In this article high-yield (77%) and high-speed (2700 cells s(-1)) single cell droplet encapsulation is described using a Dean-coupled inertial ordering of cells in a simple curved continuous microchannel. By introducing the Dean force, the particles will order to one equilibrium position after travelling less than 1 cm. We use a planar curved microchannel structure in PDMS to spatially order two types of myeloid leukemic cells (HL60 and K562 cells), enabling deterministic single cell encapsulation in picolitre drops. An efficiency of up to 77% was reached, overcoming the limitations imposed by Poisson statistics for random cell loading, which yields only 37% of drops containing a single cell. Furthermore, we confirm that > 90% of the cells remain viable. The simple planar structure and high throughput provided by this passive microfluidic approach makes it attractive for implementation in lab on a chip (LOC) devices for single cell applications using droplet-based platforms.

Thrombi that occlude blood vessels can be resolved with fibrinolytic agents that degrade fibrin, the polymer that forms between and around platelets to provide mechanical stability. Fibrinolysis rates however are often constrained by transport-limited delivery to and penetration of fibrinolytics into the thrombus. Here, these limitations are overcome with colloidal microwheel (µwheel) assemblies functionalized with the fibrinolytic tissue-type plasminogen activator (tPA) that assemble, rotate, translate, and eventually disassemble via applied magnetic fields. These microwheels lead to rapid fibrinolysis by delivering a high local concentration of tPA to induce surface lysis and, by taking advantage of corkscrew motion, mechanically penetrating into fibrin gels and platelet-rich thrombi to initiate bulk degradation. Fibrinolysis of plasma-derived fibrin gels by tPA-microwheels is fivefold faster than with 1 µg mL tPA. µWheels following corkscrew trajectories can also penetrate through 100 µm sized platelet-rich thrombi formed in a microfluidic model of hemostasis in ≈5 min. This unique combination of surface and bulk dissolution mechanisms with mechanical action yields a targeted fibrinolysis strategy that could be significantly faster than approaches relying on diffusion alone, making it well-suited for occlusions in small or penetrating vessels not accessible to catheter-based removal.

In this article, we present a microfluidic device capable of successive high-yield single-cell encapsulation in droplets, with additional droplet pairing, fusion, and shrinkage. Deterministic single-cell encapsulation is realized using Dean-coupled inertial ordering of cells in a Yin-Yang-shaped curved microchannel using a double T-junction, with a frequency over 2000 Hz, followed by controlled droplet pairing with a 100% success rate. Subsequently, droplet fusion is realized using electrical actuation resulting in electro-coalescence of two droplets, each containing a single HL60 cell, with 95% efficiency. Finally, volume reduction of the fused droplet up to 75% is achieved by a triple pitchfork structure. This droplet volume reduction is necessary to obtain close cell-cell membrane contact necessary for final cell electrofusion, leading to hybridoma formation, which is the ultimate aim of this research.

A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell-dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.

Hemostasis is the process of sealing a vascular injury with a thrombus to arrest bleeding. The type of thrombus that forms depends on the nature of the injury and hemodynamics. There are many models of intravascular thrombus formation whereby blood is exposed to prothrombotic molecules on a solid substrate. However, there are few models of extravascular thrombus formation whereby blood escapes into the extravascular space through a hole in the vessel wall. Here, we describe a microfluidic model of hemostasis that includes vascular, vessel wall, and extravascular compartments. Type I collagen and tissue factor, which support platelet adhesion and initiate coagulation, respectively, were adsorbed to the wall of the injury channel and act synergistically to yield a stable thrombus that stops blood loss into the extravascular compartment in ~7.5 min. Inhibiting factor VIII to mimic hemophilia A results in an unstable thrombus that was unable to close the injury. Treatment with a P2Y12 antagonist to reduce platelet activation prolonged the closure time two-fold compared to controls. Taken together, these data demonstrate a hemostatic model that is sensitive to both coagulation and platelet function and can be used to study coagulopathies and platelet dysfunction that result in excessive blood loss.

We present a microfluidic chip that enables electrofusion of cells in microdroplets, with exchange of nuclear components. It is shown, to our knowledge for the first time, electrofusion of two HL60 cells, inside a microdroplet. This is the crucial intermediate step for controlled hybridoma formation where a B cell is electrofused with a myeloma cell. We use a microfluidic device consisting of a microchannel structure in PDMS bonded to a glass substrate through which droplets with two differently stained HL60 cells are transported. An array of six recessed platinum electrode pairs is used for electrofusion. When applying six voltage pulses of 2–3 V, the membrane electrical field is about 1 MV/cm for 1 ms. This results in electrofusion of these cells with a fusion yield of around 5%. The operation with individual cell pairs, the appreciable efficiency and the potential to operate in high-throughput (up to 500 cells sec−1) makes the microdroplet fusion technology a promising platform for cell electrofusion, which has the potential to compete with the conventional methods. Besides, this platform is not restricted to cell fusion but is also applicable to various other cell-based assays such as single cell analysis and differentiation assays.

Platelet adhesion and aggregation, coagulation, fibrin formation, and fibrinolysis are regulated by the forces and flows imposed by blood at the site of a vascular injury. Flow chambers designed to observe these events are an indispensable part of doing hemostasis and thrombosis research, especially with human blood. Microfluidic methods have provided the flexibility to design flow chambers with complex geometries and features that more closely mimic the anatomy and physiology of blood vessels. Additionally, microfluidic systems with integrated optics and/or pressure sensors and on-board signal processing could transform what have been primarily research tools into clinical assays. In this review, we describe a historical review of how flow-based approaches have informed mechanisms in genetic bleeding disorders, challenges and potential solutions for developing models of bleeding in vitro, and outstanding issues that need to be addressed prior to their use in clinical settings.

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