Ku Chia-Jui, D'Amico Oblak Teresa, Spence Dana M
Department of Chemistry, Michigan State University, East Lansing, Michigan 48824, USA.
Anal Chem. 2008 Oct 1;80(19):7543-8. doi: 10.1021/ac801114j. Epub 2008 Aug 27.
A simple method for immobilizing endothelial cells in the channels of a microfluidic device fabricated with soft lithography is presented that requires no surface oxidation of the substrate material used in conjunction with the microfluidic device and is operable even with a reversible seal. Specifically, optimal conditions for culturing bovine pulmonary artery endothelial cells (bPAECs) to the surface of a Petri dish were investigated. The parameters investigated included fibronectin concentration, temperature, seeding density, and immobilization time. To enhance the utility of the device, all optimization studies, and studies involving platelet adhesion to the immobilized endothelium, were performed in parallel channels, thereby enabling improved throughput over a single channel device. The optimal conditions for cell immobilization included coating the Petri dish with 100 microg/mL fibronectin, a seeding cell density of 1.00 x 10(5) cells mL(-1), and an immobilization time of 90 min at 37 degrees C. The device was then employed to monitor the physical interaction (adhesion) of platelets to the immobilized endothelium in the presence of a known platelet activator (ADP) and a drug inhibitor of platelet activation. The number of platelets adhering to the endothelial cells in the channels increased from 17.0 +/- 2.3 in the absence of ADP to 63.2 +/- 2.4 in the presence of 5.00 microM ADP. Moreover, the data presented here also shows that inhibition of endothelium nitric oxide (NO) production, a recognized inhibitor of platelet adhesion to the endothelium, increased the number of platelets adhering to the surface to 35.4 +/- 1.0. In the presence of NO inhibition and 5.00 microM ADP, the affect on platelet adhesion was further increased to 127 +/- 5.2. Finally, this device was employed to investigate the effect of a drug known to inhibit platelet adhesion (clopidogrel) and, in the presence of the drug, the platelet adhesion due to activation by 5.00 microM ADP decreased to 24.0 +/- 3.8. This work is the first representation of multiple cell types physically interacting in the channels of a microfluidic device and further demonstrates the potential of these devices in the drug discovery process and drug efficacy studies.
本文介绍了一种利用软光刻技术将内皮细胞固定在微流控装置通道内的简单方法，该方法无需对与微流控装置结合使用的基底材料进行表面氧化，即使采用可逆密封也可操作。具体而言，研究了在培养皿表面培养牛肺动脉内皮细胞（bPAECs）的最佳条件。研究的参数包括纤连蛋白浓度、温度、接种密度和固定时间。为提高该装置的实用性，所有优化研究以及涉及血小板与固定化内皮细胞黏附的研究均在平行通道中进行，从而实现了比单通道装置更高的通量。细胞固定的最佳条件包括用100μg/mL纤连蛋白包被培养皿、接种细胞密度为1.00×10⁵个细胞/mL⁻¹以及在37℃下固定90分钟。然后使用该装置监测在存在已知血小板激活剂（ADP）和血小板激活药物抑制剂的情况下血小板与固定化内皮细胞的物理相互作用（黏附）。在不存在ADP时，通道内黏附在内皮细胞上的血小板数量为17.0±2.3，在存在5.00μM ADP时增加到63.2±2.4。此外，本文提供的数据还表明，抑制内皮细胞一氧化氮（NO）的产生（一种公认的血小板与内皮细胞黏附的抑制剂）会使黏附在表面的血小板数量增加到35.4±1.0。在存在NO抑制和5.00μM ADP的情况下，对血小板黏附的影响进一步增加到127±5.2。最后，使用该装置研究了一种已知可抑制血小板黏附的药物（氯吡格雷）的作用，在存在该药物的情况下，由5.00μM ADP激活引起的血小板黏附减少到24.0±3.8。这项工作首次展示了多种细胞类型在微流控装置通道内的物理相互作用，并进一步证明了这些装置在药物发现过程和药物疗效研究中的潜力。
