潜伏期，反应时间和采样时间点;并改变所述DNA模板长度和序列，其它感兴趣的变量之间的，从而有可能提供大量的数据。这转录实验也一应俱全，与提供一切必要的反应组分实惠的套装，虽然组件可以购买并单独准备。在这些实验中，我们使用已知具有高产市售的试剂盒。41 为了评估转录的抑制，我们使用两种方法：琼脂糖凝胶电泳和UV-Vis光谱。琼脂糖凝胶电泳是分离，识别和纯化0.5至25-kb的DNA和RNA片段的简单而有效的方法。42 UV-Vis光谱可用于确定浓度和RNA的纯度。43

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			<td height="21" style="height:21px;width:305px;">Name of Material/ Equipment</td>
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			<td>Store at 2-8 &#176;C , protect from light</td>
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			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">T3652</td>
			<td>Store at 2-8 &#176;C , protect from light</td>
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			<td height="25" style="height:25px;">nuclease-free H2O&#160;</td>
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			<td style="width:219px;">Sigma-Aldrich</td>
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			<td>CAUTION: For proper handling procedures of ethidium bromide, please see: http://www.sciencelab.com/msds.php?msdsId=9927667</td>
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			<td style="width:219px;">Sigma-Aldrich</td>
			<td style="width:119px;">EDS</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">mini Quick Spin RNA Columns</td>
			<td style="width:219px;">Roche Life Science</td>
			<td align="right" style="width:119px;">11814427001</td>
			<td>Store at 2-8 &#176;C , do not freeze</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:305px;">1 kb DNA Ladder</td>
			<td style="width:219px;">New England Biolabs</td>
			<td style="width:119px;">N3232S</td>
			<td>Store at &#8211;20 &#176;C&#160;</td>
		</tr>
	</tbody>
</table>

an in vitro enzymatic assay to measure transcription inhibition by gallium(iii) and h3 5,10,15-tris(pentafluorophenyl)corroles

化疗通常涉及广谱细胞毒性剂与许多副作用和有限的定位。 Corroles是一类表现出微分细胞抑制和细胞毒特性在特定细胞系中，根据不同的螯合金属和官能团的身份四吡咯大环化合物的。对特定的细胞功能化corroles独特的行为引入靶向化疗的可能性。 许多抗癌药物是通过它们抑制RNA的转录的能力进行评估。这里，我们提出一个一步一步协议用于RNA转录的已知和潜在抑制剂的存在。的转录反应的RNA产物通过凝胶电泳和UV-Vis光谱评价提供潜在的抗癌候选药物的抑制特性的信息，并与修改的测定中，更多关于它们的作用机制。 小已知大约corrole细胞毒性作用的分子机制。在这个实验中，我们考虑两个corrole化合物：镓（Ⅲ）5,10,15-（三）pentafluorophenylcorrole（镓（TPFC））和游离碱类似物5,10,15-（三）pentafluorophenylcorrole（TPFC）。一种RNA转录测定法用于检查corroles的抑制性质。五个转录反应制备：DNA用放线菌素D，雷公藤，镓（TPFC），TPFC在[复]处理：[模板DNA碱基]比为0.01，分别与未处理的对照组。 转录反应4小时后，用琼脂糖凝胶电泳和UV-Vis光谱进行分析。有明显的抑制作用嘎（TPFC），放线菌素D，和雷公藤。 该RNA转录测定法可以被修改通过改变抗癌复杂，DNA或聚合酶的浓度，以提供更详细的机理，或通过培养该DNA聚合酶或与完井XES前RNA转录;这些修改将涉及DNA或酶的抑制机制之间的区别。加入复合后的RNA转录可以被用来测试该络合物是否降解或水解的RNA。该测定也可以用于研究额外的抗癌候选。

RNA转录定性琼脂糖凝胶电泳评定 琼脂糖凝胶电泳用于图像转录的RNA。结合后溴化乙锭荧光（λEM = 605纳米，λ= 前 210纳米，285纳米）的RNA 46允许成像。在凝胶较暗条带对应于更高浓度的RNA。如果放线菌素D，雷公藤，或任corrole复杂抑制RNA的转录，产生的RNA的降低，并且带将出现更轻。使用此概念，相对抑制进行评估。 ...

