1，修改聚合物表面与recCD47 请注意:该协议是示意性地概括于图1图1A示出了产生的巯基反应性聚合物表面图1B示出了产生的巯基反应性recCD47。 <ol><li>第1天<ol><li>制备PDT-BzPh在无菌水中的溶液（1毫克/毫升）和碳酸氢钾（KHCO 3）（0.7毫克/毫升）。搅拌过夜，4℃（ 避光 ）。 </li></ol></li><li> 2天<ol><li>切管聚合成40厘米长的块（足够长的时间，以适应周围旋转的轮子）。 </li><li>泡管中hexacylpyridinium的90分钟在振荡器上或上钱德勒回路装置a的0.1％水溶液，在室温下进行。冲洗用无菌水3倍的管子后90分钟浸泡。 </li><li>加入15％的水磷酸二氢钾酸化PDT-BzPh从第1天的液（KH 2 </suB&gt; PO 4）。加入50微升的KH 2 PO 4每毫升PDT-BzPh，形成混浊的胶束溶液。 </li><li>浸泡在振荡器上或在装置中进行40分钟，在室温下将酸化的PDT-BzPh溶液的试管中。 </li><li>冲洗，用稀乙酸（1:1,000）的管一次。 </li><li>暴露管紫外线照射60分钟的总时间。旋转管¼转每15分钟照射的整个表面区域。 </li><li>泡管中的20毫克/毫升碳酸氢钾20分钟，在摇动器上或在设备中的溶液（KHCO 3）在室温下进行。 </li><li>冲洗在4℃下在无菌水中，用无菌水3x和存储管中。 </li></ol></li><li> 3天<ol><li>溶于5毫克磺基-SMCC在200微升二甲基甲酰胺（DMF）（ 注意:请在通风橱 ）。 </li><li>加入制备步骤1到recCD47聚赖氨酸溶液0.1毫克/毫升的磺基-SMCC溶液50微升，并搅拌在滚装60分钟OM温度。 </li><li>在60分钟的轰动，德加无菌贝科的磷酸盐缓冲液（DPBS），备用。 </li><li>净化磺基-S​​MCC反应recCD47使用每个制造商的指令7K分子量截止旋转脱盐柱。收集最后的流通（优质硫醇反应recCD47）并稀释至所需的涂布用DPBS该管的内部的容积。 </li><li>冲洗2天管无菌，脱气DPBS 3倍。 </li><li>反应管的改性表面用20毫克/毫升，三（2 - 羧乙基）膦盐酸盐（TCEP）在脱气的DPBS为小于2分钟的溶液中。冲洗用无菌脱气DPBS 4倍的管子。 </li><li>加入磺基-SMCC反应recCD47在管子和孵育过夜，在4℃在摇床上或装置。 </li></ol></li></ol> recCD47对表面改性2，免疫定量<ol><li>修改冲洗管从第3天机智量化ħDPBS 3倍。不使用存储区管量化在DPBS中在4℃下。 </li><li>使用4毫米的活检穿孔，使管式两份样品，并将活检拳在96孔板的孔中。 </li><li>制备阴性对照包括不与检测抗体处理的未修饰的管样品。制备抗体的控制由与检测抗体处理未修饰的管样品的。制备与检测抗体处理的改性管材的测试样品。 </li><li>块样品用0.4％牛血清白蛋白（BSA）的DPBS中，在室温下60分钟，在振荡器上。 </li><li>后粘连，吸BSA和冲洗用Tris缓冲盐水加1％吐温20（TBST）的3倍，在振荡器上，每次10分钟。 </li><li>按照制造商的建议免疫准备稀释抗体的稀释工作在0.4％BSA的。在0.4％的BSA 100的稀释:人CD47抗体B6H12-FITC 1。 </li><li>加入200μl抗体稀释至适当的水井。 IncubaTE阴性对照，只有0.4％的BSA。在室温下孵育为在振荡器上60分钟（ 避光 ）。 </li><li>冲洗用TBST 3次，每次在振荡器上10分钟。吸出最后的TBST漂洗，加入200微升的DPBS到管样品孔。 </li><li>收集最终流过，这是高质量的巯基反应性的重组CD47并稀释至所需的涂布用DPBS管子的内部体积。 </li><li>用酶标仪用FITC激发（485纳米）和发射（538 nm）的设置阅读FITC标记的信号强度。 </li><li>计算recCD47结合到聚合物表面的基于标准值，占自发荧光和非特异性抗体对照样品结合。 </li></ol> 3，钱德勒循环设备协议寻求机构审查委员会（IRB）批准的采血协议和知情同意手续之前收集人类血样的开始。获取信息Rmed指从人的献血者同意。 注:图描绘装置示于图2。 <ol><li>填写水浴，用蒸馏水，直到大约直径轮子的一半被淹没。添加足够的漂白剂的水浴中，使10％的漂白剂溶液。置水浴至37℃，并让温度达到平衡。 </li><li>收集金属的适配器（在图1B中示出），未修饰的油管和改性油管，以及装配周围装置的车轮， 如图1C所示 。确保管道舒服地适应周围的轮代替金属适配器。 </li><li>获得使用IRB批准协议30毫升血液样本，在预充2毫升柠檬酸或其他抗凝血剂的小瓶中，并颠倒混匀，以防止凝血，一旦样本已收集。 </li><li>加入约10毫升血液使用的金属阀与注射器的每个管中，留下一些空气在管内。固定在阀帽而rotate 3小时。 </li><li>沥血为用10％漂白粉溶液处理废烧杯（或所要求的体制政策）。 </li><li>轻轻冲洗管内用DPBS去除血液的所有痕迹。收集流过的废物烧杯。处置血液按照体制政策。 </li><li>消毒的装置和任何血液接触，用10％漂白剂溶液或根据机构政策表面。 </li><li>处理样品的荧光显微镜或扫描电子显微镜，如下所述。 </li></ol> 4，荧光显微镜，细胞计数<ol><li>准备了4％多聚甲醛（PFA）解决方案（ 注意:请在通风橱 ）。 </li><li>在4％的煤灰液管孵育过夜，4℃。 </li><li>隔夜培养后，除去4％的煤灰和冲洗膜轻轻地用DPBS。 </li><li>切管段，以暴露腔表面进行染色。 </li><li>染色管的几滴安装媒体与4&#39;，6 -二脒-2 -苯基吲哚（DAPI）进行30分钟，在室温下的部分（ 避光 ）。染色后，用水冲洗管轻轻地用DPBS降低背景DAPI信号。 </li><li>图片用荧光显微镜配备了DAPI过滤器和数码相机计数细胞在9盲目选择来看下放大200倍的场数。 </li><li>每个视场记录细胞进行计数，并执行适当的统计分析，以确定结果的统计意义。 </li></ol> 5，扫描电子显微镜<ol><li>准备2％戊二醛溶液（ 注意:请在通风橱 ）。 </li><li>孵育管的2％戊二醛溶液中过夜，4℃。 </li><li>隔夜培养后，轻轻冲洗管3X用DPBS。 </li><li>准备四氧化锇的1％溶液（ 注意:重度吸入性危害！使用通风柜）。 </li><li>在四氧化锇在室温下的1％溶液15分钟孵育管。 </li><li>轻轻冲洗管子3X用DPBS。 </li><li>制备了一系列的乙醇浓度（25％，50％，75％，95％和100％乙醇）。 </li><li>脱水管保温在系列乙醇浓度。孵育在25％，50％，75％，和95％乙醇各20分钟。孵育在100％乙醇中30分钟。 </li><li>临界点干燥与CO 2的管45分钟以除去所有的水分。 </li><li>安装管段上试桩用银浆或石墨。 </li><li>外套管段与金 - 钯12.5纳米。 </li><li>检查在扫描型电子显微镜。 </li></ol>

