Stanford Genome Technology Center, Stanford University, 3165 Porter Drive, Palo Alto, CA 94304, USA.
Bioengineering Department, University of Louisville, 2301 S. Third St., Paul C. Lutz Hall, # 419, Louisville, KY 40292, USA.
Lab Chip. 2022 Nov 22;22(23):4705-4716. doi: 10.1039/d2lc00555g.
We introduce a microfluidic impedance platform to electrically monitor in real-time, endothelium monolayers undergoing fluid shear stress. Our platform incorporates sensing electrodes (SEs) that measure cell behavior and cell-free control electrodes that measure cell culture media resistance simultaneously but independently from SEs. We evaluated three different cellular subpopulations sizes through 50, 100, and 200 μm diameter SEs. We tested their utility in measuring the response of human umbilical vein endothelial cells (HUVECs) at static, constant (17.6 dyne per cm), and stepped (23.7-35-58.1 dyne per cm) shear stress conditions. For 14 hours, we collected the impedance spectra (100 Hz-1 MHz) of sheared cells. Using equivalent circuit models, we extracted monolayer permeability (), cell membrane capacitance, and cell culture media resistance. Platform evaluation concluded that: (1) 50 μm SEs (∼2 cells) suffered interfacial capacitance and reduced cell measurement sensitivity, (2) 100 μm SEs (∼6 cells) was limited to measuring cell behavior only and cannot measure cell culture media resistance, and (3) 200 μm SEs (∼20 cells) detected cell behavior with accurate prediction of cell culture media resistance. Platform-based shear stress studies indicated a shear magnitude dependent increase in at the onset of acute flow. Consecutive stepped shear conditions did not alter in the same magnitude after shear has been applied. Finally, endpoint staining of VE-cadherin on the actual SEs and endpoint measurements were greater for 23.7-35-58.1 dyne per cm than 17.6 dyne per cm shear conditions.
我们介绍了一种微流控阻抗平台，用于实时电监测经受流体切应力的内皮单层。我们的平台整合了传感电极 (SEs)，可同时但独立于 SEs 测量细胞行为和无细胞培养介质电阻；我们通过 50、100 和 200 μm 直径的 SE 评估了三种不同的细胞亚群大小；我们测试了它们在测量静态、恒定 (17.6 达因/厘米) 和阶跃 (23.7-35-58.1 达因/厘米) 切应力条件下人脐静脉内皮细胞 (HUVECs) 响应的实用性。在 14 小时内，我们收集了被切细胞的阻抗谱 (100 Hz-1 MHz)。使用等效电路模型，我们提取了单层渗透率 (), 细胞膜电容和细胞培养介质电阻。平台评估得出结论：(1) 50 μm SEs (∼2 个细胞) 受到界面电容的影响，降低了细胞测量的灵敏度；(2) 100 μm SEs (∼6 个细胞) 仅局限于测量细胞行为，无法测量细胞培养介质电阻；(3) 200 μm SEs (∼20 个细胞) 检测到细胞行为，并能准确预测细胞培养介质电阻。基于平台的切应力研究表明，急性流动开始时， 随着切应力的增加而增加。在应用切应力后，连续的阶跃切应力条件不会以相同的幅度改变 。最后，实际 SEs 上 VE-cadherin 的终点染色和终点 测量对于 23.7-35-58.1 达因/厘米的切应力条件比 17.6 达因/厘米的切应力条件更大。
