Name: construction of a human aorta smooth muscle cell organ-on-a-chip model for recapitulating biomechanical strain in the aortic wall
Uploaded: 2022-07-06T14:00:00
Description: Watch this Scientific Journal Video about Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall at JoVE.com

作者承认，这项工作得到了上海市科学技术委员会（20ZR1411700）、国家自然科学基金（81771971）和上海帆船计划（22YF1406600）的资助。

经复旦大学伦理委员会中山医院批准，采用人主动脉标本进行原发性HASMC分离。B2020-158）。从复旦大学附属中山医院行升主动脉手术的患者中采集主动脉标本。参与前获得所有患者的书面知情同意。
1.原发性人主动脉平滑肌细胞分离
<ol><li>用无菌PBS清洗升主动脉的右侧区域，1x-2x。</li>
	<li>用两个眼科镊子去除组织的内膜和外膜层，并保留培养基层以收获细胞。</...

<ol>
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随着微流控技术的快速发展，近年来出现了可以在体外复制一个或多个器官的生物学功能和结构的OOC模型，用于生物学，医学和药理学15。OOC可以模拟人体生理微环境的关键功能，对于探索疾病机制和促进临床前药物翻译至关重要8，16。虽然OOC仍处于早期阶段，需要更多的投入来优化OOC的设计，但近年来已经取得了很大进展<sup c...