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研究报告本出版物中所生物医学成像与生物工程研究所在奖号R21 EB015612（SJS）和国家心脏，肺和血液研究所支持的国家中，根据奖号T32 HL007915（JBS和RJL）卫生研究所。

光活化化学（总结于图1），使得几乎任何聚合物表面具有足够的烃，以方便PDT-BzPh附着和随后的UV照射，以修改光激活的PDT-BzPh。官能聚合物与表面活性巯基可以为后续的连接各种兴趣测试的分子组成。在我们特定的研究中，我们选择了重组CD47 11-13。 ，我们使用所涉及的蛋白质的胺基团与双官能交联剂SMCC 11-13的反应的特定结合化学。这提供硫醇反应recCD47用于与巯基?...

许多临床程序，如体外循环，肾透析，需要使用聚合血导管，并经常与后并发症相关联。当与血液灌流，这些聚合物非法的异物反应（FBR），从而导致吸附血液中的蛋白和血小板，单核细胞/巨噬细胞的粘附，以及促炎细胞因子，所有这些都有助于后并发症和释放/或设备故障2,3。因此，策略来解决这一问题保持生物材料研究的重要和持续的区域。研究者已经试图通过改变血液接触的表面具有生物活性或生物惰性分子4-6来解决这个问题。研究在我们的实验室都集中在所附的重组CD47（recCD47）到聚合物的生物材料作为一种策略，以减轻FBR和提高这些材料的功效。 CD47是一种广泛表达transmembrANE蛋白与免疫逃逸时表达细胞7-10显示了一个著名的角色，授予"自我"的状态，在承诺时追加到聚合物表面11-13授予的生物相容性。信号调节蛋白α（SIRPα），同族受体CD47和免疫受体基于酪氨酸的抑制基序（ITIM）基家族的跨膜蛋白的成员，表达于骨髓来源14的细胞。我们以前表明，CD47，通过SIRPα介导的细胞信号传导，下调在体外 ， 离体 ，以聚氨酯（PU）和聚氯乙烯（PVC）的免疫反应，并且在体内模型11-13。 中央到我们的调查是一种相对较新的光活化化学，本文中所描述，其中的化学反应性硫醇基团通过管路与多官能聚合物（PDT-BZ反应共价地附于聚合管PH），2 -吡啶基二硫（PDT）组成，所述光反应性二苯甲酮（BzPh）和羧基改性聚烯丙基胺11-13。降低了共价附PDT组与三（2 -羧乙基）膦盐酸盐（TCEP）11产生一个硫醇化表面，其可接着与治疗性结构部分的反应。这里和前面详述12,13，recCD47，进一步修饰与另外的C末端多聚赖氨酸尾12,13，与磺基琥珀酰亚胺基-4- [N -maleimidomethyl]环己烷-1 -羧酸乙酯反应（磺基-SMCC） 1小时，以产生硫醇反应性基团，从而允许管道和recCD47 11之间的硫化物键的形成。的CD47官能表面的抗炎能力进行了测试， 当然VIV O，使用钱德勒回路装置与人全血，它最初是于1958年描述为血栓凝固15的体外模型。该装置依靠闭管系统部分地填充有空气和旋转马达来循环血液通过导管15。此实验模型提供了机会，检查血液接触后修饰和未修饰的表面的效果，以及这些表面修饰后，血液中的细胞的生理学效果。 recCD47可以被附加到各种使用这种光活化化学聚合的表面，并且它的抗炎能力可以通过利用临床相关的体外模型模仿血液灌注过的聚合物表面11,12进行评估。改性recCD47临床级血液导管显示显著少血小板和炎性细胞附着比未改性的聚合物时，在该装置暴露于人血。这个修改​​过程中的一步一步的说明详述如下。

