The authors have no acknowledgements.

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，样品需要充分粘附到所述衬底。不完全的粘附，非径向对称的样品或具有小撕裂或孔的样品应该避免，因为它们会显著影响干燥变形的分布和径向位移型材使用D代表模型拟合。 如果一个湿度控制系统不可用，可以使用的技术。如果没有环境控制，组织样本会干在实验室条件下12。这样，实验室环境应连续监测和维护，因为干燥行为和结果的重复性会由温度和湿度两昼夜和季节变化受到影响。 目前，该技术是有限的仅可粘附到基底和弹性体膜中诱发变形样品。而该技术可以很容易地适应于经受较小的面内位移，通过减小基板的弹性模量12个测试样品中，从样品的结果，仅仅在衬底上滑动将缺乏含义。 许多体内和离体技术，可以评估干燥性能和机械SC的性质已报道3，8，9，10，18，19，20，21。然而， 在体内的技术不能完全区分从底层的表皮和真皮层中的SC机械变化。此外， 离体技术通常只能评估每个实验一个样本。我们在这篇文章中报道的方法允许最多6 SC每个样本实验进行评估。衬底和环境室的尺寸但是可以扩大，以允许更多的样本，同时进行评估。我们估计当n = 6 SC样本，〜需要准备和测试13小时的时间尺度，不包括基板固化和组织平衡。相比较而言，我们估计单轴tensometry测试将需要更多的超过两次这个时期。显著也与那些tensometry 9（2.5厘米2）需要比较需要每个单独样品（0.28 cm 2）的更小的SC组织。通过防止蒸发从SC组织的下侧这种技术还使得更生理学相关的干燥。除了评估在SC干燥行为和力学，我们相信这种技术也可以适用于形成在干燥时有凝聚力的薄膜的聚合物或胶体体系的研究。