胸主动脉瘤和夹层 （TAAD） 是主动脉壁的局部扩张或分层，与高发病率和死亡率有关1。人主动脉平滑肌细胞（HASMC）在TAAD的发病机制中起着至关重要的作用。HASMC不是终末分化的细胞，HASMC保持高可塑性，允许它们响应不同的刺激切换表型2。HASMCs在 体内主要受到节律性拉伸应变，这是调节平滑肌形态变化、分化和生理功能的关键因素之一3，4。因此，循环应变在HASMCs研究中的作用不容忽视。然而，传统的2D细胞培养物无法复制HASMC在 体内经历的循环菌株的生物力学刺激。此外，动物TAAD模型的构建不适用于某些类型的TAAD，例如二叶式主动脉瓣（BAV）相关的TAAD。此外，临床人体研究与动物实验之间的物种差距也不容忽视。它阻碍了临床实践中的药物翻译。因此，在主动脉疾病的研究中迫切需要更复杂的生理系统来模拟 体内 生物力学环境。
用于生物医学研究和药物开发的动物实验成本高昂，耗时且在道德上存在问题。此外，动物研究的结果经常无法预测在人体临床试验中获得的结果5，6。缺乏人类临床前模型和临床试验中的高失败率导致临床有效药物很少，这增加了医疗保健成本7。因此，迫切需要寻找其他实验模型来补充动物模型。近年来，微细加工和微流控技术得到了长足的发展，为建立类器官芯片模型提供了新技术，弥补了传统2D细胞培养技术的缺点，并为生理研究和药物开发建立了更现实、低成本和高效的体外模型。使用微流体装置，建立器官芯片，在具有不同刺激的微米大小的腔室中培养活细胞，以复制组织或器官的关键功能。该系统由单个或多个微流体微通道组成，其中一种细胞在灌注室中培养，复制一种组织类型的功能，或者在多孔膜上培养不同的细胞类型以重建不同组织之间的界面。基于微流控的类器官与患者来源的细胞相结合，具有弥合小鼠和人类疾病模型之间巨大物种差异的独特优势，并克服了传统2D细胞培养在疾病机制研究和药物发现方面的缺点。随着过去几年微流控技术的快速发展，研究人员已经意识到体外器官芯片（OOC）模型复制复合体内生物学参数的有用性8。这些微流体类器官模拟体外生物力学环境，如循环应变、剪切应力和液体压力，提供三维 （3D） 细胞培养环境。迄今为止，已经建立了几个OOC模型来模拟肺9，肾脏10，肝脏11，肠12和心脏13等器官的生物力学刺激，但这些模型尚未广泛应用于人类主动脉疾病的研究。
在这项研究中，我们提出了一种人主动脉平滑肌细胞器官芯片（HASMC-OOC）模型，该模型可以控制应用于TAAD患者来源的原发性HASMC的仿生机械力和节律。该芯片由蚀刻有通道的三层聚二甲基硅氧烷（PDMS）厚板和两个商业化的高柔性PDMS膜组成。HASMC在PDMS膜上培养。芯片中间的通道充满了用于细胞培养的培养基。芯片的顶部和底部通道连接到真空压力供应系统，该系统可以控制PDMS膜机械拉伸应变的节奏和频率。HASMC所经历的节律应变可以在HASMC-OOC中模拟，复制传统2D培养系统无法实现的组织或器官的生物力学微环境。利用高分辨率、实时成像和生物力学微环境的优势，可以研究活细胞的生化、遗传和代谢活性，用于组织发育、器官生理学、疾病病因、分子机制和生物标志物鉴定、心血管疾病和主动脉疾病。结合组织特异性和患者细胞，该系统可用于药物筛选、个性化医疗和毒性测试。该HASMC-OOC模型为研究主动脉疾病的发病机制提供了一个新颖的 体外 平台。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>4% paraformaldehyde</td>
			<td>Beyotime</td>
			<td>P0099-100ml</td>
			<td>Used for cell immobilization</td>
		</tr>
		<tr>
			<td>Alexa Fluor 350-labeled Goat Anti-Rabbit IgG</td>
			<td>Beyotime</td>
			<td>A0408</td>
			<td>Antibodies used for immunostaining</td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Beyotime</td>
			<td>ST025-20g</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium AM/PI</td>
			<td>Invitrogen</td>
			<td>L3224</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture flask&#160;</td>
			<td>Corning</td>
			<td>430639</td>
			<td></td>
		</tr>
		<tr>
			<td>CNN1</td>
			<td>Abcam&#160;</td>
			<td>Ab46794</td>
			<td></td>
		</tr>
		<tr>
			<td>Commercial flexible 
			PDMS membrane</td>
			<td>Hangzhou Bald Advanced Materials</td>
			<td>KYQ-200</td>
			<td></td>
		</tr>
		<tr>
			<td>F-actin</td>
			<td>Invitrogen</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>Sigma</td>
			<td>M8318</td>
			<td></td>
		</tr>
		<tr>
			<td>Hoses</td>
			<td>Runze Fluid</td>
			<td>96410</td>
			<td>1 mm inner diameter; 3 mm outer diameter; 1 mm wall thickness; Official website address: https://www.runzefluidsystem.com</td>
		</tr>
		<tr>
			<td>Human aortic smooth 
			muscle cell line CRL1999</td>
			<td>ATCC</td>
			<td>Lot Number:70019189</td>
			<td></td>
		</tr>
		<tr>
			<td>Image J</td>
			<td>Imagej.net/fiji/downloads</td>
			<td>Free Download: https://fiji.sc</td>
			<td>Imaging platform that is used to identify fluorescence intensity</td>
		</tr>
		<tr>
			<td>Incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>Ensures that the temperature, 
			humidity, and light exposure is 
			exactly the same throughout 
			experiment.</td>
		</tr>
		<tr>
			<td>Luer</td>
			<td>Runze Fluid</td>
			<td>RH-M016</td>
			<td>Official website address: https://www.runzefluidsystem.com.</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>mouse collagen</td>
			<td>Sigma</td>
			<td>C7661</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxygen plasma&#160;</td>
			<td>Changzhou Hongming Instrument</td>
			<td>HM-Plasma5L</td>
			<td></td>
		</tr>
		<tr>
			<td>Pasteur pipette</td>
			<td>Biologix</td>
			<td>30-0138A1</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Beyotime</td>
			<td>C0221A</td>
			<td></td>
		</tr>
		<tr>
			<td>Pen-Strep</td>
			<td>Sigma</td>
			<td>P4458-100ml</td>
			<td>Antibiodics used to prevent bacterial 
			contamination of cells during culture.</td>
		</tr>
		<tr>
			<td>peristaltic pump</td>
			<td>Kamoer</td>
			<td>F01A-STP-B046</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Corning</td>
			<td>430167</td>
			<td></td>
		</tr>
		<tr>
			<td>PLC controller</td>
			<td>Zhejiang Jun Teng (BenT) CNC factory</td>
			<td>BR010-11T8X2M</td>
			<td>The detailed program setting can be found in supplementary. Official website address: files.http://www.btcnc.net</td>
		</tr>
		<tr>
			<td>polydimethylsiloxane (PDMS)</td>
			<td>Dow Corning</td>
			<td>Sylgard 184</td>
			<td></td>
		</tr>
		<tr>
			<td>SM22</td>
			<td>Abcam&#160;</td>
			<td>ab14106</td>
			<td></td>
		</tr>
		<tr>
			<td>SMCM</td>
			<td>ScienCell</td>
			<td>Cat 1101</td>
			<td></td>
		</tr>
		<tr>
			<td>solenoid valve</td>
			<td>SMC (China)</td>
			<td>VQZ300</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Becton,Dickinson and Company</td>
			<td>300841</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton-X 100</td>
			<td>Beyotime</td>
			<td>ST795</td>
			<td>To penetrate cell membranes</td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>10296010</td>
			<td>Used for RNA extraction</td>
		</tr>
		<tr>
			<td>trypsin</td>
			<td>Sigma</td>
			<td>15400054</td>
			<td></td>
		</tr>
		<tr>
			<td>vacuum filter</td>
			<td>SMC (China)</td>
			<td>ZFC5-6</td>
			<td>Official website address: https://www.smc.com.cn</td>
		</tr>
		<tr>
			<td>vacuum pump</td>
			<td>Kamoer</td>
			<td>KVP15-KL-S</td>
			<td></td>
		</tr>
		<tr>
			<td>vacuum regulator</td>
			<td>AirTAC</td>
			<td>GVR-200-06</td>
			<td></td>
		</tr>
		<tr>
			<td>Primers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Primer Name</td>
			<td>Forward (5&#8217; to 3&#8217;)</td>
			<td>Reverse (5&#8217; to 3&#8217;)</td>
			<td></td>
		</tr>
		<tr>
			<td>SM22</td>
			<td>CCGTGGAGATCCCAACTGG</td>
			<td>CCATCTGAAGGCCAATGACAT</td>
			<td></td>
		</tr>
		<tr>
			<td>CNN1</td>
			<td>CTCCATTGACTCGAACGACTC</td>
			<td>CAGGTCTGCGAAACTTCTTAGA</td>
			<td></td>
		</tr>
	</tbody>
</table>