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:107px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">16% Paraformaldehyde (PFA)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">58906</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">25% Glutaraldehyde</td>
			<td style="width:175px;">VWR</td>
			<td style="width:107px;">AAA17876-AP&#160;</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">2-pyridyldithio,benzophenone (PDT-BzPH)</td>
			<td style="width:175px;">Synthesized in lab</td>
			<td style="width:107px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">A3059-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Citrate</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">S5770-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Digital Camera</td>
			<td style="width:175px;">Leica</td>
			<td style="width:107px;">DC500</td>
			<td>Out of production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dimethylformamide (DMF)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">270547-100ML</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:175px;">Gibco/Life Technologies</td>
			<td style="width:107px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fluorescent Microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:107px;">TE300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glacial Acetic Acid</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">A38-212</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Human CD47 (B6H12) &#8211; FITC Antibody</td>
			<td style="width:175px;">Santa Cruz Biotechnology</td>
			<td style="width:107px;">SC-12730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Osmium Tetroxide</td>
			<td style="width:175px;">Acros Organics</td>
			<td style="width:107px;">197450050</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Bicarbonate (KHCO3)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">237205-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Phosphate Monobasic (KH2PO4)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">P5655-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PVC Tubing (Cardiovascular Procedure Kit)</td>
			<td style="width:175px;">Terumo Cardiovascular Systems</td>
			<td style="width:107px;">60050</td>
			<td>Most clinical-grade tubing will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>JSM-T330A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Sodium Chloride (NaCl)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:269px;">Microplate Reader</td>
			<td style="width:175px;">Molecular Devices</td>
			<td style="width:107px;">Spectramax Gemini EM&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sulfo-SMCC</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">M6035-10MG</td>
			<td>Moisture Sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">tris (2-carboxyethyl) phosphine (TCEP-HCl)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">20491</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tris Base</td>
			<td style="width:175px;">Sigma</td>
			<td>T1503-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tween-20</td>
			<td>Bio-Rad</td>
			<td>170-6531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Vectashield with DAPI</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">H-1200</td>
			<td>Light sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Zeba Spin Desalt Columns &#8211; 7K MWCO</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">89891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of the ex vivo chandler loop apparatus to assess the biocompatibility of modified polymeric blood conduits

当合成的表面被引入到主体时的异物反应。它的特征在于，吸附血液中的蛋白质，并随后附着的血小板活化，单核细胞/巨噬细胞的粘附性，和炎性细胞信号转导事件，导致后并发症。钱德勒循环设备是一个实验系统，让研究人员研究时，大量血液灌流与聚合物管道发生的分子和细胞的相互作用。为此，该装置已被用作离体模型允许的各种聚合物表面修饰的抗炎性质的评估。我们实验室已经表明，血液导管，通过光敏化学与重组CD47的共价修饰，可以赋予生物相容性的聚合物表面。附加CD47到聚合物表面可以促进聚合血液导管的疗效的有效手段。她EIN是该方法详述用于追加重组CD47对临床相关的聚合血液的导管和使用钱德勒回路作为体外实验模型研究与CD47的改性和控制导管血液相互作用的光活化的化学反应。

通过使用PDT-BzPh和TCEP，连同硫醇反应recCD47使用SMCC聚赖氨酸产生硫醇反应性聚合物表面允许recCD47到聚合物表面的附着。改性方法是示意性地概括于图1中 。这个修改过程的方便之处在于它可应用于许多不同的蛋白质和许多不同的聚合物表面，假设蛋白能够以足够的化学反应性基团，例如含胺的赖氨酸和修改该聚合物具有足够可用的烃。 先前已证明，recCD47可以附加...

Watch this Scientific Journal Video about The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits at JoVE.com

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

的使用<em&gt;前体内</em&gt;钱德勒循环装置，以评估改性聚合物血导管的生物相容性

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

the-use-ex-vivo-chandler-loop-apparatus-to-assess-biocompatibility

Research

JoVE Journal

Bioengineering

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

11.5K 次观看.  The Children's Hospital of Philadelphia. Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits. 

