Multiple three-dimensional mammalian cell aggregates formed away from solid substrata in ultrasound standing waves.

Single and multiple three-dimensional cell aggregates of human red blood cells (RBCs) and HepG2 cells were formed rapidly in low mega-Hertz ultrasound standing wave fields of different geometries. A single discoid aggregate was formed in a half-wavelength pathlength resonator at a cell concentration sufficient to produce a 3D structure. Multiple cell aggregates were formed on the axis of a cylindrical resonator with a plane transducer (discoid aggregates); in a resonator with a tubular transducer and in the cross-fields of plane and tubular transducers and two plane orthogonal transducers (all cylindrical aggregates). Mechanically strong RBC aggregates were obtained by crosslinking with wheat germ agglutinin (WGA, a lectin). Scanning electron microscopy showed aggregate surface porous structures when RBCs were mixed with WGA before sonication and tighter packing when ultrasonically preformed aggregates were subsequently exposed to a flow containing WGA. HepG2 cell aggregates showed strong accumulation of F-actin at sites of cell-cell contact consistent with increased mechanical stability. The aggregates had a porous surface, and yet confocal microscopy revealed a tight packing of cells in the aggregate's inner core.

Functional three-dimensional HepG2 aggregate cultures generated from an ultrasound trap: comparison with HepG2 spheroids.

Three-dimensional culture systems are an ideal in vitro model being capable of sustaining cell functionalities in a manner that resembles the in vivo conditions. In the present study, we developed an ultrasound trap-based technique to rapidly produce HepG2 hepatocarcinoma cell aggregates within 30 min. Enhanced junctional F-actin was observed at the points of cell-cell contact throughout the aggregates. HepG2 aggregates prepared by the ultrasound trap can be maintained in culture on a P-HEMA-coated surface for up to 3 weeks. The cells in these aggregates proliferated during the initial 3 days and cell number was consistent during the following maintenance period. Albumin secretion from these HepG2 aggregates recovered after 3 days of aggregate formation and remained relatively stable for the following 12 days. Cytochrome P450-1A1 activity was significantly enhanced after 6 days with maximal enzyme activity observed between 9 and 18 days. In addition, comparison experiments demonstrated that HepG2 aggregates generated by the ultrasound trap had comparable functional characterizations with HepG2 spheroids formed by a traditional gyrotatory-mediated method. Our results suggest that HepG2 aggregate cultures prepared through the ultrasound trap-based technique may provide a novel approach to produce in vitro models for hepatocyte functional studies.

Cell adhesion dynamics and actin cytoskeleton reorganization in HepG2 cell aggregates.

The temporal dependence of cytoskeletal remodelling on cell-cell contact in HepG2 cells has been established here. Cell-cell contact occurred in an ultrasound standing wave trap designed to form and levitate a 2-D cell aggregate, allowing intercellular adhesive interactions to proceed, free from the influences of solid substrata. Membrane spreading at the point of contact and change in cell circularity reached 50% of their final values within 2.2 min of contact. Junctional F-actin increased at the interface but lagged behind membrane spreading, reaching 50% of its final value in 4.4 min. Aggregates had good mechanical stability after 15 min in the trap. The implication of this temporal dependence on the sequential progress of adhesion processes is discussed. These results provide insight into how biomimetic cell aggregates with some liver cell functions might be assembled in a systematic, controlled manner in a 3-D ultrasound trap.

Stability of 2-D colloidal particle aggregates held against flow stress in an ultrasound trap.