Watch this Scientific Journal Video about An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles at JoVE.com

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

镓（Ⅲ）5,10,15-（三）pentafluorophenylcorrole和其游离碱类似物显示出低的微摩尔细胞的细胞毒性。这个手稿描述的RNA转录反应中，成像的RNA用溴化乙锭染色的凝胶，并用UV-Vis光谱进行定量的RNA，为了通过corroles评估转录抑制和演示评价抗癌候选属性的一个简单的方法。

一个<em&gt;在体外</em&gt;酶测定来测量转录抑制作用镓（Ⅲ）和H<sub&gt; 3</sub&gt; -5,10,15-三（五氟苯基）corroles

Chemotherapy often involves broad-spectrum cytotoxic agents with many side effects and limited targeting. Corroles are a class of tetrapyrrolic ...

an-vitro-enzymatic-assay-to-measure-transcription-inhibition

Research

JoVE Journal

Bioengineering

An In Vitro Enzymatic Assay to Measure Transcription Inhibition by Gallium(III) and H3 5,10,15-tris(pentafluorophenyl)corroles

11.5K 次观看.  California Institute of Technology. 镓（Ⅲ）5,10,15-（三）pentafluorophenylcorrole和其游离碱类似物显示出低的微摩尔细胞的细胞毒性。这个手稿描述的RNA转录反应中，成像的RNA用溴化乙锭染色的凝胶，并用UV-Vis光谱进行定量的RNA，为了通过corroles评估转录抑制和演示评价抗癌候选属性的一个简单的方法。 

视频: An In Vitro Enzymatic Assay to Measure Transcription Inhibition by GalliumIII and H3 5,10,15-trispentafluorophenylcorroles

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

使用Cell-基板阻抗和活细胞成像，以衡量矩阵修改诱导实时变化的细胞粘附和去粘附

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

科研

生物工程

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

To comply with the Seventh Amendment to the EU Cosmetics Directive and EU REACH legislation, validated non-animal alternative methods for reliable and accurate assessment of ocular toxicity in man are needed. To address this need, we have developed an eye irritation test (EIT) which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model that is based on normal human cells. The EIT is able to separate ocular&#160;irritants and corrosives (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category). The test utilizes two separate protocols, one designed for liquid chemicals and a second, similar protocol for solid test articles. The EIT prediction model uses a single exposure period (30 min for liquids, 6 hr for solids) and a single tissue viability cut-off (60.0% as determined by the MTT assay). Based on the results for 83 chemicals (44 liquids and 39 solids) EIT achieved 95.5/68.2/ and 81.8% sensitivity/specificity and accuracy (SS&#38;A) for liquids, 100.0/68.4/ and 84.6% SS&#38;A for solids, and 97.6/68.3/ and 83.1% for overall SS&#38;A. The EIT will contribute significantly to classifying the ocular irritation potential of a wide range of liquid and solid chemicals without the use of animals to meet regulatory testing requirements. The EpiOcular EIT method was implemented in 2015 into the OECD Test Guidelines as TG 492.

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

We have developed an eye irritation test which utilizes a three dimensional reconstructed human cornea-like epithelial (RhCE) tissue model. The test is able to discriminate between ocular irritant and corrosive materials (GHS Categories 1 and 2 combined) and those that do not require labeling (GHS No Category).

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 ...

Development of an In Vitro Ocular Platform to Test Contact Lenses

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic physiological tear volumes.7 The traditional model also lacks the natural tear flow component and the blinking reflex, both of which are defining factors of the ocular environment. The development of a novel model is described in this study, which consists of a unique 2-piece design, eyeball and eyelid piece, capable of mimicking physiological tear volume. The models are created from 3-D printed molds (Polytetrafluoroethylene or Teflon molds), which can be used to generate eye models from various polymers, such as polydimethylsiloxane (PDMS) and agar. Further modifications to the eye pieces, such as the integration of an explanted human or animal cornea or human corneal construct, will permit for more complex in vitro ocular studies. A commercial microfluidic syringe pump is integrated with the platform to emulate physiological tear secretion. Air exposure and mechanical wear are achieved using two mechanical actuators, of which one moves the eyelid piece laterally, and the other moves the eyeballeyepiece circularly. The model has been used to evaluate CLs for drug delivery and deposition of tear components on CLs.

