这项工作是由香港研究资助局的支持（赠款17122014，C1013-15G，719813E和17304514）。 AST皇裘槎高级研究奖学金的获得者。

1.微流体器件设计和制造<ol><li>微流控大师设计<ol><li>设计并绘制与任何计算机辅助设计（CAD）软件的微流体通道模式。 注意:通常情况下，该CAD绘图可以被发送到光掩模公司生产的光掩模。微流体设计包括三个相同的平行通道，每个通道具有以下尺寸为4mm×25毫米×250微米（宽度×长度×高度），并设置2mm的。两个信道两端分别设计有127°的角度，以促进液体的入口和出口（ 图1B）。在这个协议中使用的通道被报道在以前的出版物13。 </li><li>洗用丙酮，异丙醇超声波（500瓦/ 42千赫），和去离子水的硅晶片，每次15分钟。干燥用氮气压的晶片。加热在250℃的热板上的硅片15分钟完全干燥它。 </li><li>旋涂SU-8 2075光致抗蚀剂（125微米厚）上以1800 rpm的纺丝速度在硅晶片的层。直接烘烤光致抗蚀剂涂覆的晶片在热板上在65℃，然后在95℃下，分别以除去过量的溶剂，为5和25分钟（根据SU-8的产品手册）。 </li><li>重复步骤3和旋涂SU-8 2075光致抗蚀剂的另一层，得到250μm的最终厚度。烘烤直接在热板上在65℃下7分钟的光致抗蚀剂涂覆的晶片，然后在95℃下30分钟。慢慢冷却该晶片至室温。不要用高压空气冷却。 </li><li>对准并直接把光掩模在晶片的顶部。其暴露于UV光20秒以交联的图案。 </li><li>烘烤在65℃下用光刻胶晶片和5和12分钟95℃的加热板上，分别以除去溶剂用于聚合（根据SU-8的产品手册）。随后，冷却晶片室内温度。 </li><li>通过浸渍在SU-8显影剂将晶片25分钟除去非交联光致抗蚀剂。冲洗用异丙醇晶圆和压缩空气吹干。 注:如果用异丙醇漂洗时，表明发育不完全观察到乳状液，浸泡晶圆回SU-8开发者更长的开发时间。 </li><li>放置在180℃的热板上的晶片10分钟，以治愈潜在裂纹图案。关掉加热板和由留在热板上慢慢冷却晶片至室温。 </li><li>在小瓶帽添加三氯几滴（1H，1H，2H，2H-全氟辛基）硅烷。放入真空干燥器瓶盖。旁边放置到瓶盖晶片silanize晶片10分钟。 </li></ol></li><li> PDMS芯片制造<ol><li>裹片用铝箔创建持有者。 </li><li>混合聚二甲基硅氧烷（PDMS）基和固化剂以10:1的质量比下混合和脱气功能的离心混合器。慢慢倒入PDMS混合物到晶片至约5毫米的高度。脱气在真空下与PDMS 40分钟。固化在烘箱中的PDMS在65℃下2小时。 </li><li>仔细剥PDMS厚板关闭主并修剪沿通道的PDMS，直到它是载玻片的大小。用1毫米活检穿孔创建设备的入口和出口冲的PDMS。 </li><li>请用压缩空气和胶带PDMS表面去除灰尘和杂物PDMS。 </li><li>放置的PDMS板，与通道侧朝上，成等离子体清洁器。放置一个干净的玻璃滑动到等离子体清洁器沿着PDMS厚板。 </li><li>对待空气等离子体下的PDMS和载玻片在高RF级别1分钟。绑定PDMS到载玻片以创建一个不可逆共价键。 </li><li>放置在150℃的热板上的PDMS芯片1小时，以提高粘接强度，并返回hydrop使用前，PDMS的hobicity。 </li></ol></li></ol> 2.播种微流控芯片原发性腹膜间皮细胞（间皮细胞） <ol><li>微流体通道的纤连蛋白涂层<ol><li>用75％乙醇的30微升灭菌的微流体通道。除去乙醇并用一个枪头灭菌磷酸盐缓冲盐水的30微升（PBS）冲洗两次。在通道中充分吸PBS中。 注意:为尽量减少污染，微流体细胞培养物应仔细充分无菌条件下通过使用层流罩下进行。 </li><li>制备在无血清的M199 10微克/毫升纤连蛋白溶液:MCDB105（1:1）培养基。吸取的溶液上下，特别小心，以避免形成气泡。不要旋涡。 </li><li>慢慢地填满每个通道与纤维连接蛋白溶液30微升。密封用胶带的频道，并在4℃过夜孵育芯片。 </li></ol></li><li&gt;播种到间皮细胞微流体通道<ol><li>在10％胎牛血清（FBS）的培养基中培养的HPMC（M199:MCDB105补充有10％FBS和100U / mL青霉素/链霉素）下，5％CO 2在37℃直至80％汇合。 </li><li>冲洗用3mL的PBS的间皮细胞，并与/ 2毫升0.05％胰蛋白酶的30秒0.01％EDTA（TE）溶液trypsinize。用4mL 10％FBS的培养基中和在1000 xg离心降速细胞5分钟。 </li><li>重悬在间皮细胞2毫升10％FBS培养基。计数与血球细胞数和调节细胞浓度为3.5×10 6 /毫升。 </li><li>暖在37℃培养箱中纤连蛋白涂布的微流控芯片使用前5分钟。完全吸出纤维连接蛋白的解决方案。 </li><li>慢慢地吸取HPMC悬挂的30微升到每个通道。密封用胶带渠道和放置设备中的CO 2培养箱中过夜。 </li></ol></li></ol><ol><li>卵巢癌的球体形成<ol><li>保持人上皮卵巢癌细胞系SKOV-3中的5％FBS的培养基（M199:有5％FBS和100U / mL青霉素/链霉素MCDB105）下，5％CO 2在37℃的实验之前。 </li><li>通过煮沸溶解0.5％琼脂糖在无菌水中。分配琼脂糖溶液的50微升到96孔板各孔中。留在机罩板20分钟，以使琼脂糖固化;固化琼脂糖涂层的孔可以防止细胞粘附和指示板是可以使用了。 </li><li>冲洗SKOV-3细胞用3mL的PBS。用2ml的TE溶液3分钟Trypsinize。用4mL 5％FBS的培养基中和在1000 xg离心降速细胞5分钟。 </li><li>计数与血球SKOV-3细胞数和重悬在5％FBS的培养基中的细胞以5,000个细胞/ mL的密度。 TRansfer 200细胞悬液到每一μL的琼脂糖包被的孔，并培养，5％CO 2下在37℃下48小时。 </li></ol></li><li>癌症球体装载<ol><li>转移癌症球体到一个50毫升的离心管中预先润湿用1mL无血清培养基中并降速，在750×g离心5分钟。 注:预湿吸头和离心管可以防止意外球体附着到离心管。 </li><li>制备2.5微克/毫升的5-氯甲基二乙酸酯（CMFDA）溶液在无血清的M199:MCDB105培养基。悬浮癌症球体在1mL的CMFDA溶液孵育在37℃下30分钟。离心在750×g离心5分钟。 </li><li>冲洗癌症球体用2mL无血清培养基和离心机在750×g离心5分钟。重复此步骤两次，以从培养基中除去剩余的荧光染料。 注意:荧光信号可在球体保留时间超过96小时。 </li><li>悬浮癌症球状体在无血清的M199:MCDB105培养基和计数与血球球体数量。浓度调整到2,000球体/毫升无血清培养基。 </li><li>前球体装载，缓缓注入100微升无血清的M199中:MCDB105培养基洗去通道内的非粘附间皮细胞。 注:快速注射，吸入或气泡的形成会导致皮单层的脱离。 </li><li>负载30微升荧光标记的球体悬浮液到每个信道的。微流体芯片放置到CO 2的培养箱中。 </li></ol></li><li>灌注装配平台和联合培养<ol><li>附装用新鲜的无血清的M199注射器:MCDB105培养基为1米长的管。 </li><li>放置在注射泵，注射器和固定柱塞与注射器的主体。在相同的高度培养箱内的微流控芯片安装在水平位置的注射器泵。 ñOTE:数量和注射器的大小取决于使用的信道数和在实验的持续时间。 </li><li>按住快进键，直到媒体溢出管迅速注入与介质全管。 </li><li>运行在所需的流速注射。使用以下等式估算壁剪切应力: <img alt="式（1）" src="/files/ftp_upload/55337/eq1.jpg" /> 其中τ代表腔室，Ǫ=流速内部的壁剪应力，μ=粘度（0.01克厘米-1）; H =流动室的高度，以及w =流动室的宽度。 </li><li>管连接到该信道的入口。放置过量入口管路在孵化器，以确保在介质被加热之前注入通道。为了便于管子和通道之间的连接，相应的连接器可以选择。注意:谨防不引入任何泡到syrin通用电气，管道和连接。 </li><li>通道的出口连接到一个50毫升锥形管，收集用短输出管流出介质。 注:完整的动态共培养体系如图1D所示。 </li></ol></li></ol> 4.灌注后观察间皮细胞与癌症的球体<ol><li>观察间皮细胞和癌症球体与​​明场或荧光显微镜和灌注1小时后拍照。 </li></ol>

