Name: development of an in vitro ocular platform to test contact lenses
Uploaded: 2016-04-06T15:00:00.000+00:00
Duration: 8 min 28 s
Description: Watch this Scientific Journal Video about Development of an In Vitro Ocular Platform to Test Contact Lenses at JoVE.com

作者要感谢我们的资金来源NSERC 20/20网络高级眼科材料的发展。

所有实验均按照和遵守滑铁卢大学的动物实验伦理委员会列出所有相关指引完成。牛眼睛都慷慨地从本地屠宰场捐赠的。 1.眼模型<ol><li>模具设计13生产<ol><li>根据成人眼睛的生理平均尺寸设计眼球的机型。13 </li><li>离开的眼球和眼模型的眼睑片之间250微米的差距。设计采用计算机辅助设计（CAD）软件各自的模具。 </li><li>创建新.cad文件或文件.SLDPRT与AutoCAD或Solidworks的。创建人的眼球/眼睑的3D模型。创建模型的模具和模具保存为.STL文件。 </li><li>导入·STL文件到三维打印机的软件（ 例如，用于makeware replicator2）。指定打印参数（位置，稀疏，规模，方向，平滑度等 。 </EM&gt;）13。 </li><li>将文件保存为G代码文件的3D打印机阅读。选择的材料，如聚乳酸（聚乳酸），ABS（丙烯腈-丁二烯-苯乙烯），PC（聚碳酸酯），或它们的组合，以打印模具13。 </li><li>安装所选择的材料的期望的长丝。导入G代码文件到三维打印机读取。打印模具。 注:另外，生产用的控制（CNC）机计算机数字眼模，如果对眼睛模型光滑的表面是需要的。对于数控模具生产，对于模具材料不再局限于热塑料，而是扩大到金属，陶瓷和化学电阻聚合物如聚四氟乙烯。 </li><li>打开连接到切割钻头CNC软件接口。根据前面，顶部，侧面，以及在控制软件接口的先前构造的眼球/眼睑模型模具的透视图构建的3D模具。选择合适的参数加工（比特大小，基片材料，材料的厚度），并继续切割模具。 </li></ol></li><li>目镜使用PDMS的合成<ol><li>使用注射器，措施10毫升体积的PDMS（聚二甲基硅氧烷）基并将其填充到15-50毫升离心管中。添加10％w / v的由PDMS的总重量，弹性体溶液。用搅拌棒，混合解决方案很好。 </li><li>倒入PDMS溶液进入眼球和眼睑模具。允许PDMS到在RT O / N定居（或至少12小时）以开始聚合反应，以允许气泡以溶解出聚合物。 注:确保没有留下可能上升或扩大PDMS气泡。 </li><li>接着，把模具成75℃（167°F）的烘箱中1小时，或150℃（302°F）下5分钟。对于较软的凝胶，让PDMS坐在RT至少48小时至完全聚合。 </li><li>把样品中的几分钟的冷冻;这将缩小PDMS和简化除去从模具的样品。提取使用薄铲模具目镜。 </li><li>为的溶液进入眼球和眼睑片之间的空间内的输送，用1/16"等于腿耦合管连接器连接一个1/16"×1/8"的聚四氟乙烯管，它在配管孔连接到眼睑片。 </li></ol></li><li>用琼脂糖片眼球的合成 注:眼球片可以使用其它聚合物，例如琼脂糖来合成。下面的过程也可以被修改，以从各种琼脂的类型，如PDA（马铃薯葡萄糖琼脂）或SDA（沙氏葡萄糖琼脂）的产生眼件。 <ol><li>以产生2％（2克/ 100毫升）的凝胶，测量​​2克琼脂糖，用100ml超纯水混合。使溶液到沸腾（100℃），使得琼脂糖完全溶解。让该溶液冷却5分钟。 </li><li>将溶液倒入眼球模具，并使溶液冷却30分钟在室温。用锅铲取出眼球件。存储眼球琼脂-20 ℃冰箱供以后使用。用于微生物学研究中，消毒通过高压灭菌和/或UV照射眼球模具。 </li></ol></li><li>牛角膜对眼球PDMS掺入 注 :此协议已被改编自巴力等[14] <ol><li>在层流罩下执行在无菌条件下的牛角膜的解剖和掺入。获得眼睛和解剖它们在同一天。 </li><li>打开对在使用前10分钟的流罩并用70％的乙醇酒精消毒。确保所有的材料和仪器是无菌的通过高压灭菌器在273°F / 133℃45分钟，和位于从所述流罩入口不小于4英寸。 </li><li>浸入牛眼在含有2分钟稀释的聚维酮碘溶液的烧杯中。冲洗在烧杯眼含磷酸盐缓冲盐水（PBS）芘^ h 7.4。使用镊子轻轻地放在眼睛上的玻璃培养皿，角膜正面向上。 </li><li>通过用钝头剪刀切开巩膜附着点切断去除多余的肌肉和脂肪组织。处置多余的组织的成指定用于动物废物的无菌烧杯中。 </li><li>采用微型剪刀，从眼睛取出结膜。包住眼用无菌纱布，保持从角膜缘的至少为1厘米的距离。 </li><li>使用手术刀，从角膜缘区域大约2mm切开巩膜和表面，以避免潜在的脉络膜和玻璃体的渗透。请小心地用手术刀或夹层剪刀360°的切口不角膜从它的自然弯曲变形。 </li><li>用细镊子，取出从眼睛角膜。使用镊子，小心地取出任何秉承葡萄膜组织，用PBS冲洗角膜。 </li><li>储存于31ºC角膜与文化的无菌容器中介质（如培养基199）含有3％胎牛血清保持组织的水分和细胞滋养。 </li><li>之前的实验，搁置在PDMS眼球切除角膜，及两片用专门夹式夹紧在一起。 </li></ol></li></ol> 2.闪烁平台<ol><li>设计生产眨眼睛平台 注:闪烁平台是由三个功能部分:眼模型（在第1中所述），齿轮系统和电子系统。 <ol><li>采用CAD和3D打印，类似的眼模型（1.1节）中描述的设计和制造闪烁的平台。设计齿轮系统，使得它转换马达入目镜的横向和旋转运动的简单的转动。15 </li><li>利用小齿轮和齿轮机构，翻译步进电机的旋转运动转换成一个小齿轮，其连接到所述眼睑片的横向运动。 </li><li>使用共轭齿轮系统中，来自步进电机扩增一个旋转运动转化为三个不同的眼球片三个（或更多）的旋转运动。 </li><li>对齐两个齿轮系统，一个用于眼睑和一个用于眼球，使两者之间的距离是恒定的。组装该电子系统与微控制器，马达罩，和两个马达。 注意:使用两个步进电机提供的旋转电机，其由齿轮系统转换成闪烁动作。 </li><li>用由堆叠在微控制器的电动机罩的一个系统连接的两个步进电机。连接和配置的电子元件与开源软件产品的工作。 </li><li>编程系统来控制电机参数，例如每分钟轮（RPM），正向轮数轮向后的数目，和转动式。 注:请参阅补充"的Arduino代码文件"的说明。 </li><li>从马下载系统软件nufacturers的网站。 </li><li>安装软件并打开它。写的代码，以控制步进电机在所需的配置。该系统连接与源到电子系统供电，使电机以期望的方式由研究人员所限定移动。 注:请参阅补充"的Arduino代码文件"。 </li></ol></li><li>大会与微流体（人造泪液） <ol><li>以合成的眼球和眼睑片和防滑他们到其相应的夹式的眼睛模型。连接该接合用注射器和位于与眼睑片（部分1.2.5）微流体泵的管路。测试运行平台，并检查是否有一致的运动。 </li><li>总理管道和检查的人造泪液（ATS）源源不断。对于ATS配方曾报道16 </li><li>手动移动眼模型部件一起在水平平面上，使得该眼球和眼盖子相接触。设置微流体泵到所需值的流速。设定生理流速1-1.5微升/分。17 </li><li>启动泵和致动器，开始实验。用于药物递送的实验中，将含有药物的接触透镜在眼球片。 </li><li>允许流通的流体滴落到12孔板中。在所需的设定的时间间隔，使用普通的检测方法，如UV-Vis光谱或荧光量化分析物或药物的浓度。1,4,18 </li><li>对于研究隐形眼镜评估泪液成分沉积，放置隐形眼镜的"眼球"作品。收集流通液，其可以被丢弃。 </li><li>在所需的时间间隔后，从眼球片隐形眼镜，并准备作进一步的分析透镜如共焦显微镜。 </li></ol></li></ol>