construction of a human aorta smooth muscle cell organ-on-a-chip model for recapitulating biomechanical strain in the aortic wall

传统的二维细胞培养技术和动物模型已用于人胸主动脉瘤和夹层（TAAD）的研究。然而，人类TAAD有时不能用动物模型来表征。临床人体研究和动物实验之间存在明显的物种差距，这可能会阻碍治疗药物的发现。相比之下，传统的细胞培养模型无法模拟 体内 生物力学刺激。为此，微细加工和微流体技术近年来有了长足的发展，为建立复制生物力学微环境的芯片类器官模型提供了新技术。在这项研究中，开发了人主动脉平滑肌细胞器官芯片（HASMC-OOC）模型来模拟主动脉生物力学的病理生理参数，包括人主动脉平滑肌细胞（HASMC）经历的周期性应变的幅度和频率在TAAD中起至关重要的作用。在该模型中，HASMC的形态在形状上变得细长，垂直于应变方向对齐，并且在应变条件下呈现比静态常规条件下更具收缩性的表型。这与天然人主动脉壁中的细胞取向和表型一致。此外，使用二叶式主动脉瓣相关TAAD（BAV-TAAD）和三尖瓣主动脉瓣相关TAAD（TAV-TAAD）患者来源的原发性HASMC，我们建立了BAV-TAAD和TAV-TAAD疾病模型，这些模型复制了TAAD中的HASMC特征。HASMC-OOC模型提供了一个与动物模型互补的新型 体外 平台，用于进一步探索TAAD的发病机制和发现治疗靶点。

HASMC-OOC模型由真空控制系统、循环系统和PDMS芯片以及HASMC-OOC模型的原理图设计组成（图1）。真空控制系统由真空泵、电磁阀和 PLC 控制器组成。为了充当循环系统，使用蠕动泵来更新细胞培养基并添加药物。PDMS芯片由两个真空室和一个充满SMCM的中间室组成，用于细胞生长。根据芯片结构设计，制备PMMA模具，将3块PDMS板坯倒入模具中，在70 °C交联2 h。用血浆处理后，将三个P...

Watch this Scientific Journal Video about Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall at JoVE.com

Construction of a Human Aorta Smooth Muscle Cell Organ-On-A-Chip Model for Recapitulating Biomechanical Strain in the Aortic Wall

在这里，我们开发了一种人主动脉平滑肌细胞器官芯片模型，以复制人主动脉壁中平滑肌细胞的 体内 生物力学应变。

构建人主动脉平滑肌细胞器官芯片模型，用于概括主动脉壁中的生物力学应变

Conventional two-dimensional cell culture techniques and animal models have been used in the study of human thoracic aortic aneurysm and dissection ...