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作者什么都没有透露。

该测定提供了一个灵活的和生理学相关模型，可以与各种生物化学和基于细胞的测定，包括被结合，但不限于，粘附测定，间皮间隙测定法，和药物筛选。它可以应用到的腹腔微环境对癌症发展的效果的评估。然而，一些实验条件可能需要进行优化，这取决于计划的目标（ 例如，每信道，共培养时间等接种的HPMC和癌症球体的数量）。在腹膜内微环境的其它细胞类型，诸如成纤维细胞，内皮细胞，或免疫细胞，也可以为了研究卵巢癌球体和附近小区之间的重要的相互作用被包括在系统中。其它ECM成分，如层粘连蛋白，玻连蛋白或胶原，也可以使用。该协议可能需要考虑的一个弱点是，虽然营养补充和剪切应力的耦合使人想起在体内看出，在我们目前的系统营养补充和剪切应力的没有单独的控制。此外，将需要的技术，如片上胰蛋白酶消化，用荧光激活细胞分选组合以分别收集卵巢癌细胞和间皮细胞有关不同类型的细胞的进一步的分子研究。 当与传统方法相比，该技术具有几个优点。首先，三维共培养更好地模拟卵巢癌细胞和间皮细胞的体内细胞间通讯。第二，细胞是在流体流动培养的，但不是在静止状态下，产生有潜在代表的体内情况12,13细胞应答。此外，流体流动可以精确控制，模仿hydrodynami条件C在肿瘤进展的不同阶段。 总之，动态3D卵巢癌间皮共培养这里提出的模型提供了用于在腹膜腔建模肿瘤行为的灵活框架。用该模型，动态水动力条件下的癌细胞和间皮细胞间的相互作用可全面分析。它可以极大地推进我们的转移进程的基本机制的理解，也可能有助于治疗的发展。

