ATPSpin: A Single Microfluidic Platform that Produces Diversified ATPS-Alginate Microfibers.

Microfluidic spinning is emerging as a useful technique in the fabrication of alginate fibers, enabling applications in drug screening, disease modeling, and disease diagnostics. In this paper, by capitalizing on the benefits of aqueous two-phase systems (ATPS) to produce diverse alginate fiber forms, we introduce an ATPS-Spinning platform (ATPSpin). This ATPS-enabled method efficiently circumvents the rapid clogging challenges inherent to traditional fiber production techniques by regulating the interaction between alginate and cross-linking agents like Ba2+ ions. By varying system parameters under the guidance of a regime map, our system produces several fiber forms─solid, hollow, and droplet-filled─consistently and reproducibly from a single device. We demonstrate that the resulting alginate fibers possess distinct features, including biocompatibility. We also encapsulate HEK293 cells in the microfibers as a proof-of-concept that this versatile microfluidic fiber generation platform may have utility in tissue engineering and regenerative medicine applications.

Microfluidically-generated Encapsulated Spheroids (µ-GELS): An All-Aqueous Droplet Microfluidics Platform for Multicellular Spheroids Generation.

Spheroids are three-dimensional clusters of cells that serve as in vitro tumor models to recapitulate in vivo morphology. A limitation of many existing on-chip platforms for spheroid formation is the use of cytotoxic organic solvents as the continuous phase in droplet generation processes. All-aqueous methods do not contain cytotoxic organic solvents but have so far been unable to achieve complete hydrogel gelation on chip. Here, we describe an enhanced droplet microfluidic platform that achieves on-chip gelation of all-aqueous hydrogel multicellular spheroids (MCSs). Specifically, we generate dextran-alginate droplets containing MCF-7 breast cancer cells, surrounded by polyethylene glycol, at a flow-focusing junction. Droplets then travel to a second flow-focusing junction where they interact with calcium chloride and gel on chip to form hydrogel MCSs. On-chip gelation of the MCSs is possible here because of an embedded capillary at the second junction that delays the droplet gelation, which prevents channel clogging problems that would otherwise exist. In drug-free experiments, we demonstrate that MCSs remain viable for 6 days. We also confirm the applicability of this system for cancer drug testing by observing that dose-dependent cell death is achievable using doxorubicin.

Water-in-water droplet microfluidics: A design manual.

Droplet microfluidics is utilized in a wide range of applications in biomedicine and biology. Applications include rapid biochemical analysis, materials generation, biochemical assays, and point-of-care medicine. The integration of aqueous two-phase systems (ATPSs) into droplet microfluidic platforms has potential utility in oil-free biological and biomedical applications, namely, reducing cytotoxicity and preserving the native form and function of costly biomolecular reagents. In this review, we present a design manual for the chemist, biologist, and engineer to design experiments in the context of their biological applications using all-in-water droplet microfluidic systems. We describe the studies achievable using these systems and the corresponding fabrication and stabilization methods. With this information, readers may apply the fundamental principles and recent advancements in ATPS droplet microfluidics to their research. Finally, we propose a development roadmap of opportunities to utilize ATPS droplet microfluidics in applications that remain underexplored.

Evaporation-Driven Water-in-Water Droplet Formation.

We present new observations of aqueous two-phase system (ATPS) thermodynamic and interfacial phenomena that occur inside sessile droplets due to water evaporation. Sessile droplets that contain polymeric solutions, which are initially in equilibrium in a single phase, are observed at their three-phase liquid-solid-air contact line. As evaporation of a sessile droplet proceeds, we find that submicron secondary water-in-water (W/W) droplets emerge spontaneously at the edges of the mother sessile droplet due to the resulting phase separation from water evaporation. To understand this phenomenon, we first study the secondary W/W droplet formation process on different substrate materials, namely, glass, polycarbonate (PC), thermoplastic elastomer (TPE), poly(dimethylsiloxane)-coated glass slide (PDMS substrate), and Teflon-coated glass slide (Teflon substrate), and show that secondary W/W droplet formation arises only in lower-contact-angle substrates near the three-phase contact line. Next, we characterize the size of the emergent secondary W/W droplets as a function of time. We observe that W/W drops are formed, coalesced, aligned, and trapped along the contact line of the mother droplet. We demonstrate that this W/W multiple emulsion system can be used to encapsulate magnetic particles and blood cells, and achieve size-based separation. Finally, we show the applicability of this system for protein sensing. This is the first experimental observation of evaporation-induced secondary W/W droplet generation in a sessile droplet. We anticipate that the phenomena described here may be applicable to some biological assay applications, for example, biomarker detection, protein sensing, and point-of-care diagnostic testing.