Development of an In Vitro Ocular Platform to Test Contact Lenses

Current in vitro models for evaluating contact lenses (CLs) and other eye-related applications are severely limited. The presented ocular platform simulates physiological tear flow, tear volume, air exposure and mechanical wear. This system is highly versatile and can be applied to various in vitro analyses with CLs.

的发展<em&gt;体外</em&gt;眼平台，试验用隐形眼镜

Currently, in vitro evaluations of contact lenses (CLs) for drug delivery are typically performed in large volume vials,1-6 which fail to mimic ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

一个<em&gt;体外</em鼠类的椎间盘&gt;器官培养模型

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver engineering, including in vivo organ decellularization followed by repopulation, has emerged as a promising approach over ex vivo liver engineering. However, postoperative survival was not achieved. The aim of this study is to develop a novel surgical technique of in vivo selective liver lobe perfusion in rats as a prerequisite for in vivo liver engineering. We generate a circuit bypass only through the left lateral lobe. Then, the left lateral lobe is perfused with heparinized saline. The experiment is performed with 4 groups (n = 3 rats per group) based on different perfusion times of 20 min, 2 h, 3 h, and 4 h. Survival, as well as the macroscopically visible change of color and the histologically determined absence of blood cells in the portal triad and the sinusoids, is taken as an indicator for a successful model establishment. After selective perfusion of the left lateral lobe, we observe that the left lateral lobe, indeed, turned from red to faint yellow. In a histological assessment, no blood cells are visible in the branch of the portal vein, the central vein, and the sinusoids. The left lateral lobe turns red after reopening the blocked vessels. 12/12 rats survived the procedure for more than one week. We are the first to report a surgical model for in vivo single liver lobe perfusion with a long survival period of more than one week. In contrast to the previously published report, the most important advantage of the technique presented here is that perfusion of 70% of the liver is maintained throughout the whole procedure. The establishment of this technique provides a foundation for in vivo partial liver engineering in rats, including decellularization and recellularization.

A Novel Surgical Technique As a Foundation for In Vivo Partial Liver Engineering in Rat

We establish a novel surgical technique for an in vivo single liver lobe perfusion model in rat as a prerequisite for further studying in vivo partial liver engineering in the future.

大鼠体内部分肝工程的外科技术基础

Organ engineering is a novel strategy to generate liver organ substitutes that can potentially be used in transplantation. Recently, in vivo liver ...

Repressing Gene Transcription by Redirecting Cellular Machinery with Chemical Epigenetic Modifiers

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene expression regulation are commonly disrupted in many diseases, a locus-specific, endogenous, and reversible method to study and control these mechanisms of action has been lacking. To address this issue, we have developed and characterized novel gene-regulating bifunctional molecules. One component of the bifunctional molecule binds to a DNA-protein anchor so that it will be recruited to an allele-specific locus. The other component engages endogenous cellular chromatin-modifying machinery, recruiting these proteins to a gene of interest. These small molecules, called chemical epigenetic modifiers (CEMs), are capable of controlling gene expression and the chromatin environment in a dose-dependent and reversible manner. Here, we detail a CEM approach and its application to decrease gene expression and histone tail acetylation at a Green Fluorescent Protein (GFP) reporter located at the Oct4 locus in mouse embryonic stem cells (mESCs). We characterize the lead CEM (CEM23) using fluorescent microscopy, flow cytometry, and chromatin immunoprecipitation (ChIP), followed by a quantitative polymerase chain reaction (qPCR). While the power of this system is demonstrated at the Oct4 locus, conceptually, the CEM technology is modular and can be applied in other cell types and at other genomic loci.

Regulation of the chromatin environment is an essential process required for proper gene expression. Here, we describe a method for controlling gene expression through the recruitment of chromatin-modifying machinery in a gene-specific and reversible manner.

