Name: measuring and modeling contractile drying in human stratum corneum
Uploaded: 2017-03-01T10:30:00.000+00:00
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豁免核准（3002-13）使用依照卫生署去识别组织样本和人类服务条例开展调研，45 CFR 46.101（二）（4）获得了批准。全层皮肤从择期手术的好评。在这篇文章中，组织来源是66岁的白人女性的乳房。 1.弹性体覆盖盖玻片的制备<ol><li>在20毫升玻璃小瓶中，用5.893克碱混合0.107克的Sylgard 184固化剂。总混合物的质量是6克与碱固化55剂:1的比例。 </li><li>用玻璃棒混合以确保均匀后，将玻璃小瓶在真空室中脱气以除去所有的气泡。 </li><li>放置在旋涂机的中心的玻璃盖玻片（55毫米×25毫米）。添加〜1毫升混合物，到盖玻片的中心。使用5000微升吸管到底用剪刀剪除。旋涂在2,000rpm的盖玻片60秒。 <ol><li>重复该process创建5-6基板。 </li></ol></li><li>固化在12小时的烘箱外盖单在60℃。 </li><li>用剃刀刀片部分地由牺牲衬底取出弹性体膜。使用了不可磨灭的标记，以纪念弹性体膜和暴露的玻璃的上部。 </li><li>安装在倒置显微镜的样品，并使用远程焦点附件以记录在两个标记的焦平面之间在z高度差。这对应于弹性体衬底厚度h。 </li></ol> 2.角质层的制备<ol><li>使用水浴或加热搅拌板上以加热玻璃烧杯中半充满去离子水（DW）至60℃。内的生物安全柜，浸泡在水中的充分厚度人类皮肤4分钟。 </li><li>紧接皮肤样品转移到含有烧杯DW冷却至&lt;10℃4分钟。半填充烧杯减少生物危险品溅。 </li><li>从烧杯中取出，放置在培养皿中的皮肤上，并轻轻地隔离利用一对弯曲鼻组织镊子的表皮。 </li><li>放置的分离的表皮基底侧向下在培养皿中，用纱布衬里。确保基底层完全与纱布接触。 <ol><li>浸泡的纱布在0.25％（重量/体积）型溶解于0.1M磷酸盐缓冲的盐水，在室温下6-8ħIX-S猪胰腺的胰蛋白酶溶液。添加只够胰蛋白酶在容器湿纱布。 </li></ol></li><li>提起与组织镊子纱布浮起来部分装有DW的容器。轻轻拉动SC把它从纱布中分离出来。 </li><li>洗净角质层3-4倍DW以去除仍然连接到SC残留的表皮组织。 </li><li>浮法孤立SC在0.4％，大豆（黄豆）的胰蛋白酶抑制剂的DW解决方案。用板振荡器来搅拌组织10分钟。 </li><li>在培养皿中漂浮的SC部分充满了DW。用板振荡器来搅拌组织10分钟。 </li><li>干上的超细塑料网的分离的SC纸，在室温下48小时（25℃，40％相对湿度（RH））。 </li><li>从网格分开SC和切出用圆形打孔个人循环 - [R = 3毫米半径的样品。马克最外层的脸用了不可磨灭的标记的小螺旋痕迹的中心。这提供了用于识别SC的顶面视觉线索。 注:不可磨灭标记应在样本，其中，在干燥的变形将是最小的中心施加。这将最小化标记上记录，干燥位移轮廓的影响。 </li></ol> 3.样品处理和沉积<ol><li>搅动SC样品在含有90微升荧光标记珠15毫升DW 30分钟（515分之505纳米，直径为1微米，羧酸改性的）。这种存款珠到SC面 注意:当升沉积的在SC珠粒ARGE号码可以相对于样品略微慢干而不珠粒本12，将最大化可随后获得的面内变形场的空间分辨率。因此，珠体积增加的选择应作出临时性的 。 </li><li>除去SC样品并将它们放置在培养皿中部分地填充有DW传递。 </li><li>在15-30°的小角度部分浸泡在DW的衬底。 </li><li>在基板和水界面之间的接触线脚浮动SC样品的边缘。垂直从水中取出衬底将顺利层压SC样品到衬底没有皱褶或夹带的空气气泡。 </li><li>重复步骤3.4至放置最多6个SC样品到每个衬底。离开至少样本之间2-3毫米间隙，避免靠近衬底边缘样品层压。这可以防止的一个样本影响干燥位移干燥另一个。 </l我&gt; <li>干燥在实验室条件下安装的SC样品60分钟。这允许SC和基板之间残留的水蒸发，并确保完整组织粘连。 注:在这一时刻，SC样品可以用化学或化妆品配方7,8通过放置在基片倒置在一段必要的时间的所需溶液处理。重复步骤3.6一旦处理步骤进行。干燥后，SC样品到衬底的不完全粘附可使用透射光显微术来验证。在SC样品或脱层边缘被困气泡会形成良好定义的边缘样品中鲜明对比的变化。 </li><li>通过将培养皿部分地填充有水进入密封容器中创建一个湿度室中。 </li><li>地方基板进入室24小时平衡至99％的相对湿度。不要将基板到切赫我的菜。 </li></ol> 4.显微镜环境控制<ol><li>实现通过连接到显微镜安装灌注室中的湿度控制系统的环境条件的控制。在德语等设置在湿度调节装置的细节。 （2013年）7和刘和德国（2015年）8。 </li><li>安装在基板上的显微镜，将灌注腔的衬底上，并使用真空润滑脂密封灌注腔的边缘与弹性体。 </li><li>一旦安装之前，实验平衡内部空气到99％RH。这可以防止水分的蒸发前的实验。一旦在第5或7成像开始，减少内部的空气湿度期望值。 注:在本文中，SC样品干燥至25％RH </li></ol> 5.成像平面位移干燥<ol><li>获得使用倒置microsco SC样本图像用1X物镜PE。