Microfluidic Formulation of Topological Hydrogels for Microtissue Engineering.

Microfluidics has recently emerged as a powerful tool in generation of submillimeter-sized cell aggregates capable of performing tissue-specific functions, so-called microtissues, for applications in drug testing, regenerative medicine, and cell therapies. In this work, we review the most recent advances in the field, with particular focus on the formulation of cell-encapsulating microgels of small "dimensionalities": "0D" (particles), "1D" (fibers), "2D" (sheets), etc., and with nontrivial internal topologies, typically consisting of multiple compartments loaded with different types of cells and/or biopolymers. Such structures, which we refer to as topological hydrogels or topological microgels (examples including core-shell or Janus microbeads and microfibers, hollow or porous microstructures, or granular hydrogels) can be precisely tailored with high reproducibility and throughput by using microfluidics and used to provide controlled "initial conditions" for cell proliferation and maturation into functional tissue-like microstructures. Microfluidic methods of formulation of topological biomaterials have enabled significant progress in engineering of miniature tissues and organs, such as pancreas, liver, muscle, bone, heart, neural tissue, or vasculature, as well as in fabrication of tailored microenvironments for stem-cell expansion and differentiation, or in cancer modeling, including generation of vascularized tumors for personalized drug testing. We review the available microfluidic fabrication methods by exploiting various cross-linking mechanisms and various routes toward compartmentalization and critically discuss the available tissue-specific applications. Finally, we list the remaining challenges such as simplification of the microfluidic workflow for its widespread use in biomedical research, bench-to-bedside transition including production upscaling, further in vivo validation, generation of more precise organ-like models, as well as incorporation of induced pluripotent stem cells as a step toward clinical applications.

Stochastic Jetting and Dripping in Confined Soft Granular Flows.

We report new dynamical modes in confined soft granular flows, such as stochastic jetting and dripping, with no counterpart in continuum viscous fluids. The new modes emerge as a result of the propagation of the chaotic behavior of individual grains-here, monodisperse emulsion droplets-to the level of the entire system as the emulsion is focused into a narrow orifice by an external viscous flow. We observe avalanching dynamics and the formation of remarkably stable jets-single-file granular chains-which occasionally break, resulting in a non-Gaussian distribution of cluster sizes. We find that the sequences of droplet rearrangements that lead to the formation of such chains resemble unfolding of cancer cell clusters in narrow capillaries, overall demonstrating that microfluidic emulsion systems could serve to model various aspects of soft granular flows, including also tissue dynamics at the mesoscale.

A double-step emulsification device for direct generation of double emulsions.

In microfluidic step emulsification, the size of droplets generated in the dripping regime is predominantly determined by the nozzle's height and only weakly depends on the applied flow rates or liquid properties. While the generation of monodisperse emulsions at high throughput using step emulsifiers has been well established, the generation of double emulsions, i.e., liquid core-shell structures, is still challenging. Here, we demonstrate a novel double-step emulsification method for the direct generation of multi-core double-emulsions and provide a predictive model for the number of cores. While the mechanism of the formation of the core droplets or empty shell droplets follows the well-established scenario of simple step emulsification, the formation of double-emulsion droplets is strongly affected by the presence of the cores. Passing of the cores through the narrowing neck of the shell postpones shell pinch-off. In particular, we demonstrate that our system can be used for the generation of arbitrary large, tightly packed droplet clusters consisting of a controllable number of droplets. Finally, we discuss the options of upscaling the system for high-throughput generation of tailored double emulsions.

Translocation Dynamics of High-Internal Phase Double Emulsions in Narrow Channels.

We numerically study the translocation dynamics of double emulsion drops with multiple close-packed inner droplets within constrictions. Such liquid architectures, which we refer to as HIPdEs (high-internal phase double emulsions), consist of a ternary fluid, in which monodisperse droplets are encapsulated within a larger drop in turn immersed in a bulk fluid. Extensive two-dimensional lattice Boltzmann simulations show that if the area fraction of the internal drops is close to the packing fraction limit of hard spheres and the height of the channel is much smaller than the typical size of the emulsion, the crossing yields permanent shape deformations persistent over long periods of time. Morphological changes and rheological response are quantitatively assessed in terms of the structure of the velocity field, circularity of the emulsion, and rate of energy dissipated by viscous forces. Our results may be used to improve the design of soft mesoscale porous materials, which employ HIPdEs as templates for tissue engineering applications.

Particle clusters at fluid-fluid interfaces: equilibrium profiles, structural mechanics and stability against detachment.

