Ultrasonic standing wave (USW) manipulation is a technology that has been used for separating, sorting, enriching and trapping particles and cells in microfluidic devices including microchannels, microchambers and microwells. One application area is to use the technology for 3D cell cultures on a chip. Such USW-formed 3D cultures have been used for high content screening of tumor spheroids interacting with chemotherapeutic drugs and immune cells. For this purpose, we have developed multiwell microplates designed for high-resolution optimal microscopy. In these microplates, hundreds of tumor spheroids can be formed and shaped by the ultrasound in parallel, followed by high-quality imaging in 3D. However, in our previous work, our USW-based method was not compatible with live cell imaging. Instead, the method was based on active temperature regulation and high-power RF amplification, including bulky and expensive instrumentation. To address this, a novel transducer configuration in combination with an adaptive ultrasonic actuation method has been designed and characterized. The actuation method is applied to a chip-based high-content multi-well screening platform for USW-mediated formation of spheroids. The methodology results in better control of the shape of formed spheroids, eliminates the need for active temperature control and costly RF amplifiers, and enables live-cell microscopy-based imaging during spheroid formation and maturation.

Acoustic trapping is used in modern biophysics laboratories to study cell adhesion or aggregation, to sort particles, or to build model tissues. Here, we create an acoustic trapping setup in liquid for an undergraduate instructional laboratory that is low-cost, easy to build, and produces results in a 1-hour laboratory period. In this setup, we use a glass slide, cover slip, and double-sided tape to make the sample chamber. A piezo-electric transducer connected to a function generator serves as the acoustic source. We use this setup to measure the node spacing (millimeters) and the acoustic trap force (picoNewtons). We anticipate that the simplicity of the experimental setup, the tractability of the theoretical equations, and the richness of the research topics on the subject will lead to an undergraduate laboratory with many interesting student projects.

The health hazards of micro- and nanoplastic contaminants in drinking water has recently emerged as an area of concern to policy makers and industry. Plastic contaminants range in size from micro- (5 mm to 1 μm) to nanoplastics (<1 μm). Microfluidics provides many tools for particle manipulation at the microscale, particularly in diagnostics and biomedicine, but has in general a limited capacity to process large volumes. Drinking water and environmental samples with low-level contamination of microplastics require processing of deciliter to liter sample volumes to achieve statistically relevant particle counts. Here, we introduce the EchoGrid, an acoustofluidics device for high throughput continuous flow particle enrichment into a robust array of particle clusters. The EchoGrid takes advantage of highly efficient particle capture through the integration of a micropatterned transducer for surface displacement-based acoustic trapping in a glass and polymer microchannel. Silica seed particles were used as anchor particles to improve capture performance at low particle concentrations and high flow rates. The device was able to maintain the silica grids at a flow rate of 50 mL/min. In terms of enrichment, the device is able to double the final pellet’s microplastic concentration every 78 s for 23 μm particles and every 51 s for 10 μm particles at a flow rate of 5 mL/min. In conclusion, we demonstrate the usefulness of the EchoGrid by capturing microplastics in challenging conditions, such as large sample volumes with low microparticle concentrations, without sacrificing the potential of integration with downstream analysis for environmental monitoring.

Micro- and nanoplastics have become increasingly relevant as contaminants to be monitored due to their potential health effects and environmental impact. Nanoplastics, in particular, have been shown to be difficult to detect in drinking water, requiring new capture technologies. In this work, we applied the acoustofluidic seed particle method to capture nanoplastics in an optimized, tilted grid of silica clusters even at the high flow rate of 5 mL/min. Moreover, we achieved, using this technique, the enrichment of nanoparticles ranging from 500 nm to 25 nm as a first in the field. We employed fluorescence to observe the enrichment profiles according to size, using a washing buffer flow at 0.5 mL/min, highlighting the size-dependent nature of the silica seed particle release of various sizes of nanoparticles. These results highlight the versatility of acoustic trapping for a wide range of nanoplastic particles and allow further study into the complex dynamics of the seed particle method at these size ranges. Moreover, with reproducible size-dependent washing curves, we provide a new window into the rate of nanoplastic escape in high-capacity acoustic traps, relevant to both environmental and biomedical applications.

The development of new immunotherapeutic drugs and combinatorial strategies requires the implementation of novel methods to test their efficacy in vitro. Here, we present a series of miniaturized in vitro assays to assess immune cell cytotoxic activity, infiltration, and phenotype in renal carcinoma spheroids with the use of a recently developed multichambered microwell chip. We provide protocols for tumor spheroid formation, NK cell culture, fluorescence labelling and imaging of live or fixed cells directly in the chip together with data analysis.