利用激发光引擎与FITC滤光片（503-530 nm发射带通）荧光珠。多个样品可以按顺序在整个烘干使用自动化的xy阶段进行成像。 </li><li>记录荧光灯和使用数码相机的CCD在1392点¯x1,040像素的分辨率透射光图像。的视图中的每个图像的场为8.98点¯x6.71毫米，允许单个图像来捕获完整的SC样品。取的图像为10分钟16小时的频率。 </li></ol> 6.基板制备厚度测量<ol><li>在化学通风橱中，放置1毫升硅烷（3-氨基丙基;≥98％）在一些小的塑料盖。放置弹性体基板从部分1和在密封的容器盖5小时。不允许基材以在与硅烷直接接触来。 </li><li>在1.5mL管中;添加5毫克的EDC（≥99％的N -ethylcarbodiimide盐酸- N - （3-二甲基氨基丙基））。添加500微升DW到EDC。搅拌用涡旋混合器10秒的溶液。 </li><li>添加0.076克四硼酸钠和0.1克硼酸，以20毫升DW传递。混合使用磁力搅拌器在70℃（1小时）。添加硼酸，直至pH为7.4。 </li><li>添加20mL的硼酸缓冲液至50毫升离心管中。加1微米珠（五百七十五分之五百三十五纳米，羧化物修饰）60微升到硼酸盐缓冲液。最后，加入200μl的EDC解决方案瓶。摇管珠混合溶液中，然后倒入一个直径10厘米的培养皿。 </li><li>从容器中取出硅烷化衬底并将它们弹性体膜侧向下进入珠溶液。这样做慢慢地防止气泡被困。两个基板将适合在每个培养皿。 </li><li>离开基板在胎圈溶液漂浮45分钟。 </li><li>使用镊子从珠溶液中除去基材，再在DW冲洗，除去未结合的珠子。 </li><li>空气干燥该衬底。吹入压缩空气过弹性体膜表面减少水斑的形成。 </li><li>密封基板在一个不透明的盒子，以防止珠粒光致漂白，直到SC样品沉积。 </li></ol> 7. SC的成像厚度<ol><li>关于使用部分3.但是在基板存款SC样本，执行步骤3.1无添加荧光珠到DW。此外，在完成步骤3.6之前申请未稀释的荧光标记物珠溶液（五百十五分之五百零五纳米，直径为0.1μm），以用移液管每次敷SC样品的表面的5微升下降。 </li><li>建立使用具有40X物镜的显微镜的SC厚度的测量。使用远程焦点附件以记录在位于所述SC-衬底界面两个胎圈层焦平面和SC的顶侧之间z高度差测量SC样品随时间的厚度。 </li><li>测量每个SC样品的3个地区的超过3小时的干燥期的厚度。厚度SC样品到达这个时间框架内8稳态值。 </li></ol> 8.量化和建模组织变形<ol><li>使用粒子图像测速13在每个记录的时间步骤，以获得从所述荧光图像空间分辨的面内干燥位移。 </li><li>使用MATLAB从每个径向对称SC样品的位移场获得方位平均径向和方位角位移曲线。 注:示例数据集（题为"d.mat&#39;），并且同时执行这一步和步8.3 MATLAB代码（题为"PIV-processing.m"）已经在补充信息中提供。 </li><li>适合径向位移轮廓到模型7，8，14，15，16，描述烘干SC作为收缩线状弹性circula变化的厚度，H SC，半径R，和弹性模量，E SC，附着于弹性模量的可变形的弹性基底，大肠杆菌假定的SC的时间R盘具有明确定义和恒定泊松比，νSC = 0.4 7 8。获得使用最小最小二乘法最适合。 注:用于拟合该模型描述了在修正贝塞尔函数方面的径向位移是: <img alt="方程" src="/files/ftp_upload/55336/55336eq2.jpg" /> （1） 同<img alt="方程" src="/files/ftp_upload/55336/55336eq3.jpg" /> ， <img alt="方程" src="/files/ftp_upload/55336/55336eq4.jpg" />和<img alt="方程" src="/files/ftp_upload/55336/55336eq13.jpg" /> 期限<img alt="方程" src="/files/ftp_upload/55336/55336eq6.jpg" />对应于由下式给出一个穿透深度， <img alt="公式"SRC ="/文件/ ftp_upload / 55336 / 55336eq7.jpg"/&gt;。 <img alt="方程" src="/files/ftp_upload/55336/55336eq8.jpg" />表示基底刚性参数;时有效的样本大小是比基板厚度大得多。这里，参数<img alt="方程" src="/files/ftp_upload/55336/55336eq9.jpg" />和<img alt="方程" src="/files/ftp_upload/55336/55336eq10.jpg" />分别表示基板厚度泊松比。有机硅弹性体基片17的泊松比是ν= 0.5。 </li><li>获得模型参数α和β在从向径向位移分布拟合等式（1）的最小二乘每个时间步。 <ol><li>聘请拟合参数β获得SC弹性模量E SC，使用表达式， <img alt="方程" src="/files/ftp_upload/55336/55336eq11.jpg" /></li><li>使用拟合参数α，以获得随时间变化的收缩的干燥STRESS，PSC，使用表达式， <img alt="方程" src="/files/ftp_upload/55336/55336eq12.jpg" /></li></ol></li></ol>

