• We have developed a biomimetic microfluidic platelet bioreactor that recapitulates bone marrow and blood vessel microenvironments.• Application of shear stress in this bioreactor triggers physiological proplatelet production, and platelet release.Platelet transfusions total >2.17 million apheresis-equivalent units per year in the United States and are derived entirely from human donors, despite clinically significant immunogenicity, associated risk of sepsis, and inventory shortages due to high demand and 5-day shelf life. To take advantage of known physiological drivers of thrombopoiesis, we have developed a microfluidic human platelet bioreactor that recapitulates bone marrow stiffness, extracellular matrix composition, micro-channel size, hemodynamic vascular shear stress, and endothelial cell contacts, and it supports high-resolution live-cell microscopy and quantification of platelet production. Physiological shear stresses triggered proplatelet initiation, reproduced ex vivo bone marrow proplatelet production, and generated functional platelets. Modeling human bone marrow composition and hemodynamics in vitro obviates risks associated with platelet procurement and storage to help meet growing transfusion needs. (Blood. 2014;124(12):1857-1867) IntroductionAlthough platelets (PLTs) play critical roles in hemostasis, 1 angiogenesis, 2 and innate immunity, 3 PLT production remains poorly understood. Consequently, PLT units are derived entirely from human donors, despite serious clinical concerns owing to their immunogenicity and associated risk of sepsis. 4 More than 2.17 million apheresisequivalent PLT units are transfused yearly in the United States 5,6 at a cost of .$1 billion per year. Although demand for PLT transfusions has increased markedly in the past decade, a near-static pool of donors and a 5-day PLT unit shelf life resulting from bacterial contamination 7 and storage-related PLT deterioration, 8 have resulted in significant PLT shortages. 9 Furthermore, artificial platelet substitutes have failed to replace physiological platelet products. 10 An efficient, donorindependent PLT bioreactor capable of generating clinically significant numbers of functional human PLTs is necessary to obviate risks associated with PLT procurement and storage, and help meet growing transfusion needs. In vivo, megakaryocytes (MKs) PLT progenitors sit outside blood vessels in the bone marrow (BM) and extend long, branching cellular structures designated proPLTs into the circulation from which PLTs are released. 11-15 Nearly 100% of human adult MKs must produce ;10 3 PLTs each to account for circulating PLT counts. 16 Although functional human PLTs were first grown in vitro in 1995, 17 to date only ;10% of human MKs initiate proPLT production in culture. This results in yields of 10 122 PLTs per CD34 1 cord blood-derived or embryonic stem cell-derived MK, 18 which are themselves of limited availability, constituting a significant bottleneck in the ex vivo production of a PLT transfusion unit. Although second-generation c...

Intravascular blood clots form in an environment in which hydrodynamic forces dominate and in which fluid-mediated transport is the primary means of moving material. The clotting system has evolved to exploit fluid dynamic mechanisms and to overcome fluid dynamic challenges to ensure that clots that preserve vascular integrity can form over the wide range of flow conditions found in the circulation. Fluid-mediated interactions between the many large deformable red blood cells and the few small rigid platelets lead to high platelet concentrations near vessel walls where platelets contribute to clotting. Receptor-ligand pairs with diverse kinetic and mechanical characteristics work synergistically to arrest rapidly flowing cells on an injured vessel. Variations in hydrodynamic stresses switch on and off the function of key clotting polymers. Protein transport to, from, and within a developing clot determines whether and how fast it grows. We review ongoing experimental and modeling research to understand these and related phenomena.

To cite this article: Neeves KB, Maloney SF, Fong KP, Schmaier AA, Kahn ML, Brass LF, Diamond SL. Microfluidic focal thrombosis model for measuring murine platelet deposition and stability: PAR4 signaling enhances shear-resistance of platelet aggregates. J Thromb Haemost 2008; 6:Summary. Background: Flow chambers allow the ex vivo study of platelet response to defined surfaces at controlled wall shear stresses. However, most assays require 1-10 mL of blood and are poorly suited for murine whole blood experiments. Objective: To measure murine platelet deposition and stability in response to focal zones of prothrombotic stimuli using 100 lL of whole blood and controlled flow exposure. Methods: Microfluidic methods were used for patterning acid-soluble collagen in 100 lm · 100 lm patches and creating flow channels with a volume of 150 nL. Within 1 min of collection into PPACK and fluorescent anti-mouse CD41 mAb, whole blood from normal mice or from mice deficient in the integrin a 2 subunit was perfused for 5 min over the patterned collagen. Platelet accumulation was measured at venous and arterial wall shear rates. After 5 min, thrombus stability was measured with a Ôshear step-upÕ to 8000 s )1. Results: Wild-type murine platelets adhered and aggregated on collagen in a biphasic sheardependent manner with increased deposition from 100 to 400 s )1 , but decreased deposition at 1000 s )1. Adhesion to patterned collagen was severely diminished for platelets lacking a functional a 2 b 1 integrin. Those integrin a 2 -deficient platelets that did adhere were removed from the surface when challenged to shear step-up. PAR4 agonist (AYPGKF) treatment of the thrombus at 5 min enhanced aggregate stability during the shear step-up. Conclusions: PAR4 signaling enhances aggregate stability by mechanisms independent of other thrombindependent pathways such as fibrin formation.

Propulsion at the microscale requires unique strategies such as the undulating or rotating filaments that microorganisms have evolved to swim. These features however can be difficult to artificially replicate and control, limiting the ability to actuate and direct engineered microdevices to targeted locations within practical timeframes. An alternative propulsion strategy to swimming is rolling. Here we report that low-strength magnetic fields can reversibly assemble wheel-shaped devices in situ from individual colloidal building blocks and also drive, rotate and direct them along surfaces at velocities faster than most other microscale propulsion schemes. By varying spin frequency and angle relative to the surface, we demonstrate that microwheels can be directed rapidly and precisely along user-defined paths. Such in situ assembly of readily modified colloidal devices capable of targeted movements provides a practical transport and delivery tool for microscale applications, especially those in complex or tortuous geometries.