Acoustophoresis in polymer-based microfluidic devices: Modeling and experimental validation.

A finite-element model is presented for numerical simulation in three dimensions of acoustophoresis of suspended microparticles in a microchannel embedded in a polymer chip and driven by an attached piezoelectric transducer at MHz frequencies. In accordance with the recently introduced principle of whole-system ultrasound resonances, an optimal resonance mode is identified that is related to an acoustic resonance of the combined transducer-chip-channel system and not to the conventional pressure half-wave resonance of the microchannel. The acoustophoretic action in the microchannel is of comparable quality and strength to conventional silicon-glass or pure glass devices. The numerical predictions are validated by acoustic focusing experiments on 5-µm-diameter polystyrene particles suspended inside a microchannel, which was milled into a polymethylmethacrylate chip. The system was driven anti-symmetrically by a piezoelectric transducer, driven by a 30-V peak-to-peak alternating voltage in the range from 0.5 to 2.5 MHz, leading to acoustic energy densities of 13 J/m3 and particle focusing times of 6.6 s.

Particle Manipulation Methods in Droplet Microfluidics.

This Feature describes the different particle manipulation techniques available in the droplet microfluidics toolbox to handle particles encapsulated inside droplets and to manipulate whole droplets. We address the advantages and disadvantages of the different techniques to guide new users.

Acoustic micro-vortexing of fluids, particles and cells in disposable microfluidic chips.

We demonstrate an acoustic platform for micro-vortexing in disposable polymer microfluidic chips with small-volume (20 µl) reaction chambers. The described method is demonstrated for a variety of standard vortexing functions, including mixing of fluids, re-suspension of a pellet of magnetic beads collected by a magnet placed on the chip, and lysis of cells for DNA extraction. The device is based on a modified Langevin-type ultrasonic transducer with an exponential horn for efficient coupling into the microfluidic chip, which is actuated by a low-cost fixed-frequency electronic driver board. The transducer is optimized by numerical modelling, and different demonstrated vortexing functions are realized by actuating the transducer for varying times; from fractions of a second for fluid mixing, to half a minute for cell lysis and DNA extraction. The platform can be operated during 1 min below physiological temperatures with the help of a PC fan, a Peltier element and an aluminum heat sink acting as the chip holder. As a proof of principle for sample preparation applications, we demonstrate on-chip cell lysis and DNA extraction within 25 s. The method is of interest for automating and chip-integrating sample preparation procedures in various biological assays.

Ultrasound-controlled cell aggregation in a multi-well chip.

We demonstrate a microplate platform for parallelized manipulation of particles or cells by frequency-modulated ultrasound. The device, consisting of a silicon-glass microchip and a single ultrasonic transducer, enables aggregation, positioning and high-resolution microscopy of cells distributed in an array of 100 microwells centered on the microchip. We characterize the system in terms of temperature control, aggregation and positioning efficiency, and cell viability. We use time-lapse imaging to show that cells continuously exposed to ultrasound are able to divide and remain viable for at least 12 hours inside the device. Thus, the device can be used to induce and maintain aggregation in a parallelized fashion, facilitating long-term microscopy studies of, e.g., cell-cell interactions.

An intra-droplet particle switch for droplet microfluidics using bulk acoustic waves.

To transfer cell- and bead-assays into droplet-based platforms typically requires the use of complex microfluidic circuits, which calls for methods to switch the direction of the encapsulated particles. We present a microfluidic chip where the combination of acoustic manipulation at two different harmonics and a trident-shaped droplet-splitter enables direction-switching of microbeads and yeast cells in droplet microfluidic circuits. At the first harmonic, the encapsulated particles exit the splitter in the center daughter droplets, while at the second harmonic, the particles exit in the side daughter droplets. This method holds promises for droplet-based assays where particle-positioning needs to be selectively controlled.

Ultrasonic three-dimensional on-chip cell culture for dynamic studies of tumor immune surveillance by natural killer cells.