Immunotherapy is revolutionizing cancer therapy. The rapid development of new immunotherapeutic strategies to treat solid tumors is posing new challenges for preclinical research, demanding novel in vitro methods to test treatments. Such methods should meet specific requirements, such as enabling the evaluation of immune cell responses like cytotoxicity or cytokine release, and infiltration into the tumor microenvironment using cancer models representative of the original disease. They should allow high-throughput and high-content analysis, to evaluate the efficacy of treatments and understand immune-evasion processes to facilitate development of new therapeutic targets. Ideally, they should be suitable for personalized immunotherapy testing, providing information for patient stratification. Consequently, the application of in vitro 3-dimensional (3D) cell culture models, such as tumor spheroids and organoids, is rapidly expanding in the immunotherapeutic field, coupled with the development of novel imaging-based techniques and -omic analysis. In this paper, we review the recent advances in the development of in vitro 3D platforms applied to natural killer (NK) cell-based cancer immunotherapy studies, highlighting the benefits and limitations of the current methods, and discuss new concepts and future directions of the field.

Serial femtosecond crystallography (SFX) has become one of the standard techniques at X-ray free-electron lasers (XFELs) to obtain high-resolution structural information from microcrystals of proteins. Nevertheless, reliable sample delivery is still often limiting data collection, as microcrystals can clog both field- and flow-focusing nozzles despite in-line filters. In this study, we developed acoustic 2D focusing of protein microcrystals in capillaries that enables real-time online characterization of crystal size and shape in the sample delivery line after the in-line filter. We used a piezoelectric actuator to create a standing wave perpendicular to the crystal flow, which focused lysozyme microcrystals into a single line inside a silica capillary so that they can be imaged using a high-speed camera. We characterized the acoustic contrast factor, focus size, and the coaxial flow lines and developed a splitting union that enables up-concentration to at least a factor of five. The focus size, flow rates, and geometry may enable an upper limit of up-concentration as high as 200 fold. The novel feedback and concentration control could be implemented for serial crystallography at synchrotrons with minor modifications. It will also aid the development of improved sample delivery systems that will increase SFX data collection rates at XFELs, with potential applications to many proteins that can only be purified and crystallized in small amounts.

Acoustofluidic technologies utilize acoustic waves to manipulate fluids and particles within fluids, all in a contact-free and biocompatible manner. Over the past decade, acoustofluidic technologies have enabled new capabilities in biomedical applications ranging from the precise patterning of heterogeneous cells for tissue engineering to the automated isolation of extracellular vesicles from biofluids for rapid, point-of-care diagnostics. In this Primer, we explain the underlying physical principles governing the design and operation of acoustofluidic technologies and describe the various implementations that have been developed for biomedical applications. We aim to demystify the rapidly growing field of acoustofluidics and provide a unified perspective that will allow end users to choose the acoustofluidic technology that is best suited for their research needs. The experimental set-ups for each type of acoustofluidic device are discussed along with their advantages and limitations. In addition, we review typical types of data that are obtained from acoustofluidic experiments and describe how to model different forces acting on particles within an acoustofluidic device. We also discuss data reproducibility and the need to establish standards for the deposition of data sets within the field. Finally, we provide our perspective on how to optimize device performance and discuss areas of future development.

Here, we present a methodology based on multiplexed fluorescence screening of two-or three-dimensional cell cultures in a newly designed multichambered microwell chip, allowing direct assessment of drug or im-mune cell cytotoxic efficacy. We establish a framework for cell culture, formation of tumor spheroids, fluores-cence labeling, and imaging of fixed or live cells at various magnifications directly in the chip together with data analysis and interpretation. The methodology is demonstrated by drug cytotoxicity screening using ovarian and non-small cell lung cancer cells and by cellular cytotoxicity screening targeting tumor spheroids of renal carcinoma and ovarian carcinoma with natural killer cells from healthy donors. The miniaturized format allowing long-term cell culture, efficient screening, and high-quality imaging of small sample volumes makes this methodology promising for individualized cytotoxicity tests for precision medicine.

Acoustic trapping is a promising technique for aligning particles in two-dimensional arrays, as well as for dynamic manipulation of particles individually or in groups. The actuating principles used in current systems rely on either cavity modes in enclosures or complex arrangements for phase control. Therefore, available systems either require high power inputs and costly peripheral equipment or sacrifice flexibility. This work presents a different concept for acoustic trapping of particles and cells that enables dynamically defined trapping patterns inside a simple and inexpensive setup. Here, dynamic operation and dexterous trapping are realized through the use of a modified piezoelectric transducer in direct contact with the liquid sample. Physical modeling shows how the transducer induces an acoustic force potential where the conventional trapping in the axial direction is supplemented by surface displacement dependent lateral trapping. The lateral field is a horizontal array of pronounced potential minima with frequency-dependent locations. The resulting system enables dynamic arraying of levitated trapping sites at low power and can be manufactured at ultra-low cost, operated using low-cost electronics, and assembled in less than 5 min. We demonstrate dynamic patterning of particles and biological cells and exemplify potential uses of the technique for cell-based sample preparation and cell culture.