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Silicone elastomer base</td>
			<td>Dow-Corning</td>
			<td>1064291</td>
		</tr>
		<tr>
			<td>Silicone elastomer Curing Agent</td>
			<td>Dow-Corning</td>
			<td>1015311</td>
		</tr>
		<tr>
			<td>FluoSpheres Carboxylate 0.1 &#181;m yellow green fluorescent 505/515&#160;</td>
			<td>Thermo Fisher</td>
			<td>F8803</td>
		</tr>
		<tr>
			<td>FluoSpheres Carboxylate 1 &#181;m yellow green fluorescent 505/515&#160;</td>
			<td>Thermo Fisher</td>
			<td>F8823</td>
		</tr>
		<tr>
			<td>FluoSpheres Carboxylate 1 &#181;m nile red fluorescent 535/575&#160;</td>
			<td>Thermo Fisher</td>
			<td>F8819</td>
		</tr>
		<tr>
			<td>Trypsin from porcine pancreas</td>
			<td>Sigma-Aldrich</td>
			<td>T6567</td>
		</tr>
		<tr>
			<td>Trypsin inhibitor type II-s</td>
			<td>Sigma-Aldrich</td>
			<td>T9128</td>
		</tr>
		<tr>
			<td>(3-aminopropyl)triethoxysilane</td>
			<td>Sigma-Aldrich</td>
			<td>440140</td>
		</tr>
		<tr>
			<td>Sodium tetraborate</td>
			<td>Sigma-Aldrich</td>
			<td>221732</td>
		</tr>
		<tr>
			<td>Boric acid</td>
			<td>Sigma-Aldrich</td>
			<td>B0294</td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Sigma-Aldrich</td>
			<td>P7059</td>
		</tr>
		<tr>
			<td>N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>E7750</td>
		</tr>
		<tr>
			<td>Vortexer mixer</td>
			<td>VWR</td>
			<td>58816-123</td>
		</tr>
		<tr>
			<td>6mm diameter hole punch</td>
			<td>Sigma-Aldrich</td>
			<td>Z708860</td>
		</tr>
		<tr>
			<td>SOLA 6-LCR-SB&#160;</td>
			<td>Lummencor light engine</td>
			<td>No.3526</td>
		</tr>
		<tr>
			<td>Cfi Plan Achro Uw 1x Objective</td>
			<td>Nikon Plan UW</td>
			<td>MRL00012</td>
		</tr>
		<tr>
			<td>CFI Plan Fluor 40x Oil Objective 1.3 na - 0.20mm wd</td>
			<td>Nikon Plan Fluor</td>
			<td>MRH01401</td>
		</tr>
		<tr>
			<td>Nikon Eclipse Ti-U inverted microscope&#160;</td>
			<td>Nikon</td>
			<td>MEA53200</td>
		</tr>
		<tr>
			<td>Clara-E Camera</td>
			<td>Andor</td>
			<td>DR-328G-C02-SIL</td>
		</tr>
		<tr>
			<td>Remote Focus Attachment E-RFA Ergo Design</td>
			<td>Nikon</td>
			<td>99888</td>
		</tr>
		<tr>
			<td>Ti-S-E Motorized Stage</td>
			<td>Nikon</td>
			<td>MEC56110</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置，R / R。这里，R表示平均SC样品半径r / R = 0表示样品中心且r / R = 1表示的边缘。在每个径向位置标准偏差由围绕平均值的阴影区域表示。这些变型主要由SC 3的结构异质性引起的&lt;SUP&gt;，7，12。纵观干燥，方位角位移仍然很小。径向位移型材但是从中心单调地增加到边缘和在幅度增长，直到达到一个平衡。 <img alt="图1" src="/files/ftp_upload/55336/55336fig1.jpg" /> 图1:圆形SC样品（直径6.2毫米）粘附到弹性模量E = 16±1千帕的弹性体基材在25±1％RH环境下15小时干燥后。 （a）该SC样品突出用于沉积的荧光标记物珠的荧光图像跟踪空间分辨的面内干燥位移。覆盖在SC样品的透射光图像上空间分辨的面内干燥位移（b）的颤抖情节。 （ 三 ）方位平均径向（U R，红色实线）和样品的方位（Uθ，蓝色虚线）位移暗算因次径向位置，R / R。 ü正面值R对应于收缩的位移。围绕线阴影区域指示关于在每个径向位置的平均标准偏差。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55336/55336fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 记录在30分钟的间隔轮廓绘制在图2（a）和示出的面内位移的时间演变。平均SC厚度h SC，在图2中绘制（b）中 。干燥主要发生在第一2小时期间的SC减小。 <img alt="图2" src="/files/ftp_upload/55336/55336fig2.jpg" /> 图2:（ 一 ）径向位移曲线（U R，实红线）在暗算因次径向位置r / R为典型的SC样本25％RH条件下超过15小时的干燥时间在30分钟的间隔叠加。 ü正面值R对应于收缩的位移。 （ 二 ）平均SC样品的厚度，（H SC，N = 3），暗算干燥时间。 （ 三 ）选自（a）具有最小的最小二乘拟合覆盖径向位移型材（蓝色虚线）方程（1）对于第一和最后记录的径向位移轮廓。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55336/55336fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 嵌合位移与由等式（1）提供了进一步的深入了解干燥SC的机械特性描述的线性弹性收缩模型型材。排量配置文件<html> t各自时间步骤中使用最小最小二乘法拟合与模型， 如图2（c）所示 。收缩干燥应力，P SC，和弹性模量，E SC，随后从在每个时间步骤中的模型萃取。在这些参数（基于3个单独的SC样本）的平均变化在图3分别示出（a）和3（b）中 。这两个参数在第一个2小时的干燥期迅速增加和5小时内到达平台期。 <img alt="Figure3" src="/files/ftp_upload/55336/55336fig3.jpg" /> 图3:（ 一 ）平均每SC弹性模量，E SC，作图干燥时间超过15小时的时期。 （ 二 ）平均收缩干燥应力，P SC，暗算干燥时间超过15小时的时期。"_blank"&gt;请点击此处查看该图的放大版本。

