Interactions between multiple cell types in parallel microfluidic channels: monitoring platelet adhesion to an endothelium in the presence of an anti-adhesion drug.

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.

An altered oxidant defense system in red blood cells affects their ability to release nitric oxide-stimulating ATP.

A novel microflow technique is used to demonstrate that a weakened oxidant defense system found in diabetic erythrocytes leads to decreased levels of deformation-induced release of adenosine triphosphate (ATP) from erythrocytes. Addition of an oxidant to rabbit erythrocytes resulted in a 63% decrease in deformation-induced ATP release before eventually recovering to a value that was statistically equivalent to the initial value. Inhibition of glucose-6-phosphate dehydrogenase prevents recovery from the oxidant attack. Finally, results indicated that the ATP release from the erythrocytes of type II diabetics (91 nM +/- 10 nM) was less than half of that measured from the erythrocytes of healthy controls (190 +/- 10 nM). These data suggest that the antioxidant status of erythrocytes is a critical determinant in the ability of these cells to release ATP, a known nitric oxide stimulus.

Fluorescence monitoring of ATP-stimulated, endothelium-derived nitric oxide production in channels of a poly(dimethylsiloxane)-based microfluidic device.

Intracellular nitric oxide (NO) production in a microfluidic endothelium is detected using fluorescence microscopy. Bovine pulmonary artery endothelial cells (bPAECs) were loaded with the fluorescence probe diaminodifluorofluorescein diacetate (DAF-FM DA), and the subsequent fluorescent DAF-FM DA/NO adduct was measured. Solutions of bradykinin, a well-known stimulus of endothelium-derived NO, activated nitric oxide synthase (NOS) in the immobilized bPAECs. This activation was inhibited using l-nitro arginine methyl ester (L-NAME), a competitive inhibitor of NOS. Importantly, the NO production was also stimulated with adenosine triphosphate (ATP) using concentrations as low as 1 microM. Previous reports on stimulating NO production using an immobilized endothelium in microfluidic channels were limited by the requirement of ATP concentrations of at least 100 microM, a value that is not physiologically relevant. The ability to monitor NO production with ATP concentrations that are similar to in vivo levels of ATP in the microcirculation represents a major advance in the use of microfluidic technology as an in vitro model of the microcirculation.