<html>卵巢癌是最致命的妇科肿瘤，其特征是广泛腹膜扩散和恶性腹水1的形成。这种广泛的腹膜转移是一项重大的临床挑战，并与较差的临床结果有关。不同于通过血液转移最坚实癌，卵巢癌主要是腹腔内传播。肿瘤细胞转移2的过程中，存在作为多细胞聚集体/球体。即悬浮培养能丰富卵巢癌干/肿瘤起始细胞，这一事实进一步表明，这些球体可能与两个肿瘤侵袭性相关联，并增强化疗3,4。有在2D和3D培养，这大概有不同的分子机制5之间药物的反应差异。 _content"&gt;与间皮的基本相互作用构造为卵巢肿瘤进展的主要微环境，这些皮细胞躺在的胞外基质（ECM），其中纤连蛋白是一种普遍存在的组分。间皮细胞衍生纤连蛋白的表达和增加之间的链接肿瘤进展已经表明，纤维粘连蛋白是在恶性腹水6,7大量存在。卵巢癌细胞也能够诱导从间皮细胞纤连蛋白的分泌，以促进早期卵巢癌转移8。 新出现的证据表明，机械性刺激，包括剪切应力，可以调节细胞形态，基因表达，并且因此，肿瘤细胞9，10，11的表型。由于恶性腹水 开发和t内积聚umor进展，卵巢肿瘤细胞暴露于流体流，将所得的剪切应力。若干组，我们包括，已显示出剪切应力对卵巢癌的进展的影响，包括细胞骨架修饰，上皮至间质转变，和癌症干性12，13，14，15。因此，生理相关微环境是肿瘤腹膜转移的调查重要。然而，目前的体外流体力学培养系统对模拟和控制常数，低，生理学相关的剪切应力16，17，18，19限制。 常规体外方法侧重于无论是蜂窝或机械环境依然有限模仿适当生理相关的腹腔微环境的复杂性。 这里，为了工程师腹膜一个新的模型，以克服常规的策略的限制，以推进在癌转移腹膜内室的研究中，以控制流体流动的基于3D微流体平台的设计。在这个模型中，卵巢癌球体分别与在微流控芯片在连续的流体流动（ 图 1A）的原代人腹膜间皮细胞共培养。铺板于纤连蛋白的间皮细胞。非粘附性卵巢癌球状体接种到用由注射泵灌注连续流动介质的微流体通道。两个3D环境和动态机械力转移级联的非常重要的因素。此平台可用于在复杂的CEL方面调查腹膜内微细胞性和共培养的相互作用，以及关于动态力学线索。

<table>
	<tbody>
		<tr>
			<td>Silicon wafer</td>
			<td>University wafer</td>
			<td>#1196</td>
			<td>100mm</td>
		</tr>
		<tr>
			<td>SU-8 2075 photoresist&#160;</td>
			<td>Microchem</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>SU-8 developer&#160;</td>
			<td>Microchem</td>
			<td>108-65-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloro (1H, 1H, 2H, 2H-perfluorooctyl) silane</td>
			<td>Sigma</td>
			<td>448931</td>
			<td></td>
		</tr>
		<tr>
			<td>Sylgard 184</td>
			<td>Dow Corning</td>
			<td>1673921</td>
			<td>Polydimethylsiloxane (PDMS) + curing agent kit</td>
		</tr>
		<tr>
			<td>Biopsy punch&#160;</td>
			<td>Miltex</td>
			<td>33-31AA</td>
			<td>1 mm diameter</td>
		</tr>
		<tr>
			<td>Plasma cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC-002</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene tubing</td>
			<td>SCI</td>
			<td>BB31695-PE/5</td>
			<td>0.86mm (inner diameter)</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Terumo</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe pump</td>
			<td>Longer precision pump&#160;</td>
			<td>&#160;LSP01-2A</td>
			<td></td>
		</tr>
		<tr>
			<td>Medium 199</td>
			<td>Invitrogen</td>
			<td>31100-035</td>
			<td>Add 2.2g/L sodium bicarbonate</td>
		</tr>
		<tr>
			<td>MCDB 105 Medium</td>
			<td>Sigma</td>
			<td>M6395</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)</td>
			<td>Hyclone</td>
			<td>SH30068.02</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin&#160;</td>
			<td>Invitrogen</td>
			<td>15070-063</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin EDTA solution&#160;</td>
			<td>Gibco</td>
			<td>25300-054</td>
			<td>0.05% Trypsin -0.01% EDTA, phenol red</td>
		</tr>
		<tr>
			<td>Fibronectin human</td>
			<td>BD</td>
			<td>354008</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose&#160;</td>
			<td>Invitrogen</td>
			<td>15510-027</td>
			<td></td>
		</tr>
		<tr>
			<td>5-chloromethylfluorescein diacetate</td>
			<td>Life technologies</td>
			<td>C7025</td>
			<td>Green CMFDA</td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>SANYO</td>
			<td>MCO-18AIC</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Hitachi</td>
			<td>CT15RE</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Nikon</td>
			<td></td>
			<td>Model: 80i or ECLIPSE Ti; software: SPOT</td>
		</tr>
		<tr>
			<td>SKOV-3&#160;</td>
			<td></td>
			<td></td>
			<td>Gift from Dr. N Auersperg (University of British Columbia)</td>
		</tr>
	</tbody>
</table>

modeling ovarian cancer multicellular spheroid behavior in a dynamic 3d peritoneal microdevice

