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Abstract Autologous cell therapy depends on T lymphocyte expansion efficiency and is hindered by suboptimal interactions between T cell receptors (TCR) and peptide‐MHC molecules. Various artificial antigen presenting cell systems that enhance these interactions are often labor‐intensive, fabrication costly, highly variable, and potentially unscalable toward clinical setting. Here, 3D centrifugation‐enabled priming of T cell immune‐synapse junctions is performed to generate tight T cell–Dynabead aggregates at a rate 200‐fold faster than that of conventional 24‐h bulk shaking. Furthermore, by forming T cell–Dynabead aggregates in the starting culture, two‐ to sixfold greater T cell expansion is achieved over conventional T cell expansion for cancer patient‐derived primary T cells while limiting over‐activation. Creating 3D T cell–Dynabead aggregates as the “booster” material enables highly efficient polyclonal T cell expansion without the need for complex surface modification of artificial antigen‐presenting cells (APCs). This method can be modularly adapted to existing T cell expansion processes for various applications, including adoptive cell therapies (ACTs). 

Abstract Particle migration dynamics in viscoelastic fluids in spiral channels have attracted interest in recent years due to potential applications in the 3D focusing and label-free sorting of particles and cells. Despite a number of recent studies, the underlying mechanism of Dean-coupled elasto-inertial migration in spiral microchannels is not fully understood. In this work, for the first time, we experimentally demonstrate the evolution of particle focusing behavior along a channel downstream length at a high blockage ratio. We found that flow rate, device curvature, and medium viscosity play important roles in particle lateral migration. Our results illustrate the full focusing pattern along the downstream channel length, with side-view imaging yielding observations on the vertical migration of focused streams. Ultimately, we anticipate that these results will offer a useful guide for elasto-inertial microfluidics device design to improve the efficiency of 3D focusing in cell sorting and cytometry applications. 

Abstract Multiplexed solid‐contact ion‐selective electrodes (SCISEs) are fabricated using printed circuit board (PCB) and mesoporous carbon black (MCB) as ion‐to‐electron transducer (solid contact). Four sensor configurations were examined and showed that in addition to MCB, the sensor configuration plays crucial role in the stability of the potential response. The enhanced sensor stability was also linked with suppression of transmembrane flux of water. The sensors exhibited near‐Nernstian sensitivity (58.1 mV/dec for K⁺ISEs and −55.1 mV/dec for NO₃^‐ISEs), low detection limits (1.5–2.2 μM), and good short‐term stability (∼0.1 mV/min). Sensors can be stored dry and used without preconditioning. This work demonstrates a promising approach to combining PCB technology and carbon black for large‐scale production of low cost ISEs for point‐of‐care testing, wearables, orin situfield measurements. 

Abstract Tissues are complex mixtures of different cell subtypes, and this diversity is increasingly characterized using high-throughput single cell analysis methods. However, these efforts are hindered, as tissues must first be dissociated into single cell suspensions using methods that are often inefficient, labor-intensive, highly variable, and potentially biased towards certain cell subtypes. Here, we present a microfluidic platform consisting of three tissue processing technologies that combine tissue digestion, disaggregation, and filtration. The platform is evaluated using a diverse array of tissues. For kidney and mammary tumor, microfluidic processing produces 2.5-fold more single cells. Single cell RNA sequencing further reveals that endothelial cells, fibroblasts, and basal epithelium are enriched without affecting stress response. For liver and heart, processing time is dramatically reduced. We also demonstrate that recovery of cells from the system at periodic intervals during processing increases hepatocyte and cardiomyocyte numbers, as well as increases reproducibility from batch-to-batch for all tissues. 

Abstract A high‐throughput non‐viral intracellular delivery platform is introduced for the transfection of large cargos with dosage‐control. This platform, termed Acoustic‐Electric Shear Orbiting Poration (AESOP), optimizes the delivery of intended cargo sizes with poration of the cell membranes via mechanical shear followed by the modulated expansion of these nanopores via electric field. Furthermore, AESOP utilizes acoustic microstreaming vortices wherein up to millions of cells are trapped and mixed uniformly with exogenous cargos, enabling the delivery of cargos into cells with targeted dosages. Intracellular delivery of a wide range of molecule sizes (<1 kDa to 2 MDa) with high efficiency (>90%), cell viability (>80%), and uniform dosages (<60% coefficient of variation (CV)) simultaneously into 1 million cells min⁻¹per single chip is demonstrated. AESOP is successfully applied to two gene editing applications that require the delivery of large plasmids: i) enhanced green fluorescent protein (eGFP) plasmid (6.1 kbp) transfection, and ii) clustered regularly interspaced short palindromic repeats (CRISPR)‐Cas9‐mediated gene knockout using a 9.3 kbp plasmid DNA encoding Cas9 protein and single guide RNA (sgRNA). Compared to alternative platforms, this platform offers dosage‐controlled intracellular delivery of large plasmids simultaneously to large populations of cells while maintaining cell viability at comparable delivery efficiencies. 