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

Nanyang Technological University

Research

JoVE Journal

Bioengineering

6.5K 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

Measuring Left Ventricular Pressure in Late Embryonic and Neonatal Mice

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable around embryonic day (E) 8.51. Systolic left ventricular (LV) pressure is 2 mmHg at E9.5 and 11 mmHg at E14.52. At these mid-embryonic stages, the LV is clearly visible through the chest wall for invasive pressure measurements because the ribs and skin are not fully developed. Between E14.5 and birth (approximately E21) imaging methods must be used to view the LV. After birth, mean arterial pressure increases from 30 - 70 mmHg from postnatal day (P) 2 - 353. Beyond P20, arterial pressure can be measured with solid-state catheters (i.e. Millar or Scisense). Before P20, these catheters are too big for developing mouse arteries and arterial pressure must be measured with custom pulled plastic catheters attached to fluid-filled pressure transducers3 or glass micropipettes attached to servo null pressure transducers4.
Our recent work has shown that the greatest increase in blood pressure occurs during the late embryonic to early postnatal period in mice5-7. This large increase in blood pressure may influence smooth muscle cell (SMC) phenotype in developing arteries and trigger important mechanotransduction events. In human disease, where the mechanical properties of developing arteries are compromised by defects in extracellular matrix proteins (i.e. Marfan's Syndrome8 and Supravalvular Aortic Stenosis9) the rapid changes in blood pressure during this period may contribute to disease phenotype and severity through alterations in mechanotransduction signals. Therefore, it is important to be able to measure blood pressure changes during late embryonic and neonatal periods in mouse models of human disease.
We describe a method for measuring LV pressure in late embryonic (E18) and early postnatal (P1 - 20) mice. A needle attached to a fluid-filled pressure transducer is inserted into the LV under ultrasound guidance. Care is taken to maintain normal cardiac function during the experimental protocol, especially for the embryonic mice. Representative data are presented and limitations of the protocol are discussed.

Measuring left ventricular pressure (LV) in embryonic and neonatal mice is described. Pressure is measured by inserting a needle connected to a fluid-filled transducer into the LV under ultrasound guidance. Care must be taken to maintain normal cardiac function during the experimental protocol.

测量中晚期胚胎和新生小鼠左心室压力

Blood pressure increases significantly during embryonic and postnatal development in vertebrate animals. In the mouse, blood flow is first detectable ...

科研

生物工程

CometChip: A High-throughput 96-Well Platform for Measuring DNA Damage in Microarrayed Human Cells

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular metabolites. Ironically, at high doses DNA damaging agents are also used to treat cancer. The ability to quantify DNA damage responses is thus critical in the public health, pharmaceutical and clinical domains. Here, we describe a novel platform that exploits microfabrication techniques to pattern cells in a fixed microarray. The &#8216;CometChip&#8217; is based upon the well-established single cell gel electrophoresis assay (a.k.a. the comet assay), which estimates the level of DNA damage by evaluating the extent of DNA migration through a matrix in an electrical field. The type of damage measured by this assay includes abasic sites, crosslinks, and strand breaks. Instead of being randomly dispersed in agarose in the traditional assay, cells are captured into an agarose microwell array by gravity. The platform also expands from the size of a standard microscope slide to a 96-well format, enabling parallel processing. Here we describe the protocols of using the chip to evaluate DNA damage caused by known genotoxic agents and the cellular repair response followed after exposure. Through the integration of biological and engineering principles, this method potentiates robust and sensitive measurements of DNA damage in human cells and provides the necessary throughput for genotoxicity testing, drug development, epidemiological studies and clinical assays.

We describe here a platform that allows comet assay detection of DNA damage with unprecedented throughput. The device patterns mammalian cells into a microarray and enables parallel processing of 96 samples. The approach facilitates analysis of base level DNA damage, exposure-induced DNA damage and DNA repair kinetics.

CometChip:一种高通量的96孔平台的微阵列人类细胞测量DNA损伤

DNA damaging agents can promote aging, disease and cancer and they are ubiquitous in the environment and produced within human cells as normal cellular ...

Quantification of Global Diastolic Function by Kinematic Modeling-based Analysis of Transmitral Flow via the Parametrized Diastolic Filling Formalism