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The authors have nothing to disclose.

在本文中，我们描述了可以用来测量动态干燥行为和人SC的机械性能的技术。以前的研究已经表明，这种技术可用于量化的化妆品清洁剂和保湿剂常用于SC 7,8的动态干燥行为的环境条件和化学品的影响。有许多在该协议的关键步骤。首先，SC含水量明显膨胀;因此，SC厚度的面内位移以及测量准确预测的弹性模量和干燥应力大小是至关重要的。其次，样品?...

最外层的表皮，或角质层（SC）包括由富含脂质的基质1,2包围凝聚力角质细胞的细胞。 SC的组合物和结构的完整性对于维持正确屏障功能3，这将阻止微生物侵入和抗蚀剂既机械力和4损失过多的水是必不可少的。个人护理产品，以维持或降低皮肤屏障功能的能力是极大的兴趣，皮肤保健和化妆品行业5。的个人护理产品的日常应用是已知的，以改变SC 6，7，8的机械性能。例如，包含在化妆品清洁剂的表面活性剂可以导致弹性模量显著增加和积聚干燥应力SC，增加了组织的倾向破解7,9。包含在几乎所有的化妆品的保湿剂的甘油可以软化SC和减少干燥应力8，10，11，减少组织破裂的可能性的积聚。 在这篇文章中详细描述的方法是能够量化动态干燥行为和在控制的环境7个 SC干燥机械性能，8。以前，这种技术已被证明能够阐明关于在动态干燥行为变化和SC组织的力学性能的不同的化妆品产品的效果的。这是通过量化粘附到一个软的弹性体基板的人类SC组织的干燥引起的收缩来实现，用一个简单的嵌合干燥位移收缩模型，然后提取的弹性模量和从拟合轮廓干燥应力。当需要多个SC样品的测试，此方法提供了更快速的替代单轴tensometry，利用显著较少的组织并通过防止蒸发从样品底面提供了更多的生理学相关的干燥。