该模型刺激人类主动脉平滑肌细胞经历的生物机械刺激。结合患者来源的细胞，该系统可用于主动脉疾病的药物筛选和个性化医疗。该模型可以模拟并精确控制人主动脉平滑肌细胞的裸机械参数，为主动脉疾病的研究和发病机制提供了新的体外平台。演示该程序的将是Mieradilijiang Abudubat，我们实验室的海报医生。首先用无菌pbs清洗升主动脉的右侧区域，一次或两次。用两个眼科镊子去除组织的内膜和外膜层，并保留培养基层以收获细胞。将介质层放在 10 厘米的培养皿上，并将其切成两到三毫米的碎片。然后，向组织样品中加入五毫升含有10%fbs和1%青霉素链霉素的SMCM。用无菌滴管将小组织移动到培养瓶中，均匀地铺开组织。尽可能丢弃培养基。然后将瓶子倒置。将两毫升SMCM加入倒置培养瓶中，并将其放入37摄氏度下含有5%二氧化碳的培养箱中一至两个小时。然后慢慢地将其右面朝上，再加入两毫升SMCM。孵育五到七天后，将SMCM与四毫升新鲜SMCM交换。更换介质时慢慢丢弃并加入 SMCM。一般来说，散发的平滑肌细胞在大约两周内爬出来。将培养瓶移至显微镜站时轻轻运输。否则，组织会漂浮，细胞会缓慢生长。当细胞生长后，将它们分成两个新的培养瓶，里面有四毫升新鲜的SMCM。通过对平滑肌细胞的四种特定标记物进行免疫荧光分析来鉴定细胞。要聚合PDMS，将固化剂或B组分添加到碱或A组分中，并将络合物混合五分钟。数量将取决于研究的需要。将制备好的PDMS凝胶置于真空提取罐中30至60分钟，并将压力保持在负0.8毫帕斯卡以下。使用计算机辅助设计软件，设计模具。使用高精度计算机数控雕刻机定制制作三层模具。使用PMMA板雕刻出模具框架和微通道后，将它们粘在另一块板上。将准备好的PDMS凝胶倒在设计的模具上。然后在70摄氏度下交联一到两个小时。从模具上剥离交联的PDMS板，并将商业化的PDMS膜切割成100毫米乘40毫米的部分。使用一毫米的打孔机在三个 PDM 板上打孔，以在 PDMS 芯片上制作空气和介质微通道的入口和出口。用氧等离子体处理三个准备好的PDMS板和两个PDMS膜五分钟，以进行PDM表面活化。将室内空气设置为处理气体，压力设置为负100千帕，电流设置为180至200毫安桥墩，电压设置为200伏，处理时间为五分钟。在立体显微镜下将三个PDMS板和两个PDMS膜粘合在一起，使得顶层由空气通道PDMS板组成，然后是PDMS膜。介质通道由PDMS板组成，然后是PDM膜，底层是空气通道PDMS板。将组装好的PDMS芯片放入70摄氏度的培养箱中一小时。准备几根一毫米内径和三厘米长的乳胶软管。将一毫米外径和一厘米长的不锈钢针插入准备好的软管的一端。然后将诱饵插入软管的另一端，以创建连接到PDMS芯片的空气和介质微通道的管子。将准备好的管插入PDMS芯片上的空气和介质微通道的出口和入口。使用两毫升注射器将两毫升每毫升80毫克的小鼠胶原蛋白注入PDM芯片的培养基微通道中，并在室温下孵育30分钟。孵育后，用两毫升注射器从通道中取出胶原蛋白。将胶原蛋白编码的PDMS芯片放入60摄氏度的培养箱中过夜。将PDMS芯片放入紫外线消毒器中超过一小时。将灭菌的PDMS芯片放在超洁净工作台上，为细胞实验做准备。使用含有2%FBS和1%青霉素链霉素的SMCM在5%二氧化碳培养箱中培养4次10至5个原发性人主动脉平滑肌细胞，设定在37摄氏度。当平滑肌细胞密度达到80%时，丢弃SMCM，并用两毫升PPS洗涤细胞。使用一毫升0.25%tripsin消化细胞两分钟，并以100 RCF离心五分钟。除去上清液并将细胞沉淀重悬于一毫升新鲜SMCM中。使用8毫升SMCM在10厘米培养皿上培养细胞。使用细胞仪计算细胞数，然后以每毫升5个细胞的两倍10的最终浓度进行。将两毫升PBS缓慢倒入胶原蛋白编码和灭菌的PDMS芯片培养基微通道中，然后使用两毫升注射器丢弃。将两毫升两倍10的平滑肌细胞悬浮液缓慢倒入PDMS芯片的培养基微通道中。然后，关闭PDMS芯片入口和出口处的诱饵。将PDMS芯片放入培养箱中，设置为37摄氏度，含5%二氧化碳一天。孵育后，当细胞附着在PDMS芯片中的PDMS膜上时，将PDMS芯片上的空气微通道出口连接到真空控制器系统。当细胞附着时，将在显微镜下看到细长的正常细胞形式。与悬浮的圆形细胞形成对比。打开电磁阀和真空泵。打开真空调节器并根据应变调整压力水平。然后，将PDMS芯片和设置为37摄氏度的培养箱与5%二氧化碳放置24小时。准备几个电磁阀，真空过滤器，真空调节器，真空泵，蠕动泵和控制电磁阀的PLC。对 PLC 控制器进行编程，并将开启关闭时间间隔设置为 1 赫兹。将电磁阀连接到编程控制器。将真空泵的入口连接到真空过滤器，然后将真空过滤器的出口连接到真空调节器。将真空调节器的出口连接到电磁阀。最后，将电磁阀的出口连接到PDMS芯片的空气微通道的出口。将蠕动泵的出口连接到PDMS芯片的培养基微通道的入口，蠕动泵的入口连接到培养基微通道PDMS芯片的出口，用于培养基更换和药物处理。由调节器调整应变幅度，由微控制器调整应变频率。在PDMS芯片中，使用活和死测定在细胞培养的第三天和第5天估计人主动脉平滑肌细胞系CRL 1990的活力。在第三天和第五天发现细胞活力高于90%。在PDMS芯片中CRL 1990的细胞骨架F-肌动蛋白染色显示正常的细胞形态，并且在拉伸24小时后细胞垂直于菌株排列。与静态条件相比，收缩标志物SM22和CNN1在应变条件下的免疫荧光更高。此外，SM22和CNN1基因在平滑肌细胞的应变条件下上调。BAV和TAV的F-肌动蛋白染色，胸主动脉瘤和患者来源的原发性人主动脉平滑肌细胞的解剖，在拉伸24小时后，细胞形态正常，细胞垂直于应变方向排列。BAVTAAD和TAV胸主动脉瘤和夹层的SM22和CNN1荧光在应变条件下高于静态条件下。与SM22和CNN1的基因表达水平相似，与静态条件相比，它们在应变条件下也上调。分离原发性HASMC时应注意无菌原则。PDMS实验室和膜用等离子体处理后，表面不应弄脏。基于该协议，内皮细胞可以与平滑肌细胞共培养，并且可以将纯粹的压力给予具有循环应变的细胞以进一步模拟更多的机械力。