视频: The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

器官Ⅰ型胶原的含量可延展的平台，以评估细胞行为的一个3维的情况下

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

科研

生物工程

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

循环压力为生物反应器的设计体外主动脉心脏瓣膜的研究

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

通过胎盘的外来物质和纳米材料的运输率测定<em&gt;体外</em&gt;人胎盘灌注型号

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

改进方法对血脑屏障的人体细胞为主，接触模型的制备

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

基因转染向着文化微模式球体板细胞基因修饰的细胞移植

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected within in vivo oral biofilms. Furthermore, a system that uses natural human saliva as the nutrient source, instead of artificial media, is particularly desirable in order to support the expression of cellular and biofilm-specific properties that mimic the in vivo communities. We describe a method for the development of multi-species oral biofilms that are comparable, with respect to species composition, to supragingival dental plaque, under conditions similar to the human oral cavity. Specifically, this methods article will describe how a commercially available microfluidic system can be adapted to facilitate the development of multi-species oral biofilms derived from and grown within pooled saliva. Furthermore, a description of how the system can be used in conjunction with a confocal laser scanning microscope to generate 3-D biofilm reconstructions for architectural and viability analyses will be presented. Given the broad diversity of microorganisms that grow within biofilms in the microfluidic system (including Streptococcus, Neisseria, Veillonella, Gemella, and Porphyromonas), a protocol will also be presented describing how to harvest the biofilm cells for further subculture&#160;or DNA extraction and analysis. The limits of both the microfluidic biofilm system and the current state-of-the-art data analyses will be addressed. Ultimately, it is envisioned that this article will provide a baseline technique that will improve the study of oral biofilms and aid in the development of additional technologies that can be integrated with the microfluidic platform.

Use of a High-throughput In Vitro Microfluidic System to Develop Oral Multi-species Biofilms

The goal of this methods paper is to describe the use of a microfluidic system for the development of multi-species biofilms that contain species typically identified in human supragingival dental plaque.&#160;Methods to describe biofilm architecture, biofilm viability, and an approach to harvest biofilm for culture-dependent or culture-independent analyses are highlighted.

使用高通量的<em&gt;在体外</em&gt;微流控系统开发口服多物种生物膜

There are few high-throughput in vitro systems which facilitate the development of multi-species biofilms that contain numerous species commonly detected ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

内镜下黏膜下注射材料性能评价的一种新的体内模型

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

心脏内侧燃光导管,用于监测细胞代谢,使用渗透哺乳动物心脏的透射吸收光谱

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Mitigation of Blood Borne Cell Attachment to Metal Implants through CD47-Derived Peptide Immobilization

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, improving the biocompatibility of metal stents remains a significant challenge. The goal of this protocol is to describe a robust technique of metal surface modification by biologically active peptides to increase biocompatibility of blood contacting medical implants, including endovascular stents. CD47 is an immunological species-specific marker of self and has anti-inflammatory properties. Studies have shown that a 22 amino acid peptide corresponding to the Ig domain of CD47 in the extracellular region (pepCD47), has anti-inflammatory properties like the full-length protein. In vivo studies in rats, and ex vivo studies in rabbit and human blood experimental systems from our lab have demonstrated that pepCD47 immobilization on metals improves their biocompatibility by preventing inflammatory cell attachment and activation. This paper describes the step-by step protocol for the functionalization of metal surfaces and peptide attachment. The metal surfaces are modified using polyallylamine bisphosphate with latent thiol groups (PABT) followed by deprotection of thiols and amplification of thiol-reactive sites via reaction with polyethyleneimine installed with pyridyldithio groups (PEI-PDT). Finally, pepCD47, incorporating terminal cysteine residues connected to the core peptide sequence through a dual 8-amino-3,6-dioxa-octanoyl spacer, are attached to the metal surface via disulfide bonds. This methodology of peptide attachment to metal surface is efficient and relatively inexpensive and thus can be applied to improve biocompatibility of several metallic biomaterials.

Presented here is a protocol for appending peptide CD47 (pepCD47) to metal stents using polybisphosphonate chemistry. Functionalization of metal stents using pepCD47 prevents the attachment and activation of inflammatory cells thus improving their biocompatibility.

通过CD47衍生肽固定，缓解血系细胞附着到金属植入物

The key complications associated with bare metal stents and drug eluting stents are in-stent restenosis and late stent thrombosis, respectively. Thus, ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

血流成像与超快多普勒

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...