Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production
Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production
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DOI：
10.3791/54014
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发表时间：
2016-05-01
影响因子：
1.2
通讯作者：
He, Pingnian
中科院分区：
文献类型：
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作者：
Xu, Sulei;Li, Xiang;He, Pingnian
血管壁上的内皮细胞（ECs）在体内不断暴露于血流中，但培养的内皮细胞通常在静态条件下生长，并表现出促炎表型。虽然微流控装置的发展已经被工程师们接受了二十年，但它们的生物应用仍然有限。通过生物学应用验证的更具生理学相关性的体外微血管模型对于推进该领域并弥合体内和体外研究之间的差距非常重要。在这里，我们介绍了使用具有长期灌注能力的微流体装置开发培养微血管网络的详细程序。我们还展示了其应用于激动剂诱导的EC [Ca2+](i)和一氧化氮（NO）生产的实时定量测量，使用共聚焦和常规荧光显微镜。连续灌注形成的微血管网络显示内皮细胞间连接发育良好。与静态培养的EC单层相比，ve -钙粘蛋白的分布更接近于完整微血管中的分布。在单个细胞水平上定量测量了atp诱导的EC [Ca2+](i)和NO生成的短暂增加，验证了培养微血管的功能。这种微流体装置允许内皮细胞在良好控制的生理相关流动下生长，这使得细胞培养环境比传统的静态二维培养环境更接近体内。微通道网络设计通用性强，制作工艺简单、可重复。该装置可以很容易地集成到共聚焦或传统的显微系统，实现高分辨率成像。最重要的是，由于培养的微血管网络可以由原代人内皮细胞形成，因此该方法将作为一种有用的工具，用于研究患者样本中病理改变的血液成分如何影响人内皮细胞，并为临床问题提供见解。它也可以作为药物筛选的平台。
Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+](i) and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+](i) and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.