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>

<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 border="0" cellpadding="0" cellspacing="0" width="1129">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Arduino Uno R3 (Atmega328 - assembled)</td>
			<td style="width:125px;">Adafruit</td>
			<td style="width:451px;">50</td>
			<td style="width:273px;">Board</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Stepper motor</td>
			<td style="width:125px;">Adafruit</td>
			<td style="width:451px;">324</td>
			<td>Motor and Motor shield</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Equal Leg Coupler 1.6mm 1/16&#34;</td>
			<td style="width:125px;">VWR</td>
			<td style="width:451px;">CA11009-280</td>
			<td>50 pcs of tube connector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Tubing PT/SIL 1/16&#34;x1/8&#34;</td>
			<td style="width:125px;">VWR</td>
			<td style="width:451px;">16211-316</td>
			<td>Case of 50feet</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">PDMS</td>
			<td style="width:125px;">Dow Corning</td>
			<td style="width:451px;">Sylgard 184 Solar Cell Encapsulation</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Agarose, Type 1-A, low EEO</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:451px;">A0169-25G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">PHD UltraTM</td>
			<td style="width:125px;">Harvard Apparatus</td>
			<td style="width:451px;">703006</td>
			<td>MicroFluidic Pump</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Bovine cornea</td>
			<td style="width:125px;">Cargill, Guelph/ON</td>
			<td style="width:451px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Soldidworks</td>
			<td style="width:125px;">Dassault Systemes</td>
			<td style="width:451px;"></td>
			<td>Software</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:281px;">3-D printing</td>
			<td style="width:125px;">University of Waterloo - 3D Print Centre</td>
			<td style="width:451px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:281px;">Dissection tools</td>
			<td style="width:125px;">Fine Science Tools</td>
			<td style="width:451px;"></td>
			<td>General dissection tools</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Medium 199</td>
			<td style="width:125px;">Sigma-Aldrich</td>
			<td style="width:451px;"></td>
			<td>Culture medium storage for cornea</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:281px;">Fetal bovine serum</td>
			<td style="width:125px;">Thermo Fisher</td>
			<td style="width:451px;"></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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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.remotexs.ntu.edu.sg/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

Nanyang Technological University

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

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 ...

科研

生物工程

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or erosion. Amelogenin has been proven to be a critical protein for controlling the organized growth of apatite crystals. In this paper, we present a detailed protocol for superficial enamel reconstruction by using a novel amelogenin-chitosan hydrogel. Compared to other conventional treatments, such as topical fluoride and mouthwash, this method not only has the potential to prevent the development of dental caries but also promotes significant and durable enamel restoration. The organized enamel-like microstructure regulated by amelogenin assemblies can significantly improve the mechanical properties of etched enamel, while the dense enamel-restoration interface formed by an in situ regrowth of apatite crystals can improve the effectiveness and durability of restorations. Furthermore, chitosan hydrogel is easy to use and can suppress bacterial infection, which is the major risk factor for the occurrence of dental caries. Therefore, this biocompatible and biodegradable amelogenin-chitosan hydrogel shows promise as a biomaterial for the prevention, restoration, and treatment of defective enamel.

Development of Amelogenin-chitosan Hydrogel for In Vitro Enamel Regrowth with a Dense Interface

In this article, we describe a protocol for fabricating an amelogenin-chitosan hydrogel for superficial enamel reconstruction. Organized in situ growth of apatite crystals in the hydrogel formed a dense enamel-restoration interface, which will improve the effectiveness and durability of restorations.

釉原蛋白 - 壳聚糖水凝胶的发展<em&gt;体外</em&gt;搪瓷愈合了密集接口

Biomimetic enamel reconstruction is a significant topic in material science and dentistry as a novel approach for the treatment of dental caries or ...

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 ...