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作者什么都没有透露。

有一些需要特别注意的协议中的三个关键步骤:设计和生产模具（第1.1节），装配平台（部分2.2.1-2.2.3），并监测实验运行（部分2.2.4-2.2.7 ）。在设计和生产模具（第1.1节）的观点出发，眼球片应根据一个人的角膜的尺寸来设计。然而，它可以由眼球片可以创建完全符合商用接触透镜（CL）之前所需要的模具的多个原型。另外，当眼球和眼睑片相接触，以确保泪液整个眼睛模型顺畅地流动时，CL为存在要维持在250微米的需求。该距离可以在将来的迭代被改变，但不应该是小于150微米，以允许足够的间隔，以适应CL。该平台组件（部分2.2.1-2.2.3）需要特别注意，以使眼球和眼睑一块进入CONTA眨眼运动过程中CT。如果目镜不完美的联系人，然后闭合眼睑模拟和机械摩擦失败。操作员应观察在运动平台的几个周期，以确保两个眼球和眼睑相接触，并作为程序发生时，摩擦。当前平台被设计为一个月以上连续运行，但运行实验（部分2.2.4-2.2.7）当操作者应该总是在系统的稳定性检查每24小时。作为当前平台不具有温度或湿度控制，并且在这些参数波动可能干涸CLS的，这是很重要的。如果出现这种情况，将一个受控的湿度和温度室中的眼睛模型。此外，用于药物递送的实验中，所收集的流过流体应该分析或存储至少每2小时，以避免样品的显著蒸发。 目前所提出的两个限制眼模型。第一个限制是在问候暴露于周围的环境。目前，因为眼睛片不以受控的室封闭，的变化，如温度和湿度在工作区将影响实验的各个方面。例如，如果环境太干，则通函干涸更快，可从眼球片分开，或流过的流体可以蒸发。为了解决这个问题，未来的迭代将房子在控制温度和湿度室眼球模型。第二个限制涉及到复杂眼球件。目前，目镜很简单，无论是由PDMS或琼脂糖，两者都不真正代表角膜表面性能。未来的工作将致力于产生眼模型更接近其模仿角膜表面的结构。 体外眼研究通常被视为前述测试阶段的体内研究。然而，它要记住， 在体外研究，也可以在体内互补数据是很重要的，提供，否则不能单独从体内研究实现关键的见解。遗憾的是，目前的体外模型用于测试通函是简陋和缺乏几个关键部件，以充分模拟体内环境。例如， 在体外 CL研究是在含有2-5毫升的磷酸盐缓冲盐水，1-6大大超过生理撕裂卷在7.0±2微升小瓶进行。7此外，眼部环境的两个重要因素，天然泪液流和瞬目反射，都是从简单的静态模型小瓶缺席。常规的小瓶模型的局限性已被研究人员的认可，并且，通过包括微流体撕裂补充部件2已经尝试建立体外眼睛模型模拟眼部环境独特0-24和/或间歇性的空气暴露25,26毫不奇怪，这些实验产生的结果比用传统的小瓶模型获得的那些非常不同的，并且可以在体内数据更类似于20-25因此，开发一种错综复杂的体外眼模型来研究通函将提供与眼球表面镜片材料的相互作用的新见解，并协助推动新材料，在未来几十年通函新应用的开发。 可以说， 在体外眼模型的最有争议的问题之一是眼睛是否类似于无限下沉，当它涉及到从通函药物输送这一点尤为重要。下无限水槽的条件下，周围的溶液的体积比药物饱和量显著越高，使得药物释放不受药物的溶解度。27促进会小瓶作为附属品PTABLE眼模型认为，角膜，结膜和眼周围组织功能与无限一起沉没。虽然理论上这可能是正确的，药物必须首先溶解到泪液。此限速步骤可能不是汇的条件，并且将取决于两个泪液量和流量由我们的模型进行模拟。 该模型的唯一标识在于其效仿泪液膜的能力。通过采用两件式设计中，"角膜/巩膜"眼球部分和一个"眼睑"，有可能当两个件接触以创建整个眼球片泪膜的均匀分布的薄层。进一步模拟眼表面，机械磨损和空气暴露是通过两个机械致动器结合到模型中。由于眼睑一块横向移动，它模拟眼和间歇性暴露在空气中闭幕。眼球的旋转模拟都灵产生的机械磨损摹闪烁。该系统耦接的微流体泵，其注入眼球模型在生理流速泪液或任何其他期望的流速。泪膜是每次两片接触形成的，并且当两片分开发生泪膜破裂。 这样做的目的是创造一个万能试验平台以评价各种体外分析CLS。为了成为通用的，眼球片可以从各种聚合物，如聚二甲基硅氧烷（PDMS）或琼脂合成。对于简单的眼研究，这些聚合物，它们分别代表疏水和亲水表面上，就足够了。然而，如需要更复杂的分析，例如眼部药物渗透或毒性研究，眼睛片将需要进一步修改。这些额外的修改该模型，如纳入所示离体角膜的，都比较可行的。但是，进一步的验证研究是必需的，和未来的工作旨在通过将其与体内模型进行比较，以改善该模型的有效性。