<table><tbody><tr><td>Silicone elastomer base</td><td>Dow-Corning</td><td>1064291</td><td></td></tr><tr><td>Silicone elastomer Curing Agent</td><td>Dow-Corning</td><td>1015311</td><td></td></tr><tr><td>FluoSpheres Carboxylate 0.1 &amp;micro;m yellow green fluorescent 505/515&amp;nbsp;</td><td>Thermo Fisher</td><td>F8803</td><td></td></tr><tr><td>FluoSpheres Carboxylate 1 &amp;micro;m yellow green fluorescent 505/515&amp;nbsp;</td><td>Thermo Fisher</td><td>F8823</td><td></td></tr><tr><td>FluoSpheres Carboxylate 1 &amp;micro;m nile red fluorescent 535/575&amp;nbsp;</td><td>Thermo Fisher</td><td>F8819</td><td></td></tr><tr><td>Trypsin from porcine pancreas</td><td>Sigma-Aldrich</td><td>T6567</td><td></td></tr><tr><td>Trypsin inhibitor type II-s</td><td>Sigma-Aldrich</td><td>T9128</td><td></td></tr><tr><td>(3-aminopropyl)triethoxysilane</td><td>Sigma-Aldrich</td><td>440140</td><td></td></tr><tr><td>Sodium tetraborate</td><td>Sigma-Aldrich</td><td>221732</td><td></td></tr><tr><td>Boric acid</td><td>Sigma-Aldrich</td><td>B0294</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma-Aldrich</td><td>P7059</td><td></td></tr><tr><td>&lt;em&gt;N&lt;/em&gt;-(3-Dimethylaminopropyl)-&lt;em&gt;N&lt;/em&gt;-ethylcarbodiimide hydrochloride</td><td>Sigma-Aldrich</td><td>E7750</td><td></td></tr><tr><td>Vortexer mixer</td><td>VWR</td><td>58816-123</td><td></td></tr><tr><td>6 mm diameter hole punch</td><td>Sigma-Aldrich</td><td>Z708860</td><td></td></tr><tr><td>SOLA 6-LCR-SB&amp;nbsp;</td><td>Lummencor light engine</td><td>No.3526</td><td></td></tr><tr><td>Cfi Plan Achro Uw 1X Objective</td><td>Nikon Plan UW</td><td>MRL00012</td><td></td></tr><tr><td>CFI Plan Fluor 40X Oil Objective 1.3 na - 0.20&amp;nbsp;mm wd</td><td>Nikon Plan Fluor</td><td>MRH01401</td><td></td></tr><tr><td>Nikon Eclipse Ti-U inverted microscope&amp;nbsp;</td><td>Nikon</td><td>MEA53200</td><td></td></tr><tr><td>Clara-E Camera</td><td>Andor</td><td>DR-328G-C02-SIL</td><td></td></tr><tr><td>Remote Focus Attachment E-RFA Ergo Design</td><td>Nikon</td><td>99888</td><td></td></tr><tr><td>Ti-S-E Motorized Stage</td><td>Nikon</td><td>MEC56110</td><td></td></tr></tbody></table>

measuring and modeling contractile drying in human stratum corneum

角质层（SC）是最表面的表层。其与外部环境接触装置，该组织层进行既清洁剂，并在环境水分日变化;这两者都可以改变组织中的水含量。在严重的屏障功能障碍或低湿度的环境中的水含量减少可以改变SC刚度和引起干燥应力的积聚。在极端情况下，这些因素可能会导致组织的机械破裂。我们已经建立了量化的SC的干燥时的力学性能动态变化的高通量方法。可以采用这种技术来量化在干燥行为和改变与化妆品的清洁剂和保湿处理的SC的机械性能。这是通过测量在粘附到弹性体基体的圆形的组织样本的空间分辨的面内干燥位移动态变化来实现的。在平面径向位移ACQ干燥期间uired被方位角平均，并装配有基于线性弹性收缩模型的轮廓。在干燥应力和SC弹性模量的动态变化可以被从拟合模型轮廓萃取。