卵巢癌的特征在于丰富的腹膜转移，与恶性腹水通常发现肿瘤球。这与不良临床预后相关，目前缺乏有效的治疗方法。两个三维（3D）环境和动态机械力在此转移级联非常重要的因素。然而，传统的细胞培养不能概括这个自然肿瘤微环境。因此， 在体内样，可以效仿腹腔内环境模型的重要性显而易见。在这项研究中，腹膜的一个新的微流体平台成立转移期间以模仿卵巢癌的球体的情况在腹膜腔。一个非粘附的条件下生成的卵巢癌的球体均涂覆有经受生理学相关的剪切应力腹膜间皮细胞微流体通道中培养。总之，这个动态三维卵巢癌，间皮麦克风rofluidic平台可以在基本的癌症生物学提供了新的知识，并作为潜在的药物筛选和开发平台。

使用该协议，一个微流体平台的成立，卵巢癌的球体水动力条件下的间皮细胞模型。原代人腹膜间皮细胞在微型装置中培养16小时，明视场显微镜下观察。 如图2A所示，通道底部被成功覆盖着的HPMC的单层。要注意的纤连蛋白或HPMC图案形成过程中气泡的形成会导致信道涂层失败是重要的。通过非粘附悬浮培养，直径大约为100微米，这是在大小中的那些患者的腹水中发现类似的多细胞球状体，产生与绿色荧光染料，CMFDA标记。然后将该球体转移至HPMC包衣的微流体通道和保持在悬浮液中。在显微镜下观察细胞形态和图像被捕获（ 图2B）。荧光标记癌症球体可以由荧光显微镜（ 图2C）下的间皮细胞很容易地鉴别。 <img alt="图1" src="/files/ftp_upload/55337/55337fig1.jpg" /> 图1.动态3D腹膜微型建模卵巢癌多细胞球体行为。 （A）中使用的共同培养的卵巢癌的球体与流条件下的HPMC的微流体芯片的示意图。 （B）中示出了微流体通道的设计原理图。在协议中使用的微流控芯片（C）照片，其渠道与清晰的可视化染料溶液注入。栏= 1厘米。 （D）的照片示出的实验装置。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55337/55337fig1large.jpg" target="_blank">请点击此处查看大版本O</a>F该数字。 <img alt="图2" src="/files/ftp_upload/55337/55337fig2.jpg" /> 图2.卵巢癌球体，间皮细胞动态共培养模型的图像。 （一）在微流体通道种植的HPMC单层的形象。 （B）在涂覆有根据相差显微镜（左图）或（C）的荧光显微镜（绿色，CMFDA;右图）腹膜间皮细胞微流体通道卵巢癌球体图像栏= 100微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55337/55337fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice at JoVE.com

Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

为了研究卵巢肿瘤进展的生理相关的模型，多细胞球体下模拟流体流动的微型培养。这种动态3D模型模拟与在卵巢癌转移发生的细胞和机械部件的腹膜内环境。

模拟卵巢癌多细胞球体行为动态三维微型腹膜

Ovarian cancer is characterized by extensive peritoneal metastasis, with tumor spheres commonly found in the malignant ascites. This is associated with ...

modeling-ovarian-cancer-multicellular-spheroid-behavior-dynamic-3d

Nanyang Technological University

Research

JoVE Journal

Bioengineering

8.6K 次观看.  University of Hong Kong. 为了研究卵巢肿瘤进展的生理相关的模型，多细胞球体下模拟流体流动的微型培养。这种动态3D模型模拟与在卵巢癌转移发生的细胞和机械部件的腹膜内环境。 