的接触透镜（CL）的竞技场内的感兴趣的两个显著领域包括不适和新颖CL应用的开发。阐明底层CL不适机制是已经躲避领域几十年来一个问题。8新颖的发展，功能通函，如药物-递送装置1,3,9和生物传感器，10-12是越来越大的兴趣的区域，具有巨大潜力的市场。在这两种情况下， 体外模型精密将提供相关信息，以帮助在开发阶段选择合适的镜片材料或设计特征。不幸的是， 在体外模型用于评估CLS和其他眼部相关的应用目前都比较粗糙，不健全的。传统上， 在体外 CL研究评价泪膜沉积或药物输送含有固定的流体体积，静态的，大容量的小瓶中进行哪些格雷亚TLY超过生理量。此外，这种简单的模型缺乏天然泪液流分量和闪烁的反射，这两者都限定眼部环境的因素。 一个复杂的，生理相关的眼"模式"的发展将需要多学科的方法，并要求在体内验证可观。由于这些原因，对于我们的体外眼睛模型的基本框架是高度通用的，这样，该模型可以通过未来的升级和调制来不断改进。迄今为止，该模型能够模拟泪液量，撕裂流，机械磨损和空气暴露的。这样做的目的是创造一个体外模型，将提供有意义的结果，这是预测和免费在体内和离体观察。

<table><tbody><tr><td>Arduino Uno R3 (Atmega328 - assembled)</td><td>Adafruit</td><td>50</td><td>Board</td></tr><tr><td>Stepper motor</td><td>Adafruit</td><td>324</td><td>Motor and Motor shield</td></tr><tr><td>Equal Leg Coupler 1.6mm 1/16&quot;</td><td>VWR</td><td>CA11009-280</td><td>50 pcs of tube connector</td></tr><tr><td>Tubing PT/SIL 1/16&quot;x1/8&quot;</td><td>VWR</td><td>16211-316</td><td>Case of 50feet</td></tr><tr><td>PDMS</td><td>Dow Corning</td><td>Sylgard 184 Solar Cell Encapsulation</td><td></td></tr><tr><td>Agarose, Type 1-A, low EEO</td><td>Sigma-Aldrich</td><td>A0169-25G</td><td></td></tr><tr><td>PHD UltraTM</td><td>Harvard Apparatus</td><td>703006</td><td>MicroFluidic Pump</td></tr><tr><td>Bovine cornea</td><td>Cargill, Guelph/ON</td><td></td><td></td></tr><tr><td>Soldidworks</td><td>Dassault Systemes</td><td></td><td>Software</td></tr><tr><td>3-D printing</td><td>University of Waterloo - 3D Print Centre</td><td></td><td></td></tr><tr><td>Dissection tools</td><td>Fine Science Tools</td><td></td><td>General dissection tools</td></tr><tr><td>Medium 199</td><td>Sigma-Aldrich</td><td></td><td>Culture medium storage for cornea</td></tr><tr><td>Fetal bovine serum</td><td>Thermo Fisher</td><td></td><td>Add to culture medium, 3% total volume</td></tr></tbody></table>

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.

