Acoustic trapping based on surface displacement of resonance modes.

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.

Unravelling the Acoustic and Thermal Responses of Perfluorocarbon Liquid Droplets Stabilized with Cellulose Nanofibers.

The attractive colloidal and physicochemical properties of cellulose nanofibers (CNFs) at interfaces have recently been exploited in the facile production of a number of environmentally benign materials, e.g. foams, emulsions, and capsules. Herein, these unique properties are exploited in a new type of CNF-stabilized perfluoropentane droplets produced via a straightforward and simple mixing protocol. Droplets with a comparatively narrow size distribution (ca. 1-5 µm in diameter) were fabricated, and their potential in the acoustic droplet vaporization process was evaluated. For this, the particle-stabilized droplets were assessed in three independent experimental examinations, namely temperature, acoustic, and ultrasonic standing wave tests. During the acoustic droplet vaporization (ADV) process, droplets were converted to gas-filled microbubbles, offering enhanced visualization by ultrasound. The acoustic pressure threshold of about 0.62 MPa was identified for the cellulose-stabilized droplets. A phase transition temperature of about 22 °C was observed, at which a significant fraction of larger droplets (above ca. 3 µm in diameter) were converted into bubbles, whereas a large part of the population of smaller droplets were stable up to higher temperatures (temperatures up to 45 °C tested). Moreover, under ultrasound standing wave conditions, droplets were relocated to antinodes demonstrating the behavior associated with the negative contrast particles. The combined results make the CNF-stabilized droplets interesting in cell-droplet interaction experiments and ultrasound imaging.

Generation of tumor spheroids in microwells to study NK cell cytotoxicity, infiltration and phenotype.

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.

Miniaturized and multiplexed high-content screening of drug and immune sensitivity in a multichambered microwell chip.

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 immune cell cytotoxic efficacy. We establish a framework for cell culture, formation of tumor spheroids, fluorescence 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.

Single cell organization and cell cycle characterization of DNA stained multicellular tumor spheroids.

Multicellular tumor spheroids (MCTSs) can serve as in vitro models for solid tumors and have become widely used in basic cancer research and drug screening applications. The major challenges when studying MCTSs by optical microscopy are imaging and analysis due to light scattering within the 3-dimensional structure. Herein, we used an ultrasound-based MCTS culture platform, where A498 renal carcinoma MCTSs were cultured, DAPI stained, optically cleared and imaged, to connect nuclear segmentation to biological information at the single cell level. We show that DNA-content analysis can be used to classify the cell cycle state as a function of position within the MCTSs. We also used nuclear volumetric characterization to show that cells were more densely organized and perpendicularly aligned to the MCTS radius in MCTSs cultured for 96 h compared to 24 h. The method presented herein can in principle be used with any stochiometric DNA staining protocol and nuclear segmentation strategy. Since it is based on a single counter stain a large part of the fluorescence spectrum is free for other probes, allowing measurements that correlate cell cycle state and nuclear organization with e.g., protein expression or drug distribution within MCTSs.

Ultrasound-Based Scaffold-Free Core-Shell Multicellular Tumor Spheroid Formation.

In cancer research and drug screening, multicellular tumor spheroids (MCTSs) are a popular model to bridge the gap between in vitro and in vivo. However, the current techniques to culture mixed co-culture MCTSs do not mimic the structural architecture and cellular spatial distribution in solid tumors. In this study we present an acoustic trapping-based core-shell MCTSs culture method using sequential seeding of the core and shell cells into microwells coated with a protein repellent coating. Scaffold-free core-shell ovarian cancer OVCAR-8 cell line MCTSs were cultured, stained, cleared and confocally imaged on-chip. Image analysis techniques were used to quantify the shell thickness (23.2 ± 1.8 µm) and shell coverage percentage (91.2 ± 2.8%). We also show that the shell thickness was evenly distributed over the MCTS cores with the exception of being slightly thinner close to the microwell bottom. This scaffold-free core-shell MCTSs formation technique and the analysis tools presented herein could be used as an internal migration assay within the MCTS or to form core-shell MCTS co-cultures to study therapy response or the interaction between tumor and stromal cells.