Research

JoVE Journal

Bioengineering

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

蛋白质和细胞坐月子创建二维图案衬底

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

科研

生物工程

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

定向细胞自我组装制造，为生物力学分析与组织工程细胞衍生的组织环

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Rotating Cell Culture Systems for Human Cell Culture: Human Trophoblast Cells as a Model

旋转的人类细胞培养的细胞培养系统：作为一种模式的滋养细胞

The field of human trophoblast research aids in  understanding the complex environment established during placentation. Due to the  nature of these ...

Monitoring the Wall Mechanics During Stent Deployment in a Vessel

在一个容器的支架部署在监测的墙力学

Clinical trials have reported different restenosis rates for various stent designs1. It is speculated that stent-induced strain concentrations on the ...

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

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 ...

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

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 ...

The Encapsulation of Cell-free Transcription and Translation Machinery in Vesicles for the Construction of Cellular Mimics

无细胞的转录和翻译的机械囊泡的封装建设模拟蜂窝

As interest shifts from individual molecules to systems of molecules, an increasing number of laboratories have sought to build from the bottom up ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

适应静电过程中提供三个独特的环境为一个三层次<em&gt;在体外</em&gt;的气道壁模型

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Construction of Defined Human Engineered Cardiac Tissues to Study Mechanisms of Cardiac Cell Therapy

定义人工程心脏组织建设探讨心肌细胞疗法的机理

Human cardiac tissue engineering can fundamentally impact therapeutic discovery through the development of new species-specific screening systems that ...

Lucifer Yellow - A Robust Paracellular Permeability Marker in a Cell Model of the Human Blood-brain Barrier

路西法黄 - 人类血脑屏障细胞模型中一个健壮的细胞渗透标记

The blood-brain barrier BBB consists of endothelial cells that form a barrier between the systemic circulation and the brain to prevent the exchange of ...