从机械车间和从3-D打印获得的合成眼模具如图1所示，这些霉菌可以与各种聚合物，如PDMS和琼脂糖一起使用，以产生具有所需性质的目镜。与微流体注射器泵的眼睛模型平台的示意组件示于图2，平台经由眼球片进出眼睑片的运动的旋转，并通过横向空气曝光模拟机械磨损。泪液流体注入眼睑从在所需流速的微流体泵，并流过流体可以在一个12孔板进行收集。 对于牛晶状体解剖，并安装到PDMS目镜的步骤在图3中描绘的过量组织从眼睛中分离并弃去，然后除去结膜。角膜的去除开始于一个切口进入角膜缘附近的巩膜。 图4示出可用于各种体外分析各种目镜。表示的安装眼球片从PDMS，琼脂合成，并安装在PDMS眼球片的离体牛角膜。 图5描绘了一个研究，评估抗生素，莫西沙星的释放，从CLS。18当在传统的小瓶模型测定，药物释放前2小时，随后一平顶阶段内发生。与此相反，新的眼模型显示药物释放是缓慢和持续长达24小时。18的研究评估对通函胆固醇的沉积在图6中被示出。在研究中的胆固醇被荧光标记在NBD的形式 - 胆固醇（7-硝基苯-2-氧杂-1,3-二唑-4-基 - 胆固醇），和depos银行足球比赛是用激光扫描共聚焦显微镜成像。结果表明相比于眼睛模型有很大的差别时，在沉积研究在小瓶中进行。 <img alt="图1" src="/files/ftp_upload/53907/53907fig1.jpg" /> 图1.目镜模具。（A）从加工车间眼球件模具。从3-D打印（B）眼睑的模具。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53907/53907fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53907/53907fig2.jpg" /> 图2. 体外 眼平台。（A）圆周运动模拟机械磨损。 （B）横向运动产生间歇性的空气曝光。 （C）撕裂输液到眼睑。 （D）收集孔板。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53907/53907fig2large.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/53907/53907fig3.jpg" /> 图3.解剖和牛角膜结合。（A）多余的组织去除。 （ 二 ）拆除结膜。 （C）切口进入角膜缘区域。 （D）的角膜切除可存储或安装在PDMS眼球件。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53907/53907fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53907/53907fig4.jpg" /> 图4.示例目镜。PDMS目镜带隐形眼镜，琼脂目镜，以及样本体外牛角膜安装目镜。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53907/53907fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53907/53907fig5.jpg" /> 图5.使用体外 眼平台 的药物递送 。从日抛型隐形眼镜莫西沙星发布（A）大量静态的小瓶和（B）的眼睛模型（重新打印与来自视觉的研究协会许可，眼科）18中的所有数据均以平均值±标准差。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53907/53907fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <p类="jove_content"FO: - together.within页保留="1"&gt; <img alt="图6" src="/files/ftp_upload/53907/53907fig6.jpg" /> 图6.使用体外 眼平台 胆固醇沉积 。共聚焦图像显示依他菲康A，nelfilcon A，nesofilcon A，ocufilcon B，delefilcon A，somofilcon A，narafilcon A的横截面后在NBD胆固醇4小时培养小瓶和眼睛的模型。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/53907/53907fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Development of an In Vitro Ocular Platform to Test Contact Lenses at JoVE.com

Development of an In Vitro Ocular Platform to Test Contact Lenses

目前在体外模型来评估隐形眼镜（CLS）等眼睛相关的应用非常有限。所呈现的眼平台模拟生理泪液流，泪液量，空气暴露和机械磨损。这个系统是高度通用，并且可以被应用于各种体外根据CLS分析。

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 ...

development-of-an-in-vitro-ocular-platform-to-test-contact-lenses

Universidad San Sebastian

Research

JoVE Journal

Bioengineering

Development of an In Vitro Ocular Platform to Test Contact Lenses

10.5K 次观看.  University of Waterloo. 目前在体外模型来评估隐形眼镜（CLS）等眼睛相关的应用非常有限。所呈现的眼平台模拟生理泪液流，泪液量，空气暴露和机械磨损。这个系统是高度通用，并且可以被应用于各种体外根据CLS分析。 

视频: Development of an In Vitro Ocular Platform to Test Contact Lenses

Microfabricated Platforms for Mechanically Dynamic Cell Culture

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or fundamental cell biology studies is limited by current bioreactor technologies, which cannot simultaneously apply a variety of mechanical stimuli to cultured cells. In order to address this issue, we have developed a series of microfabricated platforms designed to screen for the effects of mechanical stimuli in a high-throughput format. In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and further demonstrate how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

In this protocol, we demonstrate the fabrication of a microactuator array of vertically displaced posts on which the technology is based, and how this base technology can be modified to conduct high-throughput mechanically dynamic cell culture in both two-dimensional and three-dimensional culture paradigms.

