Interactions between Multiple Cell Types in Parallel Microfluidic Channels: Monitoring Platelet Adhesion to an Endothelium in the Presence of an Anti-Adhesion Drug

Abstract

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 μg/mL fibronectin, a seeding cell density of 1.00 × 10 5 cells mL −1 and an immobilization time of 90 min at 37 °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 μM 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 μM 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 μM 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.

A Molecular Level Understanding of Zinc Activation of C-peptide and its Effects on Cellular Communication in the Bloodstream

“…It seems that almost all of these effects are beneficial. While previous studies have demonstrated that C-peptide affects intracellular mechanisms, its ability to stimulate ATP release from RBCs enables C-peptide to affect intercellular communication [59,60], and mechanisms. To date, our group has demonstrated that RBCderived ATP, stimulated by C-peptide, can increase NO production in endothelial cells and platelets, and affect platelet adhesion to endothelial cells.…”

Polymer-Based Microfluidic Devices for Pharmacy, Biology and Tissue Engineering

“…Studies have shown good viability of living cells when grown on cellular microenvironmental chips [359][360][361][362][363][364][365][366][367][368][369][370]. Microfluidics replaces the parallel plate chambers for cell growth, and shear stress investigations establish the need to match the length scales between the fluidic channels and the cells [359][360][361][362][363][364][365][366][367][368][369][370].…”

“…Microfluidics replaces the parallel plate chambers for cell growth, and shear stress investigations establish the need to match the length scales between the fluidic channels and the cells [359][360][361][362][363][364][365][366][367][368][369][370].…”

Microfluidic technology in vascular research : the endothelial response to shear stress

“…For example, the binding of leukocytes to endothelial cells is an essential step in inflammation, 4 while the endothelium-mediated activation of blood platelets is important in clotting and thrombosis. 6 Multiple reports have been published by groups that studied the adhesion of leukocytes 156 or platelets 58,92,127 to endothelial cells or endothelial cell-derived adhesion factors in microfluidic devices. These reports show that microfluidic cell interaction studies require less sample and reagents than similar, conventional studies.…”

“…Moreover, a number of these studies already show increased throughput by using parallel channels in one device. 58,92,156 Three-dimensional culturing An important factor in vascular cell physiology is its three-dimensional microenvironment. Cells are embedded in an environment that comprises other cells, extracellular matrix proteins, bodily fluids and blood.…”