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised the only means available, the development of noninvasive imaging modalities (echocardiography, MRI, CT) having high temporal and spatial resolution provide a new window for quantitative diastolic function assessment. Echocardiography is the agreed upon standard for diastolic function assessment, but indexes in current clinical use merely utilize selected features of chamber dimension (M-mode) or blood/tissue motion (Doppler) waveforms without incorporating the physiologic causal determinants of the motion itself.&#160;The recognition that all left ventricles (LV) initiate filling by serving as mechanical suction pumps allows global diastolic function to be assessed based on laws of motion that apply to all chambers. What differentiates one heart from another are the parameters of the equation of motion that governs filling. Accordingly, development of the Parametrized Diastolic Filling (PDF) formalism&#160;has shown that the entire range of clinically observed early transmitral flow (Doppler E-wave) patterns are extremely well fit by the laws of damped oscillatory motion.&#160;This permits analysis of individual E-waves in accordance with a causal mechanism (recoil-initiated suction) that yields three (numerically) unique lumped parameters whose physiologic analogues are chamber stiffness (k), viscoelasticity/relaxation (c), and load (xo).&#160;The recording of transmitral flow (Doppler E-waves) is standard practice in clinical cardiology and, therefore, the echocardiographic recording method is only briefly reviewed. Our focus is on determination of the PDF parameters from routinely recorded E-wave data.&#160;As the highlighted results indicate, once the PDF parameters have been obtained from a suitable number of load varying E-waves, the investigator is free to use the parameters or construct indexes from the parameters (such as stored energy 1/2kxo2, maximum A-V pressure gradient kxo, load independent index of diastolic function, etc.) and select the aspect of physiology or pathophysiology to be quantified.

Accurate, causality-based quantification of global diastolic function has been achieved by kinematic modeling-based analysis of transmitral flow via the Parametrized Diastolic Filling (PDF) formalism. PDF generates unique stiffness, relaxation,&#160;and load parameters and elucidates &#39;new&#39; physiology while providing sensitive and specific indexes of dysfunction.

全球舒张功能通过静脉流的通过参数化舒张形式主义运动学建模为基础的定量分析

Quantitative cardiac function assessment remains a challenge for physiologists and clinicians.&#160;Although historically invasive methods have comprised ...

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which facilitate the functional assessment of the seeded cells. To that end, microscale cantilever technology offers a platform with which to measure the contractile functionality of a range of cell types, including skeletal, cardiac, and smooth muscle cells, through assessment of contraction induced substrate bending. Application of multiplexed cantilever arrays provides the means to develop moderate to high-throughput protocols for assessing drug efficacy and toxicity, disease phenotype and progression, as well as neuromuscular and other cell-cell interactions. This manuscript provides the details for fabricating reliable cantilever arrays for this purpose, and the methods required to successfully culture cells on these surfaces. Further description is provided on the steps necessary to perform functional analysis of contractile cell types maintained on such arrays using a novel laser and photo-detector system. The representative data provided highlights the precision and reproducible nature of the analysis of contractile function possible using this system, as well as the wide range of studies to which such technology can be applied. Successful widespread adoption of this system could provide investigators with the means to perform rapid, low cost functional studies in vitro, leading to more accurate predictions of tissue performance, disease development and response to novel therapeutic treatment.

Utilization of Microscale Silicon Cantilevers to Assess Cellular Contractile Function In Vitro

This protocol describes the use of microscale silicon cantilevers as pliable culture surfaces for measuring the contractility of muscle cells in vitro. Cellular contraction causes cantilever bending, which can be measured, recorded, and converted into readouts of force, providing a non-invasive and scalable system for measuring contractile function in vitro.

微型硅悬臂梁的利用率，评估细胞收缩功能<em&gt;在体外</em

The development of more predictive and biologically relevant in vitro assays is predicated on the advancement of versatile cell culture systems which ...

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

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

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

三维组织工程人食管黏膜型号生产，表征和潜在用途

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

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 ...

Microhoneycomb Monoliths Prepared by the Unidirectional Freeze-drying of Cellulose Nanofiber Based Sols: Method and Extensions

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb monoliths (MHMs) with micrometer-scale channels are expected as efficient platforms for reactions and separations because of their large surface areas. Up to now, MHMs have been prepared by a unidirectional freeze-drying (UDF) method only from very limited precursors. Herein, we report a protocol from which a series of MHMs consisting of different components can be obtained. Recently, we found that cellulose nanofibers function as a distinct structure-directing agent towards the formation of MHMs through the UDF process. By mixing the cellulose nanofibers with water soluble substances which do not yield MHMs, a variety of composite MHMs can be prepared. This significantly enriches the chemical constitution of MHMs towards versatile applications.

Here, we present a general protocol to prepare a variety of microhoneycomb monoliths (MHMs) in which fluid can pass through with an extremely low pressure drop. MHMs obtained are expected to be used as filters, catalyst supports, flow-type electrodes, sensors and scaffolds for biomaterials.