微机械动态细胞培养平台

The ability to systematically probe in vitro cellular response to combinations of mechanobiological stimuli for tissue engineering, drug discovery or ...

科研

生物工程

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to follow and manipulate the wound response ex vivo in an environment that closely mimics that of epithelial tissue injury in vivo. This issue was addressed by creating a clinically relevant epithelial ex vivo injury-repair model based on cataract surgery. In this culture model, the response of the lens epithelium to wounding can be followed live in the cells&#8217; native microenvironment, and the molecular mediators of wound repair easily manipulated during the repair process. To prepare the cultures, lenses are removed from the eye and a small incision is made in the anterior of the lens from which the inner mass of lens fiber cells is removed. This procedure creates a circular wound on the posterior lens capsule, the thick basement membrane that surrounds the lens. This wound area where the fiber cells were attached is located just adjacent to a continuous monolayer of lens epithelial cells that remains linked to the lens capsule during the surgical procedure. The wounded epithelium, the cell type from which fiber cells are derived during development, responds to the injury of fiber cell removal by moving collectively across the wound area, led by a population of vimentin-rich repair cells whose mesenchymal progenitors are endogenous to the lens1. These properties are typical of a normal epithelial wound healing response. In this model, as in vivo, wound repair is dependent on signals supplied by the endogenous environment that is uniquely maintained in this ex vivo culture system, providing an ideal opportunity for discovery of the mechanisms that regulate repair of an epithelium following wounding.

Establishment of a Clinically Relevant Ex Vivo Mock Cataract Surgery Model for Investigating Epithelial Wound Repair in a Native Microenvironment

Described here is the establishment of a clinically relevant ex vivo mock cataract surgery model that can be used to investigate mechanisms of the injury response of epithelial tissues within their native microenvironment.

建立临床相关<em&gt;离体</em&gt;调查上皮创伤修复的微环境本地人模拟白内障手术模型

The major impediment to understanding how an epithelial tissue executes wound repair is the limited availability of models in which it is possible to ...

发育生物学

Synthesis of Soft Polysiloxane-urea Elastomers for Intraocular Lens Application

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses (a-IOLs). Aminopropyl-terminated polydimethylsiloxanes (PDMS) were previously prepared via the ring-chain equilibration of the cyclic siloxane octamethylcyclotetrasiloxane (D4) and 1,3-bis(3-aminopropyl)-tetramethyldisiloxane (APTMDS). Phenyl groups were introduced into the siloxane backbone via the copolymerization of D4 and 2,4,6,8-tetramethyl-2,4,6,8-tetraphenyl-cyclotetrasiloxane (D4Me,Ph). These polydimethyl-methyl-phenyl-siloxane-block copolymers were synthesized for increasing the refractive indices of polysiloxanes. For applications as an a-IOL, the refractive index of the polysiloxanes must be equivalent to that of a young human eye lens. The polysiloxane molecular weight is controlled by the ratio of the cyclic siloxane to the endblocker APTMDS. The transparency of the PSU elastomers is examined by the transmittance measurement of films between 200 and 750 nm, using a UV-Vis spectrophotometer. Transmittance values at 750 nm (upper end of the visible spectrum) are plotted against the PDMS molecular weight, and &#62; 90% of the transmittance is observed until a molecular weight of 18,000 g&#183;mol&#8722;1. Mechanical properties of the PSU elastomers are investigated using stress-strain tests on die-cut dog-bone-shaped specimens. For evaluating mechanical stability, mechanical hysteresis is measured by repeatedly stretching (10x) the specimens to 5% and 100% elongation. Hysteresis considerably decreases with the increase in the PDMS molecular weight. In vitro cytotoxicity of some selected PSU elastomers is evaluated using an MTS cell viability assay. The methods described herein permit the synthesis of a soft, transparent, and noncytotoxic PSU elastomer with a refractive index approximately equal to that of a young human eye lens.