A novel abrasive water jet machining technique for rapid fabrication of three-dimensional microfluidic components.

Microfluidic lab-on-a-chip devices are usually fabricated using replica molding, with poly(dimethylsiloxane) (PDMS) casting on a mold. Most common techniques used to fabricate microfluidic molds, such as photolithography and soft lithography, require costly facilities such as a cleanroom, and complicated steps, especially for the fabrication of three-dimensional (3D) features. For example, an often-desired 3D microchannel feature consists of intersecting channels with depth variations. This type of 3D flow focusing geometry has applications in flow cytometry and droplet generation. Various manufacturing techniques have recently been developed for the rapid fabrication of such 3D microfluidic features. In this paper, we describe a new method of mold fabrication that utilizes water jet cutting technology to fabricate free-standing structures on mild steel sheets to make a mold for PDMS casting. As a proof-of-concept, we use this fabrication technique to make a PDMS chip that has a 3D flow focusing junction, an inlet for the sample fluid, two inlets for the sheath fluid, and an outlet. The flow focusing junction is patterned into the PDMS slab with an abrupt, nearly stepwise change to the depth of the microchannel junction. We use confocal microscopy to visualize the 3D flow focusing of a sample flow using this geometry, and we also use the same geometry to generate water-in-oil droplets. This alternative approach to create microfluidic molds is versatile and may find utility in reducing the cost and complexity involved in fabricating 3D features in microfluidic devices.

Microfluidic Generation of All-Aqueous Double and Triple Emulsions.

Higher order emulsions are used in a variety of different applications in biomedicine, biological studies, cosmetics, and the food industry. Conventional droplet generation platforms for making higher order emulsions use organic solvents as the continuous phase, which is not biocompatible and as a result, further washing steps are required to remove the toxic continuous phase. Recently, droplet generation based on aqueous two-phase systems (ATPS) has emerged in the field of droplet microfluidics due to their intrinsic biocompatibility. Here, a platform to generate all-aqueous double and triple emulsions by introducing pressure-driven flows inside a microfluidic hybrid device is presented. This system uses a conventional microfluidic flow-focusing geometry coupled with a coaxial microneedle and a glass capillary embedded in flow-focusing junctions. The configuration of the hybrid device enables the focusing of two coaxial two-phase streams, which helps to avoid commonly observed channel-wetting problems. It is shown that this approach achieves the fabrication of higher-order emulsions in a poly(dimethylsiloxane)-based microfluidic device, and controls the structure of the all-aqueous emulsions. This hybrid microfluidic approach allows for facile higher-order biocompatible emulsion formation, and it is anticipated that this platform will find utility for generating biocompatible materials for various biotechnological applications.

Microneedle-assisted microfluidic flow focusing for versatile and high throughput water-in-water droplet generation.

Microdroplets have been utilized for a wide range of applications in biomedicine and biological studies. Despite the importance of such droplets, their fabrication is associated with difficulties in practice that emerge from the incompatible nature of chemicals, such as surfactants and organic solvents, with biological environments. Therefore, microfluidic methods have recently emerged that create biocompatible water-in-water droplets based on aqueous two-phase systems (ATPS), most commonly composed of water and incompatible polymers, dextran (DEX) and polyethylene glycol (PEG). However, so far, DEX- and PEG-based water-in-water droplet generation schemes have been plagued with low throughput, and most systems can only generate DEX-in-PEG droplets; PEG-in-DEX droplets have been elusive due to chemical interactions between the polymers and channel walls. Here, we describe a simple approach to generate water-in-water microdroplets passively at a high throughput of up to 850 Hz, and obtain both DEX-in-PEG and PEG-in-DEX droplets. Specifically, our method involves a simple modification to the conventional microfluidic flow focusing geometry, by the insertion of a microneedle to the flow focusing junction, which causes three-dimensional (3D) flow focusing of the dispersed phase fluid. We observe that the 3D flow focusing of the dispersed phase enables excellent control of droplet diameters, ranging from 5 to 65 µm, and achieves a high throughput. Moreover, we report the passive microfluidic generation of PEG-in-DEX droplets for the first time, because in our system the 3D flow focusing of the disperse phase separates the disperse PEG phase from the channel walls, negating the commonly observed wall wetting issues of the PEG phase. We expect this microfluidic approach to be useful in increasing the versatility and throughput of water-in-water droplet microfluidics, and help enable future biotechnological applications, such as microparticle-based drug delivery, cell encapsulation for single cell analysis, and immunoisolation for cell transplantation.