视频: Modeling Ovarian Cancer Multicellular Spheroid Behavior in a Dynamic 3D Peritoneal Microdevice

A Method for Ovarian Follicle Encapsulation and Culture in a Proteolytically Degradable 3 Dimensional System

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro follicle techniques provide a tool to model follicle development in order to investigate basic biology, and are further being developed as a technique to preserve fertility in the clinic1-4. Our in vitro culture system employs hydrogels in order to mimic the native ovarian environment by maintaining the 3D follicular architecture, cell-cell interactions and paracrine signaling that direct follicle development 5. Previously, follicles were successfully cultured in alginate, an inert algae-derived polysaccharide that undergoes gelation with calcium ions6-8. Alginate hydrogels formed at a concentration of 0.25% w/v were the most permissive for follicle culture, and retained the highest developmental competence 9. Alginate hydrogels are not degradable, thus an increase in the follicle diameter results in a compressive force on the follicle that can impact follicle growth10. We subsequently developed a culture system based on a fibrin-alginate interpenetrating network (FA-IPN), in which a mixture of fibrin and alginate are gelled simultaneously. This combination provides a dynamic mechanical environment because both components contribute to matrix rigidity initially; however, proteases secreted by the growing follicle degrade fibrin in the matrix leaving only alginate to provide support. With the IPN, the alginate content can be reduced below 0.25%, which is not possible with alginate alone 5. Thus, as the follicle expands, it will experience a reduced compressive force due to the reduced solids content. Herein, we describe an encapsulation method and an in vitro culture system for ovarian follicles within a FA-IPN. The dynamic mechanical environment mimics the natural ovarian environment in which small follicles reside in a rigid cortex and move to a more permissive medulla as they increase in size11. The degradable component may be particularly critical for clinical translation in order to support the greater than 106-fold increase in volume that human follicles normally undergo in vivo .

A new method for ovarian follicle encapsulation in a 3D fibrin-alginate interpenetrating network is described. This system combines structural support with proteolytic degradation to support the development of immature follicles to produce mature oocytes. This method may be applied to culture cell aggregates to maintain cell-cell contacts without limiting expansion.

卵泡在蛋白质降解的3维系统的封装和文化的一种方法

The ovarian follicle is the functional unit of the ovary that secretes sex hormones and supports oocyte maturation. In vitro  follicle techniques provide ...

科研

生物工程

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

有针对性的稳定同位素稀释液相色谱 - 质谱法分析细胞脂质提取

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Correlative Microscopy for 3D Structural Analysis of Dynamic Interactions

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, direct visualization of individual viral complexes in their host cellular environment with cryoET is challenging2, due to the infrequent and dynamic nature of viral entry, particularly in the case of HIV-1. While time-lapse live-cell imaging has yielded a great deal of information about many aspects of the life cycle of HIV-13-7, the resolution afforded by live-cell microscopy is limited (~ 200 nm). Our work was aimed at developing a correlation method that permits direct visualization of early events of HIV-1 infection by combining live-cell fluorescent light microscopy, cryo-fluorescent microscopy, and cryoET. In this manner, live-cell and cryo-fluorescent signals can be used to accurately guide the sampling in cryoET. Furthermore, structural information obtained from cryoET can be complemented with the dynamic functional data gained through live-cell imaging of fluorescent labeled target.
In this video article, we provide detailed methods and protocols for structural investigation of HIV-1 and host-cell interactions using 3D correlative high-speed live-cell imaging and high-resolution cryoET structural analysis. HeLa cells infected with HIV-1 particles were characterized first by confocal live-cell microscopy, and the region containing the same viral particle was then analyzed by cryo-electron tomography for 3D structural details. The correlation between two sets of imaging data, optical imaging and electron imaging, was achieved using a home-built cryo-fluorescence light microscopy stage. The approach detailed here will be valuable, not only for study of virus-host cell interactions, but also for broader applications in cell biology, such as cell signaling, membrane receptor trafficking, and many other dynamic cellular processes.