图1（a）示出了涂覆有荧光珠（第3节）的SC样品的代表性荧光图像。样品的相应的透射光图像在图1（b）覆盖有后在25％RH下16小时干燥形成空间分辨干燥位移的颤动情节由于样品的圆形对称性所示，这些位移可方位角平均。 图1（c）所示的径向（U R，红色实线）和方位角（Uθ，蓝色虚线）位移曲线绘制了无量纲径向位?...

Watch this Scientific Journal Video about Measuring and Modeling Contractile Drying in Human Stratum Corneum at JoVE.com

Measuring and Modeling Contractile Drying in Human Stratum Corneum

本文介绍定量通过测量空间分辨粘附到弹性体基体的圆形组织样品的面内干燥位移动态干燥行为和角质层的机械性能的方法。这种技术可以用于测量化学处理如何不同改变干燥和组织的机械性能。

测量与建模干燥收缩人类角质层

Stratum corneum (SC) is the most superficial skin layer. Its contact with the external environment means that this tissue layer is subjected to both ...

measuring-and-modeling-contractile-drying-in-human-stratum-corneum

Research

JoVE Journal

Bioengineering

6.6K 次观看.  Binghamton University. 本文介绍定量通过测量空间分辨粘附到弹性体基体的圆形组织样品的面内干燥位移动态干燥行为和角质层的机械性能的方法。这种技术可以用于测量化学处理如何不同改变干燥和组织的机械性能。 

视频: Measuring and Modeling Contractile Drying in Human Stratum Corneum

Resolving Water, Proteins, and Lipids from In Vivo Confocal Raman Spectra of Stratum Corneum through a Chemometric Approach

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in human subjects. This information is very important for skin-related diseases and characterizing skin care product performance. This protocol illustrates a method for confocal Raman spectra collection and the subsequent analysis of the spectral dataset leveraging chemometrics. The goal of this method is to establish a standard protocol for data collection and provide general guidance for data analysis. Preprocessing (e.g., removal of outlier spectra) is a critical step when processing large datasets from clinical studies. As an example, we provide guidance based on prior knowledge of a dataset to identify the types of outliers and develop specific strategies to remove them. A principal component analysis is performed, and the loading spectra are compared with spectra from reference materials to select the number of components used in the final multivariate curve resolution (MCR) analysis. This approach is successful for extracting meaningful information from a large spectral dataset.

Here, we present a protocol for collection of confocal Raman spectra from human subjects in clinical studies combined with chemometric approaches for spectral outlier removal and the subsequent extraction of key features.

通过化学测量方法，从活体共聚焦拉曼光谱中解决水、蛋白质和脂质

Development of this in vivo confocal Raman spectroscopic method enables the direct measurement of water, proteins, and lipids with depth resolution in ...

科研

化学

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

提高2D和3D皮肤<em&gt;体外</em&gt;模型使用大分子拥挤

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

生物工程

Methods for the Self-integration of Megamolecular Biopolymers on the Drying Air-LC Interface

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and macro-structures, as seen in the cases of moving water in vascular bundles and moisturizing water in skin layers. In this study, we developed a method for assessing the effect of aqueous liquid crystalline (LC) solutions composed of biopolymers on drying. As LC biopolymers have megamolecular weight, we chose to study polysaccharides, cytoskeletal proteins, and DNA. The observation of biopolymer solutions during drying under polarized light reveals milliscale self-integration starting from the unstable air-LC interface. The dynamics of the aqueous LC biopolymer solutions can be monitored by evaporating water from a one-side-open cell. By analyzing the images taken using cross-polarized light, it is possible to recognize the spatio-temporal changes in the orientational order parameter. This method can be useful for the characterization of not only artificial materials in various fields, but also natural living tissues. We believe that it will provide an evaluation method for soft materials in the biomedical and environmental fields.

A method for the drying-induced self-integration of megamolecular biopolymers on the air-liquid crystalline interface is provided here. This methodology will be useful not only for understanding the macroscopic potentials of biopolymers, but also as an evaluation method for soft materials in biomedical and environmental fields.