Fast, nondestructive three-dimensional (3D) imaging of live suspension cells remains challenging without substrate treatment or fixation, precluding scalable single-cell morphometry with minimal alterations. While optical sectioning techniques achieve 3D live cell imaging, lateral versus depth resolution differences further complicate analysis. We present a scalable microfluidic method capable of 3D fluorescent isotropic imaging of live, nonadherent cells suspended inside picoliter droplets with high-speed single-cell volumetric readout (800 to 1,200 slices in 5 to 8 s) and near-diffraction limit resolution (~216 nm). The platform features a droplet trap array that leverages flow-induced droplet interfacial shear to generate intradroplet microvortices, which rotate single cells on their axis to enable optical projection tomography (OPT)-based imaging. This allows gentle (~1 mPa shear stress) observation of cells encapsulated inside nontoxic isotonic buffer droplets, facilitating scalable OPT acquisition by simultaneous spinning of hundreds of cells. We demonstrate 3D imaging of live myeloid and lymphoid cells in suspension, including K562 cells, as well as naive and activated T cells—small cells prone to movement in their suspended phenotype. Our fully suspended, orientation-independent cell morphometry, driven by isotropic imaging and spherical harmonic analysis, enabled the study of primary T cells across various immunological activation states. This approach unveiled six distinct nuclear content distributions, contrasting with conventional 2D images that typically portray spheroid and bean-like nuclear shapes associated with lymphocytes. Our arrayed-droplet OPT technology is capable of isotropic, single live-cell 3D imaging, with the potential to perform large-scale morphometry of immune cell effector function states while providing compatibility with microfluidic droplet operations. 

We conducted a systematic numerical investigation of spherical, prolate and oblate particles in an inertial shear flow between two parallel walls, using smoothed particle hydrodynamics (SPH). It was previously shown that above a critical Reynolds number, spherical particles experience a supercritical pitchfork bifurcation of the equilibrium position in shear flow between two parallel walls, namely that the central equilibrium position becomes unstable, leading to the emergence of two new off-centre stable positions (Foxet al.,J. Fluid Mech., vol. 915, 2021). This phenomenon was unexpected given the symmetry of the system. In addition to confirming this finding, we found, surprisingly, that ellipsoidal particles can also return to the centre position from the off-centre positions when the particle Reynolds number is further increased, while spherical particles become unstable under this increased Reynolds number. By utilizing both SPH and the finite element method for flow visualization, we explained the underlining mechanism of this reverse of bifurcation by altered streamwise vorticity and symmetry breaking of pressure. Furthermore, we expanded our investigation to include asymmetric particles, a novel aspect that had not been previously modelled, and we observed similar trends in particle dynamics for both symmetric and asymmetric ellipsoidal particles. While further validation through laboratory experiments is necessary, our research paves the road for development of new focusing and separation methods for shaped particles. 

Dielectrophoresis (DEP) is a powerful tool for label-free sorting of cells, even those with subtle differences in morphological and dielectric properties. Nevertheless, a major limitation is that most existing DEP techniques can efficiently sort cells only at low throughputs (<1 mL h−1). Here, we demonstrate that the integration of a three-dimensional (3D) coupled hydrodynamic-DEP cell pre-focusing module upstream of the main DEP sorting region enables cell sorting with a 10-fold increase in throughput compared to conventional DEP approaches. To better understand the key principles and requirements for high-throughput cell separation, we present a comprehensive theoretical model to study the scaling of hydrodynamic and electrostatic forces on cells at high flow rate regimes. Based on the model, we show that the critical cell-to-electrode distance needs to be ≤10 µm for efficient cell sorting in our proposed microfluidic platform, especially at flow rates ≥ 1 mL h−1. Based on those findings, a computational fluid dynamics model and particle tracking analysis were developed to find optimum operation parameters (e.g., flow rate ratios and electric fields) of the coupled hydrodynamic-DEP 3D focusing module. Using these optimum parameters, we experimentally demonstrate live/dead K562 cell sorting at rates as high as 10 mL h−1 (>150,000 cells min−1) with 90% separation purity, 85% cell recovery, and no negative impact on cell viability. 

We derived equations and closed-form solutions of transit time for a viscous droplet squeezing through a small circular pore with a finite length at microscale under constant pressures. Our analyses were motivated by the vital processes of biological cells squeezing through small pores in blood vessels and sinusoids and droplets squeezing through pores in microfluidics. First, we derived ordinary differential equations (ODEs) of a droplet squeezing through a circular pore by combining Sampson flow, Poiseuille flow, and Young–Laplace equations and took into account the lubrication layer between the droplet and the pore wall. Second, for droplets wetting the wall with small surface tension, we derived the closed-form solutions of transit time. For droplets with finite surface tension, we solved the original ODEs numerically to predict the transit time. After validations against experiments and finite element simulations, we studied the effects of pressure, viscosity, pore/droplet dimensions, and surface tension on the transit time. We found that the transit time is inversely linearly proportional to pressure when the surface tension is low compared to the critical surface tension for preventing the droplet to pass and becomes nonlinear when it approaches the critical tension. Remarkably, we showed that when a fixed percentage of surface tension to critical tension is applied, the transit time is always inversely linearly proportional to pressure, and the dependence of transit time on surface tension is nonmonotonic. Our results provided a quick way of quantitative calculations of transit time for designing droplet microfluidics and understanding cells passing through constrictions. 

We introduce μVAST, a high-throughput acoustic microstreaming platform using second-order microstreaming to induce fluid transport and measure the viscosity of 16 samples, automating process flows in drug development, materials manufacturing and production.