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
Summary
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。
Abstract
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。
Introduction
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。
Protocol
Representative Results
Discussion
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| 4% paraformaldehyde | Beyotime | P0099-100ml | Used for cell immobilization |
| Alexa Fluor 350-labeled Goat Anti-Rabbit IgG | Beyotime | A0408 | Antibodies used for immunostaining |
| Bovine serum albumin | Beyotime | ST025-20g | |
| Calcium AM/PI | Invitrogen | L3224 | |
| Cell culture flask | Corning | 430639 | |
| CNN1 | Abcam | Ab46794 | |
| Commercial flexible PDMS membrane |
Hangzhou Bald Advanced Materials | KYQ-200 | |
| F-actin | Invitrogen | R415 | |
| FBS | Sigma | M8318 | |
| Hoses | Runze Fluid | 96410 | 1 mm inner diameter; 3 mm outer diameter; 1 mm wall thickness; Official website address: https://www.runzefluidsystem.com |
| Human aortic smooth muscle cell line CRL1999 |
ATCC | Lot Number:70019189 | |
| Image J | Imagej.net/fiji/downloads | Free Download: https://fiji.sc | Imaging platform that is used to identify fluorescence intensity |
| Incubator | Thermo Fisher Scientific | Ensures that the temperature, humidity, and light exposure is exactly the same throughout experiment. |
|
| Luer | Runze Fluid | RH-M016 | Official website address: https://www.runzefluidsystem.com. |
| Microscope | Olympus | ||
| mouse collagen | Sigma | C7661 | |
| Oxygen plasma | Changzhou Hongming Instrument | HM-Plasma5L | |
| Pasteur pipette | Biologix | 30-0138A1 | |
| PBS | Beyotime | C0221A | |
| Pen-Strep | Sigma | P4458-100ml | Antibiodics used to prevent bacterial contamination of cells during culture. |
| peristaltic pump | Kamoer | F01A-STP-B046 | |
| Petri dish | Corning | 430167 | |
| PLC controller | Zhejiang Jun Teng (BenT) CNC factory | BR010-11T8X2M | The detailed program setting can be found in supplementary. Official website address: files.http://www.btcnc.net |
| polydimethylsiloxane (PDMS) | Dow Corning | Sylgard 184 | |
| SM22 | Abcam | ab14106 | |
| SMCM | ScienCell | Cat 1101 | |
| solenoid valve | SMC (China) | VQZ300 | |
| Syringe | Becton,Dickinson and Company | 300841 | |
| Triton-X 100 | Beyotime | ST795 | To penetrate cell membranes |
| Trizol | Invitrogen | 10296010 | Used for RNA extraction |
| trypsin | Sigma | 15400054 | |
| vacuum filter | SMC (China) | ZFC5-6 | Official website address: https://www.smc.com.cn |
| vacuum pump | Kamoer | KVP15-KL-S | |
| vacuum regulator | AirTAC | GVR-200-06 | |
| Primers | |||
| Primer Name | Forward (5’ to 3’) | Reverse (5’ to 3’) | |
| SM22 | CCGTGGAGATCCCAACTGG | CCATCTGAAGGCCAATGACAT | |
| CNN1 | CTCCATTGACTCGAACGACTC | CAGGTCTGCGAAACTTCTTAGA |
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