This study describes synthetic routes for aminopropyl-terminated polydimethylsiloxanes and polydimethyl-methyl-phenyl-siloxane-block copolymers and for soft polysiloxane-based urea (PSU) elastomers. It presents the application of PSUs as accommodating an intraocular lens. An evaluation method for in vitro cytotoxicity is also described.

软聚聚硅氧烷-尿素弹性体在人工晶体中的应用

This study discusses a synthesis route for soft polysiloxane-based urea (PSU) elastomers for their applications as accommodating intraocular lenses ...

化学

Anterior Segment Organ Culture Platform for Tracking Open Globe Injuries and Therapeutic Performance

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and medical intervention in rural environments and military medicine applications where ophthalmic care is not readily available. Untreated injuries are susceptible to infection after the eye has lost its watertight seal, as well as loss of tissue viability due to intraocular hypotension. Therapeutics to temporarily seal open globe injuries, if properly developed, may be able to restore intraocular pressure and prevent infection until proper ophthalmic care is possible. To facilitate product development, detailed here is the use of an anterior segment organ culture open globe injury platform for tracking therapeutic performance for at least 72 h post-injury. Porcine anterior segment tissue can be maintained in custom-designed organ culture dishes and held at physiological intraocular pressure. Puncture injuries can be created with a pneumatic-powered system capable of generating injury sizes up to 4.5 mm in diameter, similar to military-relevant injury sizes. Loss of intraocular pressure can be observed for 72 h post-injury confirming proper injury induction and loss of the eye&#39;s watertight seal. Therapeutic performance can be tracked by application to the eye after injury induction and then tracking intraocular pressure for multiple days. Further, the anterior segment injury model is applicable to widely used methods for functionally and biologically tracking anterior segment physiology, such as assessing transparency, ocular mechanics, corneal epithelium health, and tissue viability. Overall, the method described here is a necessary next step toward developing biomaterial therapeutics for temporarily sealing open globe injuries when ophthalmic care is not readily available.

Open globe eye injuries may go untreated for multiple days in rural or military-relevant scenarios, resulting in blindness. Therapeutics are needed to minimize loss of vision. Here, we detail an organ culture open globe injury model. With this model, potential therapeutics for stabilizing these injuries can be properly evaluated.

跟踪开放全球伤害和治疗性能的前段器官文化平台

Open globe injuries have poor visual outcomes, often resulting in permanent loss of vision. This is partly due to an extended delay between injury and ...

自动压缩猪晶状体的生物力学表征

Automated Compression Testing of the Ocular Lens

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically with age in the human lens, resulting in a loss of near vision called presbyopia. However, the mechanisms of these changes remain unknown. Lens compression offers a relatively simple method for assessing the lens' biomechanical stiffness in a qualitative sense and, when coupled with appropriate analytical techniques, can help quantify biomechanical properties. A variety of lens compression tests have been performed to date, including both manual and automated, but these methods inconsistently apply key aspects of biomechanical testing such as preconditioning, loading rates, and time between measurements. This paper describes a fully automated lens compression test wherein a motorized stage is synchronized with a camera to capture the force, displacement, and shape of the lens throughout a preprogrammed loading protocol. A characteristic elastic modulus may then be calculated from these data. While demonstrated here using porcine lenses, the approach is appropriate for the compression of lenses of any species.

作者聚焦：增进对与年龄相关的晶状体僵硬变化的理解 

We present an automated method for characterizing the effective elastic modulus of an ocular lens using a compression test.

人工晶状体的自动压缩测试

The biomechanical properties of the ocular lens are essential to its function as a variable power optical element. These properties change dramatically ...

Development of a Noninvasive, Laser-Assisted Experimental Model of Corneal Endothelial Cell Loss

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the optical breakdown at the laser focus. Acoustic shock waves and cavitation bubbles are generated, causing tissue rupture. Bubble sizes and pressure amplitudes vary with pulse energy and position of the focal point. In this study, enucleated porcine eyes were positioned in front of a commercially available Nd:YAG laser. Variable pulse energies as well as different positions of the focal spots posterior to the cornea were tested. Resulting lesions were evaluated by two-photon microscopy and histology to determine the best parameters for an exclusive detachment of corneal endothelial cells (CEC) with minimum collateral damage. The advantages of this method are the precise ablation of CEC, reduced collateral damage, and above all, the non-contact treatment.

