Contactless three-dimensional trapping and manipulation of cells can be useful for various biological studies. Such manipulation is typically performed using dielectrophoresis – in which a nonuniform electric field exerts force on the cell – or using optical trapping. However, commercially available dielectrophoresis systems can damage cells after about 30 minutes, so their use is limited if long-term, gentle cell handling with high precision is required, such as for calcium signaling or protein trafficking studies. Optical trapping has similar limitations.Researchers at Royal Institute of Technology and at Karolinska Institute, both in Stockholm, Sweden, wanted to develop a system that could be applied to long-term monitoring of cells. They knew that standing-wave ultrasound had been used successfully for large-scale batch handling of cells for several days using, for example, the BioSep system from Applikon Biotechnology BV of Schiedam, the Netherlands.“We were interested to investigate whether a microscale ultrasound-based system could be designed with similar performance and manipulation precision as a dielectrophoresis-based system, and with similar biocompatibility as the BioSep system,” said investigator Martin Wiklund.In the Aug. 11 issue of Applied Physics Letters, the researchers, led by Wiklund, reported a tool that enables 3-D manipulation of individual microparticles and cells using an ultrasonic cage integrated into a microfluidic chip. The system is compatible with high-resolution transillumination microscopy as well as confocal or epifluorescence microscopy, so it also facilitates online observation of a range of intracellular parameters.Researchers have reported an ultrasonic cage that provides gentle contactless manipulation of cells and particles with high precision. When the cage is excited at 2.50 and 6.89 MHz, particles entering the cage (from the right) are three-dimensionally trapped and retained in the center (a) and then collected along a line (b). They enter an intermediate state when the beads rearrange into a more compact 3-D aggregate (c), and, finally, enter a state in which approximately 100 beads are in the aggregate (d). In these images, 10-m fluorescent beads were used.The chip comprises a 14.75 × 50-mm2 glass-silicon-glass stack with the microchannel dry-etched into the silicon layer. Wiklund and colleagues designed the 0.30 × 0.30 × 0.11-mm3 cage as a 3-D resonant box. Otto Manneberg, the first author of the paper, said, “We investigated several different geometries of the resonator – that is, the shape of the ‘cage.’ Initially, we swiped the design from laser resonators, but, finally, the filleted square box turned out to be the most efficient cage design.” They achieved precise control of cells by exciting the resonant box with two wedge transducers at frequencies of 2.50 and 6.89 MHz, which correspond to half-wave resonances in three orthogonal directions.The ultrasonic cage is compatible with high-resolution optical microscopy, enabling visualization of the trapped cells. Shown are confocal fluorescence microscopy images of a single caged B cell. The cell is labeled with the green-fluorescent viability indicator calcein-AM (left) and with the red-fluorescent membrane probe DiD (right). Reprinted with permission of Applied Physics Letters.Using this chip, the researchers performed 3-D caging, enrichment and aggregation of cells by tuning the relative actuation voltages at the two frequencies. In addition, they showed that, because actuation occurs at two frequencies, they could transform aggregates reversibly between 2-D monolayers and 3-D structures. Monolayers can be useful when using high-resolution optical microscopy, for example, and transforming between monolayer and multilayer structures can aid studies of cell-cell interactions by precisely controlling the number of neighbors for each cell.A gentle touchThe ultrasonic cage offers a number of advantages over other contactless manipulation tools, such as optical tweezers and dielectrophoresis, the clearest of which, Wiklund said, is the gentleness. “We have previously shown that a 75-minute ultrasonic exposure does not affect the state of health of mammalian cells trapped in a microfluidic chip, and work is in progress on longer-term cell handling experiments.” He added that the technique is simple and inexpensive.The primary disadvantage is that the method is still somewhat “coarse” with respect to the alternatives: “It still feels a bit like we have to sort marbles wearing boxing gloves,” Manneberg explained.Nonetheless, the ultrasonic cage holds promise for a range of applications. Besides being useful for studying dynamic processes such as calcium signaling and trafficking of proteins, it could help advance the study of intracellular interactions within small- or medium-size cell clusters. Here, it could help determine the “dimensionality” of a cluster by allowing researchers to control the number of neighbors for each cell in the cluster and to arrange cells, for example, as a 2-D monolayer or as a 3-D spheroid.The technique also might contribute to studies of how cell behavior is regulated by surface contact. Does the cell’s response to stimulation change depending on whether it is attached to a solid support or is floating? And can this response be controlled by changing the cell’s polarization, for instance? Björn Önfelt, another author of the study, noted: “This could be relevant for cells such as immune cells circulating in the body spending time in different types of tissue; for example, blood, lymph, sites of infection. Are cells changing polarization and migration behavior only due to the environment, or could a changed polarization target them to the right environment?”The researchers already are working to apply the ultrasonic cage. They are especially interested in monitoring intercellular interactions of natural killer (NK) or T cells with their targets in a controlled manner. The technique will allow them to vary the number of NK/T or target cells, as well as their relative orientation, to facilitate study of intercellular communication in small clusters. In addition, by adding fluid flow from various inlet channels on the microfluidic chip, they can combine these imaging studies with stimuli with various soluble agents, or perhaps fixation followed by labeling with fluorescent antibodies inside the chip.“Here dynamic processes could be correlated with treatment – for example, with drugs or cytokines or [with] distributions of proteins that are difficult to study in live cells,” Önfelt said.