纤维素碳纤维溶胶单向冷冻干燥制备 Microhoneycomb 石柱: 方法和外延

Monolithic honeycomb structures have been attractive to multidisciplinary fields due to their high strength-to-weight ratio. Particularly, microhoneycomb ...

Measuring Global Cellular Matrix Metalloproteinase and Metabolic Activity in 3D Hydrogels

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) culture systems. However, measurement of cell function in 3D culture is often more challenging. Many biological assays require retrieval of cellular material which can be difficult in 3D cultures. One way to address this challenge is to develop new materials that enable measurement of cell function within the material. Here, a method is presented for measurement of cellular matrix metalloproteinase (MMP) activity in 3D hydrogels in a 96-well format. In this system, a poly(ethylene glycol) (PEG) hydrogel is functionalized with a fluorogenic MMP cleavable sensor. Cellular MMP activity is proportional to fluorescence intensity and can be measured with a standard microplate reader. Miniaturization of this assay to a 96-well format reduced the time required for experimental set up by 50% and reagent usage by 80% per condition as compared to the previous 24-well version of the assay. This assay is also compatible with other measurements of cellular function. For example, a metabolic activity assay is demonstrated here, which can be conducted simultaneously with MMP activity measurements within the same hydrogel. The assay is demonstrated with human melanoma cells encapsulated across a range of cell seeding densities to determine the appropriate encapsulation density for the working range of the assay. After 24 h of cell encapsulation, MMP and metabolic activity readouts were proportional to cell seeding density. While the assay is demonstrated here with one fluorogenic degradable substrate, the assay and methodology could be adapted for a wide variety of hydrogel systems and other fluorescent sensors. Such an assay provides a practical, efficient and easily accessible 3D culturing platform for a wide variety of applications.

Here, a protocol is presented for encapsulating and culturing cells in poly(ethylene glycol) (PEG) hydrogels functionalized with a fluorogenic matrix metalloproteinase (MMP)-degradable peptide. Cellular MMP and metabolic activity are measured directly from the hydrogel cultures using a standard microplate reader.

三维水凝胶中全球细胞基质金属蛋白酶和代谢活性的测定

Three-dimensional (3D) cell culture systems often more closely recapitulate in vivo cellular responses and functions than traditional two-dimensional (2D) ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

用于刺激生物组织的电动和磁场设备

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Creation of Patient-Specific Silicone Cardiac Models with Applications in Pre-surgical Plans and Hands-on Training

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In particular, 3D models can play an important role in the field of cardiology, where complex congenital heart diseases occur. While many 3D printers can provide anatomically correct and detailed models, existing 3D printing materials fail to replicate myocardial tissue properties and can be extremely costly. This protocol aims to develop a process for the creation of patient-specific models of complex congenital heart defects using a low-cost silicone that more closely matches cardiac muscle properties. With improved model fidelity, actual surgical procedural training could occur in advance of the procedure. Successful creation of cardiac models begins with the segmentation of radiologic images to generate a virtual blood pool (blood that fills the chambers of the heart) and myocardial tissue mold. The blood pool and myocardial mold are 3D printed in acrylonitrile butadiene styrene (ABS), a plastic dissolvable in acetone. The mold is assembled around the blood pool, creating a negative space simulating the myocardium. Silicone with a shore hardness of 2A is poured into the negative space and allowed to cure. The myocardial mold is removed, and the remaining silicone/blood pool model is submerged in acetone. The described process results in a physical model in which all cardiac features, including intra-cardiac defects, are represented with more realistic tissue properties and are more closely approximated than a direct 3D printing approach. The successful surgical correction of a model with a ventricular septal defect (VSD) using a GORE-TEX patch (standard surgical intervention for defect) demonstrates the utility of the method.

Patient-specific models improve surgeon and fellow confidence when developing or learning surgical plans. Three-dimensional (3D) printers generate adequate detail for surgical preparation, but fail to replicate tissue haptic fidelity. A protocol is presented detailing the creation of patient-specific, silicone cardiac models, combining 3D printing precision with simulated silicone tissue.

创建患者专用硅胶心脏模型，应用于术前计划和实践培训

Three dimensional&#160;models can be a valuable tool for surgeons as they develop surgical plans and medical fellows as they learn about complex cases. In ...