Here, we present a protocol to detach corneal endothelial cells (CEC) from Descemet&#8217;s membrane (DM) using a neodymium:YAG (Nd:YAG) laser as an ex vivo disease model for bullous keratopathy (BK).

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

Nd:YAG lasers have been used to perform noninvasive intraocular surgery, such as capsulotomy for several decades now. The incisive effect relies on the ...

医学

Microfabrication of Implantable Optics Integrated in a Microstructured Imaging Window for Advanced In Vivo Imaging

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable integrated imaging window. The micro-device comprises a sophisticated system of microlenses coupled with micro-scaffolds specifically designed for in vivo quantification of the immune response using advanced non-linear excitation microscopy. The protocol is based on two-photon polymerization (2PP) of the biocompatible photoresist SZ2080, which enables the fabrication of micro-scaffolds and micro-lenses in a continuous sequence to enhance manufacturing efficiency and precision. To further improve speed, accuracy, and structural integrity, a hybrid optics fabrication approach was implemented, involving the 2PP of the microlens outer shell followed by UV bulk crosslinking of the inner core. This innovative technique optimizes the optical properties of the microlenses while streamlining the production process. The resulting micro-device demonstrates high reproducibility and mechanical stability, making it an effective method for prototyping microscale optical systems for a range of biomedical applications.

This protocol describes the fabrication of an implantable integrated imaging window using 3D laser printing. The window consists of a system of microlenses coupled with micro-scaffolds. The method involves two-photon polymerization (2PP) of the biocompatible photoresist SZ2080 in a continuous sequence, optimizing manufacturing efficiency and alignment between the different components.

集成在微结构成像窗口中的植入式光学元件的微纳加工，用于先进的 体内 成像

In the context of biomaterials and drug testing in animal models, this study presents a streamlined protocol for fabricating a novel implantable ...

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical exposures, among other environmental factors, are an established cause of cataracts. Ocular toxicity testing can assess whether pharmaceuticals and their components may contribute to lens damage that may lead to cataracts or aid the treatment of cataracts. 
In vitro studies and in vivo animal testing can be used for assessing the safety of chemicals prior to clinical studies. The Draize test-the current in vivo standard for ocular toxicity and irritancy testing-has been criticized for lack of sensitivity and objective measurements of determining ocular toxicity. In vitro cell-based assays are limited as cell cultures cannot appropriately model an intact functional lens. 
The method described here is a sensitive in vitro alternative to animal testing, designed to evaluate the response of the intact bovine lens to treatment at both the cellular activity level and for overall refractive performance. The non-toxic reagent resazurin is metabolized in proportion to the level of cell activity. The lens laser-scanner assay measures the ability of the lens to refract incident beams of light to a single point with minimal error, directly relevant to its natural function. The method may be used to determine both acute and delayed changes in the lens, as well as the recovery of the lens from chemical or environmental exposures.

The goal of this protocol is to evaluate changes in metabolic activity and refractive function of the lens in response to experimental treatment.

As the leading cause of blindness, cataracts are a significant burden for the tens of millions of people affected globally by this condition. Chemical ...

的发展

摘要

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此视频中的章节

微机械动态细胞培养平台

建立临床相关

软聚聚硅氧烷-尿素弹性体在人工晶体中的应用

跟踪开放全球伤害和治疗性能的前段器官文化平台

人工晶状体的自动压缩测试

人工晶状体的自动压缩测试

人工晶状体的自动压缩测试

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

集成在微结构成像窗口中的植入式光学元件的微纳加工，用于先进的体内成像

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals

的发展

摘要

探索更多视频

此视频中的章节

微机械动态细胞培养平台

建立临床相关

软聚聚硅氧烷-尿素弹性体在人工晶体中的应用

跟踪开放全球伤害和治疗性能的前段器官文化平台

人工晶状体的自动压缩测试

人工晶状体的自动压缩测试

人工晶状体的自动压缩测试

角膜内皮细胞损失非侵入性激光辅助实验模型的发展

集成在微结构成像窗口中的植入式光学元件的微纳加工，用于先进的 体内 成像

Organ Culture System for Assessing the Toxicity of Intraocular Treatment Excipients and Pharmaceuticals

集成在微结构成像窗口中的植入式光学元件的微纳加工，用于先进的体内成像