We investigate clustering of particles at an initially flat fluid-fluid interface of surface tension γ under an external force f directed perpendicular to the interface. We employ analytical theory, numerical energy minimization (Surface Evolver) and computational fluid dynamics (the Lattice-Boltzmann method) to study the equilibrium deformation of the interface and structural mechanics of the clusters, in particular at the onset of instability. In the case of incompressible clusters, we find that the equilibrium 3D interface profiles are uniquely determined by the length scale γ/(fn0), where n0 is the particle surface number density, and a non-dimensional shape parameter f2Nn0/γ2. The scaling remains valid in the whole regime of forces f, i.e., even close to the stability limit fcrit. In the cases with an initial hexagonal arrangement of the particles, upon f approaching fcrit, our simulations additionally reveal the emergence of curvature-induced defects and 2D stress anisotropy. We develop stability diagrams in terms of f, N (we study 7 ≤ N ≤ 61), and the contact angle θp at the particles and identify three unstable regimes corresponding to (i) collective detachment of the whole cluster from the interface, (ii) ejection of individual particles, and (iii) both detachment and ejection. We also discuss possible metastable states. Altogether, our results may help in better understanding and controlling the particle interfacial instabilities with potential uses in synthesis of new materials, environmental sciences and microfluidics.

3D-Printing of Functionally Graded Porous Materials Using On-Demand Reconfigurable Microfluidics.

Tailoring the morphology of macroporous structures remains one of the biggest challenges in material synthesis. Herein, we present an innovative approach for the fabrication of custom macroporous materials in which pore size varies throughout the structure by up to an order of magnitude. We employed a valve-based flow-focusing junction (vFF) in which the size of the orifice can be adjusted in real-time (within tens of milliseconds) to generate foams with on-line controlled bubble size. We used the junction to fabricate layered and smoothly graded porous structures with pore size varying in the range of 80-800 µm. Additionally, we mounted the vFF on top of an extrusion printer and 3D-printed constructs characterized by a predefined 3D geometry and a controlled, spatially varying internal porous architecture, such as a model of a bone. The presented technology opens new possibilities in macroporous material synthesis with potential applications ranging from tissue engineering to aerospace industry and construction.

Droplet clusters: exploring the phase space of soft mesoscale atoms.

We report three-dimensional structures--mesoscale "atoms"--comprising up to N=8 aqueous droplets compressed in a liquid shell. In contrast to hard colloids that self-assemble into structures unique for a given N, we observe multiple metastable states. We attribute this unexpected richness of metastable structures to the deformability of the cores that introduces irreducible many-body interactions between the droplets. These exotic, often highly anisotropic, structures are locally stable. The structures displaying highly nonoptimum packing-and hence interfacial energy much higher than that of the lowest-energy state-exhibit finite energy barriers that prevent restructuring and relaxation of energy.

Long-term day-by-day tracking of microvascular networks sprouting in fibrin gels: From detailed morphological analyses to general growth rules.

Understanding and controlling of the evolution of sprouting vascular networks remains one of the basic challenges in tissue engineering. Previous studies on the vascularization dynamics have typically focused only on the phase of intense growth and often lacked spatial control over the initial cell arrangement. Here, we perform long-term day-by-day analysis of tens of isolated microvasculatures sprouting from endothelial cell-coated spherical beads embedded in an external fibrin gel. We systematically study the topological evolution of the sprouting networks over their whole lifespan, i.e., for at least 14 days. We develop a custom image analysis toolkit and quantify (i) the overall length and area of the sprouts, (ii) the distributions of segment lengths and branching angles, and (iii) the average number of branch generations-a measure of network complexity. We show that higher concentrations of vascular endothelial growth factor (VEGF) lead to earlier sprouting and more branched networks, yet without significantly affecting the speed of growth of individual sprouts. We find that the mean branching angle is weakly dependent on VEGF and typically in the range of 60°-75°, suggesting that, by comparison with the available diffusion-limited growth models, the bifurcating tips tend to follow local VEGF gradients. At high VEGF concentrations, we observe exponential distributions of segment lengths, which signify purely stochastic branching. Our results-due to their high statistical relevance-may serve as a benchmark for predictive models, while our new image analysis toolkit, offering unique features and high speed of operation, could be exploited in future angiogenic drug tests.

Gravity-driven microfluidic assay for digital enumeration of bacteria and for antibiotic susceptibility testing.

The alarming dynamics of antibiotic-resistant infections calls for the development of rapid and point-of-care (POC) antibiotic susceptibility testing (AST) methods. Here, we demonstrated the first completely stand-alone microfluidic system that allowed the execution of digital enumeration of bacteria and digital antibiograms without any specialized microfluidic instrumentation. A four-chamber gravity-driven step emulsification device generated ∼2000 monodisperse 2 nanoliter droplets with a coefficient of variation of 8.9% of volumes for 95% of droplets within less than 10 minutes. The manual workload required for droplet generation was limited to the sample preparation, the deposition into the sample inlet of the chip and subsequent orientation of the chip vertically without an additional pumping system. The use of shallow chambers imposing a 2D droplet arrangement provided superior stability of the droplets against coalescence and minimized the leakage of the reporter viability dye between adjacent droplets during long-term culture. By using resazurin as an indicator of the growth of bacteria, we were also able to reduce the assay time to ∼5 hours compared to 20 hours using the standard culture-based test.