The formation of a two-dimensional aggregate of 25 microm latex particles in a 1.5 MHz ultrasound standing wave (USW) field and its disintegration in a flow were studied. The aggregate was held in the pressure node plane, which allowed continuous microscope observation and video recording of the processes. The trajectories and velocities of the particles approaching the formation site were analyzed by particle image velocimetry (PIV). Since the direct radiation force on the particles dominated the drag due to acoustic streaming, the acoustic pressure profile in the vicinity of the aggregate was quantifiable. The drag coefficients D(coef) for 2- to 485-particle aggregates were estimated from the balance of the drag force FD and the buoyancy-corrected gravitational force during sedimentation on termination of the ultrasound when the long axis of the aggregate was in the vertical plane. D(coef) were calculated from FD as proportional to the aggregate velocity. Experiments on particle detachment by flow (in-plane velocity measured by PIV) from horizontal aggregates suspended in deionized water and CaCl2 solution of different concentrations showed that the mechanical strength of the aggregates depended on the acoustic pressure amplitude P0 and ionic strength of the solution. In deionized water the flow velocity required to detach the first single particle from an aggregate increased from 1 mm s-1 at P0 = 0.6 MPa to 4.2 mm s-1 at P0 = 1.4 MPa. The balance of forces acting on particles in a USW trap is discussed. The magnitude of the shear stress employed ( approximately 0.05 Pa) and separation forces suggests that this technique can be applied to studying the mechanical responses of cell aggregates to hydrodynamic flow, where cell-cell interaction can be separated from the effects of solid substrata.

Applications of ultrasound streaming and radiation force in biosensors.

Direct radiation force (DRF) and acoustic streaming provide the main influences on the behaviour of particles in aqueous suspension in an ultrasound standing wave (USW). The direct radiation force, which drives suspended particles towards and concentrates them in acoustic pressure node planes, has been applied to rapidly transfer cells in small scale analytical separators. The DRF also significantly increased the sensitivity of latex agglutination test (LAT) by concentrating the particles of an analytical sample in the pressure node positions and hence greatly increasing the antibody-antigen encounter rate. Capture of biotinylated particles and spores on a coated acoustic reflector in a quarter wavelength USW resonator was DRF-enhanced by 70- and 100-fold, respectively compared to the situation in the absence of ultrasound. Acoustic streaming has been successfully employed for mixing small analytical samples. Cavitation micro-streaming substantially enhanced, through mixing, DNA hybridization and the capture efficiency of Escherichia coli K12 on the surface of immunomagnetic beads. Acoustic streaming induced in longitudinal standing wave and flexural plate wave immuno-sensors increased the detection of antigens by a factor of five and three times, respectively. Combined DRF and acoustic streaming effects enhanced the rate of the reaction between suspended mixture of cells and retroviruses. The examples of a biochip and an ultrasonic immuno-sensor implementing the DRF and acoustic streaming effects are also described in the review.

Sub-micron particle behaviour and capture at an immuno-sensor surface in an ultrasonic standing wave.

The capture of 200 nm biotinylated latex beads from suspensions of concentration 10(7) to 2.5 x 10(8) particle/ml on an immuno-coated surface of the acoustic reflector in an ultrasound standing wave (USW) resonator has been studied while the acoustic pathlength was less than one half wavelength (lambda/2). The particles were delivered to the reflector's surface by acoustically induced flow. The capture dependencies on suspension concentration, duration of experiments and acoustic pressure have been established at 1.09, 1.46 and 1.75 MHz. Five-fold capture increase has been obtained at 1.75 MHz in comparison to the control (no ultrasound) situation. The contrasting behaviours of 1, 0.5 and 0.2 mum fluorescent latex beads in a lambda/4 USW resonator at 1.46 MHz have been characterized. The particle movements were observed with an epi-fluorescent microscope and the velocities of the particles were measured by particle image velocimetry (PIV). The experiments showed that whereas the trajectories of 1 mum particles were mainly affected by the direct radiation force, 0.5 mum particles were influenced both by the radiation force and acoustic streaming. The 0.2 mum latex beads followed acoustic streaming in the chamber and were not detectably affected by the radiation force. The streaming-associated behaviour of the 200 nm particles has implications for enhanced immunocapture of viruses and macromolecules (both of which are also too small to experience significant acoustic radiation force).

Spore and micro-particle capture on an immunosensor surface in an ultrasound standing wave system.