We demonstrate a simple method for three-dimensional (3D) cell culture controlled by ultrasonic standing waves in a multi-well microplate. The method gently arranges cells in a suspension into a single aggregate in each well of the microplate and, by this, nucleates 3D tissue-like cell growth for culture times between two and seven days. The microplate device is compatible with both high-resolution optical microscopy and maintenance in a standard cell incubator. The result is a scaffold- and coating-free method for 3D cell culture that can be used for controlling the cellular architecture, as well as the cellular and molecular composition of the microenvironment in and around the formed cell structures. We demonstrate the parallel production of one hundred synthetic 3D solid tumors comprising up to thousands of human hepatocellular carcinoma (HCC) HepG2 cells, we characterize the tumor structure by high-resolution optical microscopy, and we monitor the functional behavior of natural killer (NK) cells migrating, docking and interacting with the tumor model during culture. Our results show that the method can be used for determining the collective ability of a given number of NK cells to defeat a solid tumor having a certain size, shape and composition. The ultrasound-based method itself is generic and can meet any demand from applications where it is advantageous to monitor cell culture from production to analysis of 3D tissue or tumor models using microscopy in one single microplate device.

Temperature-controlled MPa-pressure ultrasonic cell manipulation in a microfluidic chip.

We study the temperature-independent impact on cell viability of relevant physical parameters during long-term, high-acoustic-pressure ultrasonic exposure in a microfluidic chip designed for ultrasonic-standing-wave trapping and aggregation of cells. We use a light-intensity method and 5 µm polymer beads for accurate acoustic pressure calibration before injecting cells into the device, and we monitor the viability of A549 lung cancer cells trapped during one hour in an ultrasonic standing wave with 1 MPa pressure amplitude. The microfluidic chip is actuated by a novel temperature-controlled ultrasonic transducer capable of keeping the temperature stable around 37 °C with an accuracy better than ±0.2 °C, independently on the ultrasonic power and heat produced by the system, thereby decoupling any temperature effect from other relevant effects on cells caused by the high-pressure acoustic field. We demonstrate that frequency-modulated ultrasonic actuation can produce acoustic pressures of equally high magnitudes as with single-frequency actuation, and we show that A549 lung cancer cells can be exposed to 1 MPa standing-wave acoustic pressure amplitudes for one hour without compromising cell viability. At this pressure level, we also measure the acoustic streaming induced around the trapped cell aggregate, and conclude that cell viability is not affected by streaming velocities of the order of 100 µm s(-1). Our results are important when implementing acoustophoresis methods in various clinical and biomedical applications.

Live cell imaging in a micro-array of acoustic traps facilitates quantification of natural killer cell heterogeneity.

Natural killer (NK) cells kill virus-infected or cancer cells through the release of cytotoxic granules into a tight intercellular contact. NK cell populations comprise individual cells with varying sensitivity to distinct input signals, leading to disparate responses. To resolve this NK cell heterogeneity, we have designed a novel assay based on ultrasound-assisted cell-cell aggregation in a multiwell chip allowing high-resolution time-lapse imaging of one hundred NK-target cell interactions in parallel. Studying human NK cells' ability to kill MHC class I deficient tumor cells, we show that approximately two thirds of the NK cells display cytotoxicity, with some NK cells being particularly active, killing up to six target cells during the assay. We also report that simultaneous interaction with several susceptible target cells increases the cytotoxic responsiveness of NK cells, which could be coupled to a previously unknown regulatory mechanism with implications for NK-mediated tumor elimination.

Acoustofluidics 14: Applications of acoustic streaming in microfluidic devices.

In part 14 of the tutorial series "Acoustofluidics--exploiting ultrasonic standing wave forces and acoustic streaming in microfluidic systems for cell and particle manipulation", we provide a qualitative description of acoustic streaming and review its applications in lab-on-a-chip devices. The paper covers boundary layer driven streaming, including Schlichting and Rayleigh streaming, Eckart streaming in the bulk fluid, cavitation microstreaming and surface-acoustic-wave-driven streaming.