对干燥空气-LC接口Megamolecular生物大分子的自我整合方法

Living organisms that use water are always prone to drying in the environment. Their activities are driven by biopolymer-based micro- and ...

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

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

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

大规模培育三维重建人类表皮

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

Evaluation of Cardiac Contractility Modulation Therapy in 2D Human Stem Cell-Derived Cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used in regulatory submissions. Here, we extend their use to cardiac medical device safety or performance assessments. We developed a novel method to evaluate cardiac medical device contractile properties in robustly contracting 2D hiPSC-CMs monolayers plated on a flexible extracellular matrix (ECM)-based hydrogel substrate. This tool enables the quantification of the effects of cardiac electrophysiology device signals on human cardiac function (e.g., contractile properties) with standard laboratory equipment. The 2D hiPSC-CM monolayers were cultured for 2-4 days on a flexible hydrogel substrate in a 48-well format.
The hiPSC-CMs were exposed to standard cardiac contractility modulation (CCM) medical device electrical signals and compared to control (i.e., pacing only) hiPSC-CMs. The baseline contractile properties of the 2D hiPSC-CMs were quantified by video-based detection analysis based on pixel displacement. The CCM-stimulated 2D hiPSC-CMs plated on the flexible hydrogel substrate displayed significantly enhanced contractile properties relative to baseline (i.e., before CCM stimulation), including an increased peak contraction amplitude and accelerated contraction and relaxation kinetics. Furthermore, the utilization of the flexible hydrogel substrate enables the multiplexing of the video-based cardiac-excitation contraction coupling readouts (i.e., electrophysiology, calcium handling, and contraction) in healthy and diseased hiPSC-CMs. The accurate detection and quantification of the effects of cardiac electrophysiological signals on human cardiac contraction is vital for cardiac medical device development, optimization, and de-risking. This method enables the robust visualization and quantification of the contractile properties of the cardiac syncytium, which should be valuable for nonclinical cardiac medical device safety or effectiveness testing. This paper describes, in detail, the methodology to generate 2D hiPSC-CM hydrogel substrate monolayers.

Here, we demonstrate a non-invasive cardiac medical device contractility evaluation method using 2D human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) monolayers, plated on a flexible substrate, coupled with video-based microscopy. This tool will be useful for the in vitro evaluation of the contractile properties of cardiac electrophysiology devices.

2D人干细胞来源心肌细胞中心脏收缩力调节疗法的评估

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) are currently being explored for multiple in vitro applications and have been used ...

<meta charset="utf8"></head><body>高效的 3D 皮肤模型，用于灵活的研究应用

Building Up Skin Models for Numerous Applications - from Two-Dimensional (2D) Monoculture to Three-Dimensional (3D) Multiculture

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical industries. In the European Union, there has been a total ban on testing cosmetic products and their ingredients on animals. In the case of medicine and pharmaceuticals, this possibility is also constantly limited. In accordance with the 3Rs principle, it is becoming more and more common to test individual compounds as well as entire formulations on artificially created models. The cheapest and most widely used are the 2D models, which consist of a cell monolayer but do not reflect the real interactions between the cells in the tissue. Although the commercially available 3D models provide a better representation of the tissue, they are not used on a large scale. This is because they are expensive, the waiting time is quite long, and the available models are frequently limited to only those typically used. 
In order to move the conducted research to a higher level, we have optimized the procedures of various 3D skin model preparations. The described procedures are cheap and simple to prepare as they can be applied in numerous laboratories and by researchers with different experiences in cell culture.

<meta charset="utf8"></head><body>作者聚焦：使用经济高效的 3D 细胞模型增强皮肤模型的多样性

Here, we present inexpensive and simple procedures to introduce various 3D skin models for routine research in a cell culture laboratory. Researchers can create models tailored to their needs without relying on commercially available models.

为众多应用构建皮肤模型 - 从二维 （2D） 单一培养到三维 （3D） 多培养

Due to the complex structure and important functions of the skin, it is an interesting research model for the cosmetic, pharmaceutical, and medical ...