Microfluidic diamagnetic water-in-water droplets: a biocompatible cell encapsulation and manipulation platform.

Droplet microfluidics enables cellular encapsulation for biomedical applications such as single-cell analysis, which is an important tool used by biologists to study cells on a single-cell level, and understand cellular heterogeneity in cell populations. However, most cell encapsulation strategies in microfluidics rely on random encapsulation processes, resulting in large numbers of empty droplets. Therefore, post-sorting of droplets is necessary to obtain samples of purely cell-encapsulating droplets. With the recent advent of aqueous two-phase systems (ATPS) as a biocompatible alternative of the conventional water-in-oil droplet systems for cellular encapsulation, there has also been a focus on integrating ATPS with droplet microfluidics. In this paper, we describe a new technique that combines ATPS-based water-in-water droplets with diamagnetic manipulation to isolate single-cell encapsulating water-in-water droplets, and achieve a purity of 100% in a single pass. We exploit the selective partitioning of ferrofluid in an ATPS of polyethylene glycol-polypropylene glycol-polyethylene glycol triblock copolymer (PEG-PPG-PEG) and dextran (DEX), to achieve diamagnetic manipulation of water-in-water droplets. A cell-triggered Rayleigh-Plateau instability in the dispersed phase thread results in a size distinction between the cell-encapsulating and empty droplets, enabling diamagnetic separation and sorting of the cell-encapsulating droplets from empty droplets. This is a simple and biocompatible all-aqueous platform for single-cell encapsulation and droplet manipulation, with applications in single-cell analysis.

Controlled Electrospray Generation of Nonspherical Alginate Microparticles.

Electrospraying is a technique used to generate microparticles in a high throughput manner. For biomedical applications, a biocompatible electrosprayed material is often desirable. Using polymers, such as alginate hydrogels, makes it possible to create biocompatible and biodegradable microparticles that can be used for cell encapsulation, to be employed as drug carriers, and for use in 3D cell culturing. Evidence in the literature suggests that the morphology of the biocompatible microparticles is relevant in controlling the dynamics of the microparticles in drug delivery and 3D cell culturing applications. Yet, most electrospray-based techniques only form spherical microparticles, and there is currently no widely adopted technique for producing nonspherical microparticles at a high throughput. Here, we demonstrate the generation of nonspherical biocompatible alginate microparticles by electrospraying, and control the shape of the microparticles by varying experimental parameters such as chemical concentration and the distance between the electrospray tip and the particle-solidification bath. Importantly, we show that these changes to the experimental setup enable the synthesis of different shaped particles, and the systematic change in parameters, such as chemical concentration, result in monotonic changes to the particle aspect ratio. We expect that these results will find utility in many biomedical applications that require biocompatible microparticles of specific shapes.

Experimental Investigation of Left Ventricular Flow Patterns After Percutaneous Edge-to-Edge Mitral Valve Repair.

Mitral valve percutaneous edge-to-edge repair (PEtER) is a viable solution in high-risk patients with severe symptomatic mitral regurgitation. However, the generated double-orifice configuration poses challenges for the evaluation of the hemodynamic performance of the mitral valve and may alter flow patterns in the left ventricle (LV) during diastole. This in vitro study aims to evaluate the hemodynamic modifications following a simulated PEtER. A custom-made mitral valve was developed, and two configurations were tested: (i) a single-orifice valve with mitral regurgitation and (ii) a double-orifice mitral valve configuration following PEtER. The hemodynamic performance of the valve was evaluated using Doppler echocardiography and catheterization, while the flow patterns in the LV were investigated using particle image velocimetry (PIV). The tests were run at a stroke volume of 65 mL and a heart rate of 70 bpm. PEtER was found to significantly reduce the regurgitant volume (15 vs. 34 mL). There was a good agreement between Doppler and catheter transmitral pressure gradients (peak gradient: 9 vs. 7 mm Hg; mean gradient: 4 vs. 3 mm Hg) as well as an excellent agreement between maximal velocity measured by Doppler and PIV (1.60 vs. 1.58 m/s). Vortex development in the LV during diastole was significantly different after repair. PEtER significantly increased the amplitude of Reynolds and viscous shear stresses, as well as the number of high shear regions in the LV, potentially promoting thromboembolism events.