The capture of Bacillus subtilis var. niger spores on an antibody-coated surface can be enhanced when that coated surface acts as an acoustic reflector in a quarter wavelength ultrasonic (3 MHz) standing wave resonator. Immunocapture in such a resonator has been characterised here for both spores and 1 microm diameter biotinylated fluorescent microparticles. A mean spatial acoustic pressure amplitude of 460 kPa and a frequency of 2.82 MHz gave high capture efficiencies. It was shown that capture was critically dependent on reflector thickness. The time dependence of particle deposition on a reflector in a batch system was broadly consistent with a calculated time of 35 s to bring 95% of particles to the coated surface. A suspension flow rate of 0.1 ml/min and a reflector thickness of 1.01 mm gave optimal capture in a 2 min assay. The enhancement of particle detection compared with the control (no ultrasound) situation was x 70. The system detects a total of five particles in 15 fields of view in a 2 min assay when the suspending phase concentration was 10(4) particles/ml. A general expression for the dependence of minimum concentration detectable on; number of fields examined, sample volume flowing through the chamber and assay time shows that, for a practical combination of these variables, the threshold detection concentration can be two orders of magnitude lower.

Physical enviroment of 2-D animal cell aggregates formed in a short pathlength ultrasound standing wave trap.

2-D mammalian cell aggregates can be formed and levitated in a 1.5 MHz single half wavelength ultrasound standing wave trap. The physical environment of cells in such a trap has been examined. Attention was paid to parameters such as temperature, acoustic streaming, cavitation and intercellular forces. The extent to which these factors might be intrusive to a neural cell aggregate levitated in the trap was evaluated. Neural cells were exposed to ultrasound at a pressure amplitude of 0.54 MPa for 30 s; a small aggregate had been formed at the center of the trap. The pressure amplitude was then decreased to 0.27 MPa for 2 min, at which level the aggregation process continued at a slower rate. The pressure amplitude was then decreased to 0.06 MPa for 1 h. Temperature measurements that were conducted in situ with a 200 microm thermocouple over a 30 min period showed that the maximum temperature rise was less than 0.5 K. Acoustic streaming was measured by the particle image velocimetry method (PIV). It was shown that the hydrodynamic stress imposed on cells by acoustic streaming is less than that imposed by gentle preparative centrifugation procedures. Acoustic spectrum analysis showed that cavitation activity does not occur in the cell suspensions sonicated at the above pressures. White noise was detected only at a pressure amplitude of 1.96 MPa. Finally, it was shown that the attractive acoustic force between ultrasonically agglomerated cells is small compared with the normal attractive van der Waals force that operates at close cell surface separations. It is concluded that the standing wave trap operates only to concentrate cells locally, as in tissue, and does not modify the in vitro expression of surface receptor interactions.

Cavitation bubble-driven cell and particle behavior in an ultrasound standing wave.

The behavior of human erythrocytes and 1-microm-diameter fluorescent latex beads in the presence of Optison contrast agent in a single half-wavelength (lambda/2) ultrasound standing wave (USSW) resonator has been studied. The particle movements were observed with an epi-fluorescent microscope and the velocity of the particles and cells was measured by particle image velocimetry (PIV). Acoustic emissions were monitored with a microphone and a spectrum analyzer. Optison contrast agent disintegrated immediately on exposure to ultrasound of 0.98-MPa acoustic pressure amplitude or higher in a chamber driven at its resonance frequency of 1.56 MHz. A discrete cloud of active microbubbles, detected at the pressure node plane, disappeared gradually and was completely lost within 15 s. The microscopy showed three-dimensional regions of circulation of both 1-microm tracer particles and erythrocytes in planes perpendicular to the pressure node plane. A numerical simulation showed that, for parameters that conform to the experimental conditions, a bubble of a subresonance size moves towards and translates about a pressure node plane. This result is in agreement with the experimental observation that the particle and cell circulation is induced by the presence and/or translational motion of microbubbles at the pressure node plane.