Understanding the complex 3D tumor microenvironment is important in cancer research. This microenvironment can be modelled in vitro by culturing multicellular tumor spheroids (MCTS). Key challenges when using MCTS in applications such as high-throughput drug screening are overcoming imaging and analytical issues encountered during functional and structural investigations. To address these challenges, we use an ultrasonic standing wave (USW) based MCTS culture platform for parallel formation, staining and imaging of 100 whole MCTS. A protein repellent amphiphilic polymer coating enables flexible production of high quality and unanchored MCTS. This enables high-content multimode analysis based on flow cytometry and in situ optical microscopy. We use HepG2 hepatocellular carcinoma, A498 and ACHN renal carcinoma, and LUTC-2 thyroid carcinoma cell lines to demonstrate (i) the importance of the ultrasound–coating combination, (ii) bright field image based automatic characterization of MTCS, (iii) detailed deep tissue confocal imaging of whole MCTS mounted in a refractive index matching solution, and (iv) single cell functional analysis through flow cytometry of single cell suspensions of disintegrated MTCS. The USW MCTS culture platform is customizable and holds great potential for detailed multimode MCTS analysis in a high-content manner.

A novel, simple and robust route for binary separation of viable and dead cells using a density modified medium which takes advantage of the compromised cell membrane of dead cells.

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 hours 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.

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.

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.

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.