RESUMEN

We present an easy-to-assemble microfluidic system for synthesizing cell-loaded dextran/alginate (DEX/ALG) hydrogel spheres using an aqueous two-phase system (ATPS) for templated fabrication of multicellular tumor spheroids (MTSs). An audio speaker driven by an amplified output of a waveform generator or smartphone provides acoustic modulation to drive the breakup of an ATPS into MTS template droplets within microcapillary fluidic devices. We apply extensions of Plateau-Rayleigh theory to help define the flow and frequency parameter space necessary for acoustofluidic ATPS droplet formation in these devices. This method provides a simple droplet microfluidic approach using off-the-shelf acoustic components for quickly initiating MTSs and subsequent 3D cell culture.

RESUMEN

Acoustophoresis has gained increasing attention as a gentle, non-contact, and high-throughput cell and particle separation technique. It is conveniently used to isolate and enrich particles that are greater than 2 µm; however, its use in manipulating particles smaller than 2 µm is limited. In this work, we present an alternative way of using acoustic forces to manipulate sub-micrometer particles in continuous flow fashion. It has been shown that acoustic forces can be employed to relocate parallel laminar flow streams of two impedance-mismatched fluids. We demonstrate the separation of sub-micron particles from micron particles by the combination of acoustophoresis and acoustic fluid relocation. The micron particles are focused into the middle of the flow channel via primary acoustic forces while sub-micron particles are moved to the side via drag forces created by the relocating fluid. We demonstrate the proof of the concept using binary mixtures of particles comprised of sub-micron/micron particles, micron/micron particles, and bovine red blood cells with E. coli. The efficiency of the particle enrichment is determined via flow cytometry analysis of the collected streams. This study demonstrates that by combining acoustic fluid relocation with acoustophoresis, sub-micron particles can be effectively separated from micron particles at high flow rates and it can be further implemented to separate binary mixtures of micron particles if the volumetric ratio of two particles is greater than 10 and the larger particle diameter is about 10 µm. The combined method is more appropriate to use than acoustophoresis in situations where acoustic streaming and differences in acoustic impedance of fluids can be of concern. Graphical abstract In the presence of a resonance acoustic field, the clean high-density fluid (dark gray) and the low-density sample fluid are relocated. During this process, E. coli are separated from the red blood cells (RBCs).

Asunto(s)

RESUMEN

Flow cytometry provides highly sensitive multiparameter analysis of cells and particles but has been largely limited to the use of a single focused sample stream. This limits the analytical rate to ∼50K particles/s and the volumetric rate to ∼250 µL/min. Despite the analytical prowess of flow cytometry, there are applications where these rates are insufficient, such as rare cell analysis in high cellular backgrounds (e.g., circulating tumor cells and fetal cells in maternal blood), detection of cells/particles in large dilute samples (e.g., water quality, urine analysis), or high-throughput screening applications. Here we report a highly parallel acoustic flow cytometer that uses an acoustic standing wave to focus particles into 16 parallel analysis points across a 2.3 mm wide optical flow cell. A line-focused laser and wide-field collection optics are used to excite and collect the fluorescence emission of these parallel streams onto a high-speed camera for analysis. With this instrument format and fluorescent microsphere standards, we obtain analysis rates of 100K/s and flow rates of 10 mL/min, while maintaining optical performance comparable to that of a commercial flow cytometer. The results with our initial prototype instrument demonstrate that the integration of key parallelizable components, including the line-focused laser, particle focusing using multinode acoustic standing waves, and a spatially arrayed detector, can increase analytical and volumetric throughputs by orders of magnitude in a compact, simple, and cost-effective platform. Such instruments will be of great value to applications in need of high-throughput yet sensitive flow cytometry analysis.

Asunto(s)