Tendon Tissue Engineering: Effects of Mechanical and Biochemical Stimulation on Stem Cell Alignment on Cell-Laden Hydrogel Yarns.

Fiber-based approaches hold great promise for tendon tissue engineering enabling the possibility of manufacturing aligned hydrogel filaments that can guide collagen fiber orientation, thereby providing a biomimetic micro-environment for cell attachment, orientation, migration, and proliferation. In this study, a 3D system composed of cell-laden, highly aligned hydrogel yarns is designed and obtained via wet spinning in order to reproduce the morphology and structure of tendon fascicles. A bioink composed of alginate and gelatin methacryloyl (GelMA) is optimized for spinning and loaded with human bone morrow mesenchymal stem cells (hBM-MSCs). The produced scaffolds are subjected to mechanical stretching to recapitulate the strains occurring in native tendon tissue. Stem cell differentiation is promoted by addition of bone morphogenetic protein 12 (BMP-12) in the culture medium. The aligned orientation of the fibers combined with mechanical stimulation results in highly preferential longitudinal cell orientation and demonstrates enhanced collagen type I and III expression. Additionally, the combination of biochemical and mechanical stimulations promotes the expression of specific tenogenic markers, signatures of efficient cell differentiation towards tendon. The obtained results suggest that the proposed 3D cell-laden aligned system can be used for engineering of scaffolds for tendon regeneration.

Microfluidic platform for reproducible self-assembly of chemically communicating droplet networks with predesigned number and type of the communicating compartments.

We report a microfluidic system for individually tailored generation and incubation of core-shell liquid structures with multiple cores that chemically communicate with each other via lipid membranes. We encapsulate an oscillating reaction-diffusion Belousov-Zhabotinsky (BZ) medium inside the aqueous droplets and study the propagation of chemical wave-fronts through the membranes. We further encapsulate the sets of interconnected BZ-droplets inside oil-lipid shells in order to i) chemically isolate the structures and ii) confine them via tunable capillary forces which leads to self-assembly of predesigned topologies. We observe that doublets (pairs) of droplets encapsulated in the shell exhibit oscillation patterns that evolve in time. We collect statistical data from tens of doublets all created under precisely controlled, almost identical conditions from which we conclude that the different types of transitions between the patterns depend on the relative volumes of the droplets within a chemically coupled pair. With this we show that the volume of the compartment is an important control parameter in designing chemical networks, a feature previously appreciated only by theory. Our system not only allows for new insights into the dynamics of geometrically complex and interacting chemical systems but is also suitable for generating autonomous chemically interconnected microstructures with possible future use, e.g., as smart biosensors or drug-release capsules.

Highly ordered and tunable polyHIPEs by using microfluidics.

We demonstrate how to generate highly ordered porous matrices from dextran-methacrylate (DEX-MA) using microfluidics. We use a flow focusing device to inject an aqueous solution of DEX-MA and surfactant to break the flow of an organic solvent (cyclohexane) into monodisperse droplets at a high volume fraction (above 74% v/v) to form an ordered high internal phase emulsion (HIPE). We collect the crystalline HIPE structure and freeze it by gelling. The resulting polyHIPEs are characterized by an interconnected and ordered morphology. The size of pores and interconnects ranges between hundreds and tens of micrometers, respectively. The technique that we describe allows for precise tuning of all the structural parameters of the matrices, including their porosity, the size of the pores and the lumen of interconnects between the pores. The resulting ordered and precisely tailored HIPE gels represent a new class of scaffolds for applications in tissue engineering.

Custom tailoring multiple droplets one-by-one.

We report automated generation of arbitrary sequences of multiple microdroplets with online and individual control over the number of cores and volumes of all the constituents (cores and shells) of each of the multiple droplets. We show that a given sequence of volumes of the cores always folds to the same final three-dimensional architecture. The method presents the first proof-of-concept for the ability to design the three-dimensional structure of multiple droplets. We discuss the potential use of the technique in the formulation of predetermined distribution of drug release capsules and for automated generation of functional chemical microdroplet networks.

Automated high-throughput generation of droplets.

We report a microfluidic technique for high-throughput generation of droplets of nanolitre volume in parallel channels with online control of the volumes, volume fraction and distribution of droplet volumes with the use of two external valves.