We describe a correlative microscopy method that combines high-speed 3D live-cell fluorescent light microscopy and high-resolution cryo-electron tomography. We demonstrate the capability of the correlative method by imaging dynamic, small HIV-1 particles interacting with host HeLa cells.

Cryo-electron tomography (cryoET) allows 3D visualization of cellular structures at molecular resolution in a close-to-physiological state1. However, ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

组织工程:建设多细胞三维支架的层状细胞片交货

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

3D胶原凝胶和微通道3D相互作用的研究准备<em&gt;在体内</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell lines using conventional two-dimensional (2D) cell culture. These challenges can be overcome by using bioprinting techniques to build heterogeneous three-dimensional (3D) tumor models whereby different types of cells are embedded. Alginate and gelatin are two of the most common biomaterials employed in bioprinting due to their biocompatibility, biomimicry, and mechanical properties. By combining the two polymers, we achieved a bioprintable composite hydrogel with similarities to the microscopic architecture of a native tumor stroma. We studied the printability of the composite hydrogel via rheology and obtained the optimal printing window. Breast cancer cells and fibroblasts were embedded in the hydrogels and printed to form a 3D model mimicking the in vivo microenvironment. The bioprinted heterogeneous model achieves a high viability for long-term cell culture (&#62; 30 days) and promotes the self-assembly of breast cancer cells into multicellular tumor spheroids (MCTS). We observed the migration and interaction of the cancer-associated fibroblast cells (CAFs) with the MCTS in this model. By using bioprinted cell culture platforms as co-culture systems, it offers a unique tool to study the dependence of tumorigenesis on the stroma composition. This technique features a high-throughput, low cost, and high reproducibility, and it can also provide an alternative model to conventional cell monolayer cultures and animal tumor models to study cancer biology.

Bioprintable Alginate/Gelatin Hydrogel 3D In Vitro Model Systems Induce Cell Spheroid Formation

We developed a heterogeneous breast cancer model consisting of immortalized tumor and fibroblast cells embedded in a bioprintable alginate/gelatin bioink. The model recapitulates the in vivo tumor microenvironment and facilitates the formation of multicellular tumor spheroids, yielding insight into the mechanisms driving tumorigenesis.

Bioprintable 藻酸盐/明胶水凝胶 3D体外模型诱导细胞球形形成

The cellular, biochemical, and biophysical heterogeneity of the native tumor microenvironment is not recapitulated by growing immortalized cancer cell ...

Construction of a Multilayered Mesenchymal Stem Cell Sheet with a 3D Dynamic Culture System

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low cell retention and poor survival within the target zone. However, during the in vitro construction process, a solution for maintaining stem cell bioactivity and increasing the cell amount within the cell sheet is urgently needed. Here, this protocol presents a method for constructing a multilayered cell sheet with favorable stem cell bioactivity and optimal operability. Decellularized porcine pericardium (DPP) is prepared by phospholipase A2 (PLA2) decellularization method as the cell sheet scaffold, and rat bone marrow mesenchymal stem cells (BMSCs) are isolated and expanded as the seeded cells. The temporary multilayered cell sheet structure is constructed by using RAD16-I peptide hydrogel. Finally, the cell sheet is cultured with a dynamic perfusion system to stabilize the three-dimensional (3D) structure, and the cell sheet could be obtained following a 48-hour culture in vitro. This protocol provides an efficient and feasible method for constructing a multilayered stem cell sheet, and the cell sheet could be developed as a favorable stem cell therapy product in the future.

This article provides an efficient and feasible method for constructing multilayered stem cell sheets with favorable stem cell property.

3D 动态培养系统构建多层间充质干细胞片材

Stem cell therapy shows a promising future in regenerating injured organ and tissues, and the cell sheet technique has been developed to improve the low ...

Environmental Dynamic Mechanical Analysis to Predict the Softening Behavior of Neural Implants

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior of these materials. In the past, it has not been possible to satisfactorily measure the softening of thin films under conditions mimicking body environment without substantial effort. This publication presents a new and simple method that allows dynamic mechanical analysis (DMA) of polymers in solutions, such as phosphate buffered saline (PBS), at relevant temperatures. The use of environmental DMA allows measurement of the softening effects of polymers due to plasticization in various media and temperatures, which therefore allows a prediction of the materials behavior under in vivo conditions.

