(577f) Multilayer Microfluidics For Studying Thrombosis

Neeves, K. B., University of Pennsylvania
Diamond, S. L., University of Pennsylvania

In spite of the identification of thrombin generation as a critical event in forming clots, there is still a critical gap in the knowledge base that centers on how thrombin stabilizes clots under flow via platelet activator versus fibrin generation. The objective of this work was to understand how the generation of thrombin at the surface of platelets stabilizes blood clots. To achieve this objective we developed a multilayer microfluidic device which mimics the release of soluble factors (thrombin, ADP, thromboxane A2) at a defined mass flux from the platelet surface into flowing plasma or blood.

Microfluidic devices were fabricated using standard soft lithography techniques. A multilayer device consisted of two sets intersecting of channels (100 micrometers X 100 micrometers) separated by a membrane and held together by vacuum assisted bonding. Upon completion of an experiment, the vacuum was released, and the fibrin clots were further analyzed by confocal microscopy. The flux of soluble agonists was controlled by varying the concentration of the agonist (concentration gradient), the relative flow rate of intersecting channels (pressure gradient), and the pore size of a polycarbonate membrane. We validated the device using a simple two-component system in which thrombin was introduced at a flux of 10-21-10-19 mol/m2-sec into fibrinogen (3 mg/ml) flowing at venous shear rates (50-300 1/sec). This configuration allows the generation of a fibrin deposition phase diagram where the growth rate of fibrin on the membrane surface can be determined as a function of thrombin flux and wall shear rate. While membranes have been used for endothelial permeability studies, this is one of the first reports of their use in thrombosis studies with a defined flux at the interface.