Effect of Artificial Tear Formulations on the Metabolic Activity of Human Corneal Epithelial Cells after Exposure to Desiccation

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear formulations that prevent cell desiccation will result in ocular surface protection and the maintenance of cell metabolic activity. During dehydration, cells undergo the process of loss of metabolic activity and subsequently cell death. This work describes a method for assessing the efficacy of artificial tear formulations. The metabolic dye (i.e., alamarBlue) changes from a low fluorescent molecule resazurin to a fluorescent molecule resorufin in viable cells. The biological performance of an artificial tear formulation is measured as the ability of the formulation to (a) maintain cell viability and (b) provide cell protection from desiccation. Growth media and saline are used as controls for the cell viability/desiccation tests. Cells are incubated with test solutions for 30 min and then desiccated for 0 or 5 min at 37 &#176;C and 45% relative humidity. Cell metabolic activity after initial exposure and after cell desiccation is then determined. The results show the comparative effects of eye drop formulations on cell metabolic activity and desiccation protection. This method can be used to test dry eye formulations that are designed to treat individuals with evaporative dry eye.

The purpose of this protocol is to evaluate if different artificial tear formulations can protect human corneal epithelial cells from desiccation using an in vitro model. After exposure to artificial tear formulations and desiccation, human corneal epithelial cells are assessed for metabolic activity.

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

Artificial lipid-containing tear formulations are developed to reduce tear evaporation by the restoration of a deficient tear lipid layer. Artificial tear ...

医学

Isolation, Culture, and Characterization of Primary Schwann Cells, Keratinocytes, and Fibroblasts from Human Foreskin

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid enzymatic dissociation of skin. Primary keratinocytes, fibroblasts, and Schwann cells are all harvested from the human newborn foreskin, which is available following standard of care procedures. The removed skin is disinfected, and the subcutaneous fat and muscle are removed using a scalpel. The method consists of enzymatic and mechanical separation of epidermal and dermal layers, followed by additional enzymatic digestion to obtain single-cell suspensions from each of these skin layers. Finally, single cells are grown in appropriate cell culture media following standard cell culture protocols to maintain growth and viability over weeks. Together, this simple protocol allows isolation, culturing, and characterization of all three cell types from a single piece of skin for in vitro evaluation of skin-nerve models. Additionally, these cells can be used together in co-cultures to gauge their effects on each other and their responses to in vitro trauma in the form of scratches performed robotically in the culture associated with wound healing.

The study of wound healing associated with musculoskeletal injury often requires the assessment of in vitro interactions between Schwann cells (SCs), keratinocytes, and fibroblasts. This protocol describes the isolation, culturing, and characterization of these primary cells from the human foreskin.

从人包皮中分离、培养和表征原代施旺细胞、角质形成细胞和成纤维细胞

This protocol describes isolation methods, culturing conditions, and characterization of human primary cells with high yield and viability using rapid ...

测量与建模干燥收缩人类角质层

摘要

探索更多视频

此视频中的章节

通过化学测量方法，从活体共聚焦拉曼光谱中解决水、蛋白质和脂质

提高2D和3D皮肤

对干燥空气-LC接口Megamolecular生物大分子的自我整合方法

大规模培育三维重建人类表皮

2D人干细胞来源心肌细胞中心脏收缩力调节疗法的评估

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

从人包皮中分离、培养和表征原代施旺细胞、角质形成细胞和成纤维细胞

测量与建模干燥收缩人类角质层

摘要

探索更多视频

此视频中的章节

通过化学测量方法，从活体共聚焦拉曼光谱中解决水、蛋白质和脂质

提高2D和3D皮肤

对干燥空气-LC接口Megamolecular生物大分子的自我整合方法

大规模培育三维重建人类表皮

2D人干细胞来源心肌细胞中心脏收缩力调节疗法的评估

为众多应用构建皮肤模型 - 从二维 （2D） 单一培养到三维 （3D） 多培养

为众多应用构建皮肤模型 - 从二维 （2D） 单一培养到三维 （3D） 多培养

为众多应用构建皮肤模型 - 从二维 （2D） 单一培养到三维 （3D） 多培养

人工撕裂制剂对暴露于干燥后人类角膜上皮细胞代谢活动的影响

从人包皮中分离、培养和表征原代施旺细胞、角质形成细胞和成纤维细胞

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养

为众多应用构建皮肤模型 - 从二维（2D）单一培养到三维（3D）多培养