To allow reliable predictions of the softening of polymeric substrates for neural implants in an in vivo environment, it is important to have a reliable in vitro method. Here, the use of dynamic mechanical analysis in phosphate buffered saline at body temperature is presented.

预测神经植入物软化行为的环境动力力学分析

When using dynamically softening substrates for neural implants, it is important to have a reliable in vitro method to characterize the softening behavior ...

Ovarian Cancer Detection Using Photoacoustic Flow Cytometry

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies for the detection of CTCs include flow cytometry, microfluidic devices, and real-time polymerase chain reaction (RT-PCR). Despite recent advances, methods for the detection of early ovarian cancer metastasis still lack the sensitivity and specificity required for clinical translation. Here, a novel method is presented for the detection of ovarian circulating tumor cells by photoacoustic flow cytometry (PAFC) utilizing a custom three dimensional (3D) printed system, including a flow chamber and syringe pump. This method utilizes folic acid-capped copper sulfide nanoparticles (FA-CuS NPs) to target SKOV-3 ovarian cancer cells by PAFC. This work demonstrates the affinity of these contrast agents for ovarian cancer cells. The results show NP characterization, PAFC detection, and NP uptake by fluorescence microscopy, thus demonstrating the potential of this novel system to detect ovarian CTCs at physiologically relevant concentrations.

A protocol is presented to detect circulating ovarian tumor cells utilizing a custom-made photoacoustic flow system and targeted folic acid-capped copper sulfide nanoparticles.

使用光声流细胞测定卵巢癌检测

Many studies suggest that the enumeration of circulating tumor cells (CTCs) may show promise as a prognostic tool for ovarian cancer. Current strategies ...

Generating Controlled, Dynamic Chemical Landscapes to Study Microbial Behavior

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the microscale&#8213;to create micro-environments for microbial chemotaxis experiments. To create chemical pulses, we developed a system to introduce amino acid sources near-instantaneously by photolysis of caged amino acids within a polydimethylsiloxane (PDMS) microfluidic chamber containing a bacterial suspension. We applied this method to the chemotactic bacterium, Vibrio ordalii, which can actively climb these dynamic chemical gradients while being tracked by video microscopy. Amino acids, rendered biologically inert (&#39;caged&#39;) by chemical modification with a photoremovable protecting group, are uniformly present in the suspension but not available for consumption until their sudden release, which occurs at user-defined points in time and space by means of a near-UV-A focused LED beam. The number of molecules released in the pulse can be determined by a calibration relationship between exposure time and uncaging fraction, where the absorption spectrum after photolysis is characterized by using UV-Vis spectroscopy. A nanoporous polycarbonate (PCTE) membrane can be integrated into the microfluidic device to allow the continuous removal by flow of the uncaged compounds and the spent media. A strong, irreversible bond between the PCTE membrane and the PDMS microfluidic structure is achieved by coating the membrane with a solution of 3-aminopropyltriethoxysilane (APTES) followed by plasma activation of the surfaces to be bonded. A computer-controlled system can generate user-defined sequences of pulses at different locations and with different intensities, so as to create resource landscapes with prescribed spatial and temporal variability. In each chemical landscape, the dynamics of bacterial movement at the individual scale and their accumulation at the population level can be obtained, thereby allowing the quantification of chemotactic performance and its effects on bacterial aggregations in ecologically relevant environments.

A protocol for the generation of dynamic chemical landscapes by photolysis within microfluidic and millifluidic setups is presented. This methodology is suitable to study diverse biological processes, including the motile behavior, nutrient uptake, or adaptation to chemicals of microorganisms, both at the single cell and population level.

生成受控动态化学景观，研究微生物行为

We demonstrate a method for the generation of controlled, dynamic chemical pulses&#8213;where localized chemoattractant becomes suddenly available at the ...