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards vascular disease pathologies. Unhealthy arteries often present with wall stiffening, scarring, or partial stenosis which may all affect fluid flow rates, and the magnitude of pulsatile flow, or pulsatility index. Replication of various flow conditions is the result of tuning a flow pressure damping chamber downstream of a blood pump. Introduction of air within a closed flow system allows for a compressible medium to absorb pulsatile pressure from the pump, and therefore vary the pulsatility index. The method described herein is simply reproduced, with highly controllable input, and easily measurable results. Some limitations are recreation of the complex physiological pulse waveform, which is only approximated by the system. Endothelial cells, smooth muscle cells, and fibroblasts are affected by the blood flow through the artery. The dynamic component of blood flow is determined by the cardiac output and arterial wall compliance. Vascular cell mechano-transduction of flow dynamics may trigger cytokine release and cross-talk between cell types within the artery. Co-culture of vascular cells is a more accurate picture reflecting cell-cell interaction on the blood vessel wall and vascular response to mechanical signaling. Contribution of flow dynamics, including the cell response to the dynamic and mean (or steady) components of flow, is therefore an important metric in determining disease pathology and treatment efficacy. Through introducing an in vitro co-culture model and pressure damping downstream of blood pump which produces simulated cardiac output, various arterial disease pathologies may be investigated.

In Vitro Model of Physiological and Pathological Blood Flow with Application to Investigations of Vascular Cell Remodeling

This protocol replicates physiological or pathological blood flow in vitro to aid in determining cell response in disease pathologies. By introducing a pressure damping chamber downstream of a blood pump, blood flow across the vasculature can be recapitulated and imposed on a monolayer of vascular endothelium or a mimetic co-culture.

在生理体外模型和病理血流应用的血管细胞重塑调查

Vascular disease is a common cause of death within the United States. Herein, we present a method to examine the contribution of flow dynamics towards ...

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 ...

Assembly and Tracking of Microbial Community Development within a Microwell Array Platform

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial distribution and activities of community members. We have developed a microwell array platform that can be used to rapidly assemble and track thousands of bacterial communities in parallel. This protocol highlights the utility of the platform and describes its use for optically monitoring the development of simple, two-member communities within an ensemble of arrays within the platform. This demonstration uses two mutants of Pseudomonas aeruginosa, part of a series of mutants developed to study Type VI secretion pathogenicity. Chromosomal inserts of either mCherry or GFP genes facilitate the constitutive expression of fluorescent proteins with distinct emission wavelengths that can be used to monitor community member abundance and location within each microwell. This protocol describes a detailed method for assembling mixtures of bacteria into the wells of the array and using time-lapse fluorescence imaging and quantitative image analysis to measure the relative growth of each member population over time. The seeding and assembly of the microwell platform, the imaging procedures necessary for the quantitative analysis of microbial communities within the array, and the methods that can be used to reveal interactions between microbial species area all discussed.

The development of microbial communities depends on a combination of factors, including environmental architecture, member abundance, traits, and interactions. This protocol describes a synthetic, microfabricated environment for the simultaneous tracking of thousands of communities contained in femtoliter wells, where key factors such as niche size and confinement can be approximated.

微孔阵列平台中微生物群落发展的组装与跟踪

The development of microbial communities depends on a combination of complex deterministic and stochastic factors that can dramatically alter the spatial ...