Measuring the Compressibility of Cellulose Nanofiber-Stabilized Microdroplets Using Acoustophoresis.

Droplets with a liquid perfluoropentane core and a cellulose nanofiber shell have the potential to be used as drug carriers in ultrasound-mediated drug delivery. However, it is necessary to understand their mechanical properties to develop ultrasound imaging sequences that enable in vivo imaging of the vaporization process to ensure optimized drug delivery. In this work, the compressibility of droplets stabilized with cellulose nanofibers was estimated using acoustophoresis at three different acoustic pressures. Polyamide particles of known size and material properties were used for calibration. The droplet compressibility was then used to estimate the cellulose nanofiber bulk modulus and compare it to experimentally determined values. The results showed that the acoustic contrast factor for these droplets was negative, as the droplets relocated to pressure antinodes during ultrasonic actuation. The droplet compressibility was 6.6-6.8 ×10-10 Pa-1, which is higher than for water (4.4×10-10 Pa-1) but lower than for pure perfluoropentane (2.7×10-9 Pa-1). The compressibility was constant across different droplet diameters, which was consistent with the idea that the shell thickness depends on the droplet size, rather than being constant.

Acoustic separation of living and dead cells using high density medium.

The acoustic radiation force, originating from ultrasonic standing waves and utilized in numerous cell oriented acoustofluidic applications, is dependent on the acoustic contrast factor which describes the relationship between the acousto-mechanical properties of a particle and its surrounding medium. The acousto-mechanical properties of a cell population are known to be heterogeneously distributed but are often assumed to be constant over time. In this paper, we use microchannel acoustophoresis to show that the cell state within a cell population, in our case living and dead cells, influences the mechanical phenotype. By investigating the trapping location of viable and dead K562, MCF-7 and A498 cells as a function of the suspension medium density, we observed that beyond a specific medium density the viable cells were driven to the pressure anti-node while the dead cells were retained in the pressure node. Using this information, we were able to calculate the effective acoustic impedance of viable K562 and MCF-7 cells. The spatial separation between viable and dead cells along the channel width demonstrates a novel acoustophoresis approach for binary separation of viable and dead cells in a cell-size independent and robust manner.

Ultrasonic Based Tissue Modelling and Engineering.

Systems and devices for in vitro tissue modelling and engineering are valuable tools, which combine the strength between the controlled laboratory environment and the complex tissue organization and environment in vivo. Device-based tissue engineering is also a possible avenue for future explant culture in regenerative medicine. The most fundamental requirements on platforms intended for tissue modelling and engineering are their ability to shape and maintain cell aggregates over long-term culture. An emerging technology for tissue shaping and culture is ultrasonic standing wave (USW) particle manipulation, which offers label-free and gentle positioning and aggregation of cells. The pressure nodes defined by the USW, where cells are trapped in most cases, are stable over time and can be both static and dynamic depending on actuation schemes. In this review article, we highlight the potential of USW cell manipulation as a tool for tissue modelling and engineering.

Microchip Screening Platform for Single Cell Assessment of NK Cell Cytotoxicity.

Here, we report a screening platform for assessment of the cytotoxic potential of individual natural killer (NK) cells within larger populations. Human primary NK cells were distributed across a silicon-glass microchip containing 32,400 individual microwells loaded with target cells. Through fluorescence screening and automated image analysis, the numbers of NK and live or dead target cells in each well could be assessed at different time points after initial mixing. Cytotoxicity was also studied by time-lapse live-cell imaging in microwells quantifying the killing potential of individual NK cells. Although most resting NK cells (≈75%) were non-cytotoxic against the leukemia cell line K562, some NK cells were able to kill several (≥3) target cells within the 12-h long experiment. In addition, the screening approach was adapted to increase the chance to find and evaluate serial killing NK cells. Even if the cytotoxic potential varied between donors, it was evident that a small fraction of highly cytotoxic NK cells were responsible for a substantial portion of the killing. We demonstrate multiple assays where our platform can be used to enumerate and characterize cytotoxic cells, such as NK or T cells. This approach could find use in clinical applications, e.g., in the selection of donors for stem cell transplantation or generation of highly specific and cytotoxic cells for adoptive immunotherapy.