Benam Kambez H, Mazur Marc, Choe Youngjae, Ferrante Thomas C, Novak Richard, Ingber Donald E
Wyss Institute for Biologically Inspired Engineering, Center for Life Science Building, 5th Floor, 3 Blackfan Circle, Boston, MA, 02115, USA.
University Medical Center Utrecht, Utrecht, 3584, CX, Netherlands.
Methods Mol Biol. 2017;1612:345-365. doi: 10.1007/978-1-4939-7021-6_25.
Organs-on-chips are microfluidic cell culture devices created using microchip manufacturing techniques that contain hollow microchannels lined by living cells, which recreate specialized tissue-tissue interfaces, physical microenvironments, and vascular perfusion necessary to recapitulate organ-level physiology in vitro. Here we describe a protocol for fabrication, culture, and operation of a human lung "small airway-on-a-chip," which contains a differentiated, mucociliary bronchiolar epithelium exposed to air and an underlying microvascular endothelium that experiences fluid flow. First, microengineering is used to fabricate a multilayered microfluidic device that contains two parallel elastomeric microchannels separated by a thin rigid porous membrane; this requires less than 1 day to complete. Next, primary human airway bronchiolar epithelial cells isolated from healthy normal donors or patients with respiratory disease are cultured on the porous membrane within one microchannel while lung microvascular endothelial cells are cultured on the opposite side of the same membrane in the second channel to create a mucociliated epithelium-endothelium interface; this process take about 4-6 weeks to complete. Finally, culture medium containing neutrophils isolated from fresh whole human blood are flowed through the microvascular channel of the device to enable real-time analysis of capture and recruitment of circulating leukocytes by endothelium under physiological shear; this step requires less than 1 day to complete. The small airway-on-a-chip represents a new microfluidic tool to model complex and dynamic inflammatory responses of healthy and diseased lungs in vitro.
芯片器官是利用微芯片制造技术制造的微流控细胞培养装置，其中包含由活细胞排列的中空微通道，可重建体外再现器官水平生理学所需的特殊组织-组织界面、物理微环境和血管灌注。在此，我们描述了一种人肺“芯片上的小气道”的制造、培养和操作方案，该装置包含暴露于空气中的分化的、具有黏液纤毛的细支气管上皮以及经历流体流动的底层微血管内皮。首先，利用微工程技术制造一个多层微流控装置，该装置包含两个由薄刚性多孔膜隔开的平行弹性微通道；这一步骤不到1天即可完成。接下来，将从健康正常供体或呼吸系统疾病患者中分离出的原代人气道细支气管上皮细胞培养在一个微通道内的多孔膜上，同时将肺微血管内皮细胞培养在第二个通道中同一膜的另一侧，以创建一个黏液纤毛上皮-内皮界面；这个过程大约需要4-6周才能完成。最后，将含有从新鲜全血中分离出的中性粒细胞的培养基流经该装置的微血管通道，以便在生理剪切力下实时分析内皮细胞对循环白细胞的捕获和募集；这一步骤不到1天即可完成。芯片上的小气道代表了一种新的微流控工具，可在体外模拟健康和患病肺的复杂动态炎症反应。