Development of an Electrochemical DNA Biosensor to Detect a Foodborne Pathogen

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, significantly affecting the rate of human mortality and morbidity. Conventional methods for the detection of V. parahaemolyticus such as culture-based methods, immunological assays, and molecular-based methods require complicated sample handling and are time-consuming, tedious, and costly. Recently, biosensors have proven to be a promising and comprehensive detection method with the advantages of fast detection, cost-effectiveness, and practicality. This research focuses on developing a rapid method of detecting V. parahaemolyticus with high selectivity and sensitivity using the principles of DNA hybridization. In the work, characterization of synthesized polylactic acid-stabilized gold nanoparticles (PLA-AuNPs) was achieved using X-ray Diffraction (XRD), Ultraviolet-visible Spectroscopy (UV-Vis), Transmission Electron Microscopy (TEM), Field-emission Scanning Electron Microscopy (FESEM), and Cyclic Voltammetry (CV). We also carried out further testing of stability, sensitivity, and reproducibility of the PLA-AuNPs. We found that the PLA-AuNPs formed a sound structure of stabilized nanoparticles in aqueous solution. We also observed that the sensitivity improved as a result of the smaller charge transfer resistance (Rct) value and an increase of active surface area (0.41 cm2). The development of our DNA biosensor was based on modification of a screen-printed carbon electrode (SPCE) with PLA-AuNPs and using methylene blue (MB) as the redox indicator. We assessed the immobilization and hybridization events by differential pulse voltammetry (DPV). We found that complementary, non-complementary, and mismatched oligonucleotides were specifically distinguished by the fabricated biosensor. It also showed reliably sensitive detection in cross-reactivity studies against various food-borne pathogens and in the identification of V. parahaemolyticus in fresh cockles.

A protocol for the development of an electrochemical DNA biosensor comprising a polylactic acid-stabilized, gold nanoparticles-modified, screen-printed carbon electrode to detect Vibrio parahaemolyticus is presented.

电化学 DNA 生物传感器检测食源性致病菌的研究进展

Vibrio parahaemolyticus (V. parahaemolyticus) is a common foodborne pathogen that contributes to a large proportion of public health problems globally, ...

An Ultrahigh-throughput Microfluidic Platform for Single-cell Genome Sequencing

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of cellular heterogeneity in biological systems. However, single-cell sequencing of large populations has been hampered by limitations in processing genomes for sequencing. In this paper, we describe a method for single-cell genome sequencing (SiC-seq) which uses droplet microfluidics to isolate, amplify, and barcode the genomes of single cells. Cell encapsulation in microgels allows the compartmentalized purification and tagmentation of DNA, while a microfluidic merger efficiently pairs each genome with a unique single-cell oligonucleotide barcode, allowing &gt;50,000 single cells to be sequenced per run. The sequencing data is demultiplexed by barcode, generating groups of reads originating from single cells. As a high-throughput and low-bias method of single-cell sequencing, SiC-seq will enable a broader range of genomic studies targeted at diverse cell populations.

Single-cell sequencing reveals genotypic heterogeneity in biological systems, but current technologies lack the throughput necessary for the deep profiling of community composition and function. Here, we describe a microfluidic workflow for sequencing &gt;50,000 single-cell genomes from diverse cell populations.

单细胞基因组测序的超高通量微流控平台

Sequencing technologies have undergone a paradigm shift from bulk to single-cell resolution in response to an evolving understanding of the role of ...

Rodent Behavioral Testing to Assess Functional Deficits Caused by Microelectrode Implantation in the Rat Motor Cortex

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes demonstrate the ability to record action potentials from individual or small groups of neurons. Such recorded signals have successfully been used to allow patients to interface with or control&#160;computers, robotic limbs, and their own limbs. However, previous animal studies have shown that a microelectrode implantation in the brain not only damages the surrounding tissue but can also result in functional deficits. Here, we discuss a series of behavioral tests to quantify potential motor impairments following the implantation of intracortical microelectrodes into the motor cortex of a rat. The methods for open field grid, ladder crossing, and grip strength testing provide valuable information regarding the potential complications resulting from a microelectrode implantation. The results of the behavioral testing are correlated with endpoint histology, providing additional information on the pathological outcomes and impacts of this procedure on the adjacent tissue.

We have shown that a microelectrode implantation in the motor cortex of rats causes immediate and lasting motor deficits. The methods proposed herein outline a microelectrode implantation surgery and three rodent behavioral tasks to elucidate potential changes in the fine or gross motor function due to implantation-caused damage to the motor cortex.

大鼠运动皮质微电极植入所致功能缺损的啮齿动物行为检测

Medical devices implanted in the brain hold tremendous potential. As part of a Brain Machine Interface (BMI) system, intracortical microelectrodes ...

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 ...