用化学表观修饰因子重定向细胞机械抑制基因转录

Regulation of chromatin compaction is an important process that governs gene expression in higher eukaryotes. Although chromatin compaction and gene ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

用利穆鲁布莱巴细胞裂解液 (lal) 法检测纳米配方中的内毒素

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

An Automated Method to Perform The In Vitro Micronucleus Assay using Multispectral Imaging Flow Cytometry

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and introduces uncertainty in results due to variability between scorers. To remedy this, automated slide-scanning microscopy as well as conventional flow cytometry methods have been introduced in an attempt to remove scorer bias and improve throughput. However, these methods have their own inherent limitations such as inability to visualize the cytoplasm of the cell and the lack of visual MN verification or image data storage with flow cytometry. Multispectral Imaging Flow Cytometry (MIFC) has the potential to overcome these limitations. MIFC combines the high resolution fluorescent imagery of microscopy with the statistical robustness and speed of conventional flow cytometry. In addition, all collected imagery can be stored in dose-specific files. This paper describes the protocol developed to perform a fully automated version of the MN assay on MIFC. Human lymphoblastoid TK6 cells were enlarged using a hypotonic solution (75 mM KCl), fixed with 4% formalin and the nuclear content was stained with Hoechst 33342. All samples were run in suspension on the MIFC, permitting acquisition of high resolution images of all key events required for the assay (e.g. binucleated cells with and without MN as well as mononucleated and polynucleated cells). Images were automatically identified, categorized and enumerated in the MIFC data analysis software, allowing for automated scoring of both cytotoxicity and genotoxicity. Results demonstrate that using MIFC to perform the in vitro MN assay allows statistically significant increases in MN frequency to be detected at several different levels of cytotoxicity when compared to solvent controls following exposure of TK6 cells to Mitomycin C and Colchicine, and that no significant increases in MN frequency are observed following exposure to Mannitol.

The in vitro micronucleus assay is a well-established method for evaluating genotoxicity and cytotoxicity but scoring the assay using manual microscopy is laborious and suffers from subjectivity and inter-scorer variability. This paper describes the protocol developed to perform a fully automated version of the assay using multispectral imaging flow cytometry.

使用多光谱成像流细胞测定法执行体外微核测定的自动化方法

The in vitro micronucleus (MN) assay is often used to evaluate cytotoxicity and genotoxicity but scoring the assay via manual microscopy is laborious and ...

Standardized and Scalable Assay to Study Perfused 3D Angiogenic Sprouting of iPSC-derived Endothelial Cells In Vitro

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current in vitro screening models that have sufficient throughput only have limited physiological relevance, which hinders the translation of findings from in vitro to in vivo. On the other hand, microfluidic cell culture platforms have shown unparalleled physiological relevancy in vitro, but often lack the required throughput, scalability and standardization. We demonstrate a robust platform to study angiogenesis of endothelial cells derived from human induced pluripotent stem cells (iPSC-ECs) in a physiological relevant cellular microenvironment, including perfusion and gradients. The iPSC-ECs are cultured as 40 perfused 3D microvessels against a patterned collagen-1 scaffold. Upon the application of a gradient of angiogenic factors, important hallmarks of angiogenesis can be studied, including the differentiation into tip- and stalk cell and the formation of perfusable lumen. Perfusion with fluorescent tracer dyes enables the study of permeability during and after anastomosis of the angiogenic sprouts. In conclusion, this method shows the feasibility of iPSC-derived ECs in a standardized and scalable 3D angiogenic assay that combines physiological relevant culture conditions in a platform that has the required robustness and scalability to be integrated within the drug screening infrastructure.

This method describes the culture of iPSC-derived endothelial cells as 40 perfused 3D microvessels in a standardized microfluidic platform. This platform enables the study of gradient-driven angiogenic sprouting in 3D, including anastomosis and stabilization of the angiogenic sprouts in a scalable and high-throughput manner.

标准化和可扩展的测定，以研究iPSC衍生内皮细胞在Vitro的渗透3D血管生成发芽

Pre-clinical drug research of vascular diseases requires in vitro models of vasculature that are amendable to high-throughput screening. However, current ...

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 ...