Modelling condensation and the initiation of chondrogenesis in chick wing bud mesenchymal cells levitated in an ultrasound trap.

Chick wing bud mesenchymal cell micromass culture allows the study of a variety of developmental mechanisms, ranging from cell adhesion to pattern formation. However, many cells remain in contact with an artificial substratum, which can influence cytoskeletal organisation and differentiation. An ultrasound standing wave trap facilitates the rapid formation of 2-D monolayer cell aggregates with a defined zero time-point, independent from contact with a surface. Aggregates formed rapidly (within 2 min) and intercellular membrane spreading occurred at points of contact. This was associated with an increase in peripheral F-actin within 10 min of cell-cell contact and aggregates had obtained physical integrity by 30 min. The chondrogenic transcription factor Sox9 could be detected in cells in the ultrasound trap within 3 h (ultrasound exposure alone was not responsible for this effect). This approach facilitates the study of the initial cell-cell interactions that occur during condensation formation and demonstrates that a combination of cell shape and cytoskeletal organisation is required for the initiation and maintenance of a differentiated phenotype, which is lost when these phenomena are influenced by contact with an artificial substrate.

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.

NCAM and PSA-NCAM dependent membrane spreading and F-actin reorganization in suspended adhering neural cells.

Mediation of synchronous cell-cell interactions by NCAM and PSA-NCAM is examined here in aggregates (monolayers) of C6 polysialylated embryonic neural cells, formed rapidly (within 30 s) in suspension in an ultrasound trap. These cells express all three main isoforms of neural cell adhesion molecule (NCAM). The rate of extension of perimeter contact (i.e., membrane spreading) between closely adjacent cells and the temporal reinforcement of the Filamentous (F)-actin cytoskeleton at those regions were measured. Enzymatic removal of the cell-cell repelling polysialic acid (PSA) increases the rate of NCAM-induced membrane spreading, while removal of NCAM-120 had no detectable effect. Competitive peptide inhibition of the third immunoglobulin domain of NCAM significantly reduced the rate of membrane spreading, while NCAM siRNA transfected cells lost their ability to spread. It is argued that NCAM induced contact is the initial requirement for membrane spreading and facilitates conditions for subsequent cytoskeletal reorganization in these neural cells.

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.

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.

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.

Gap junctional intercellular communication and cytoskeletal organization in chondrocytes in suspension in an ultrasound trap.

Particles or cells suspended in an appropriately designed ultrasound standing wave field can be aggregated at a node to form a single monolayer in a plane that can be interrogated microscopically. The approach is applied here to investigate the temporal development of F-actin and Cx43 distribution and of gap junctional intercellular communication in 2-D chondrocyte aggregates (monolayers) rapidly and synchronously formed and held in suspension in an ultrasound trap. Development of the F-actin cytoskeleton in the confluent single layer of 'cuboidal' cells forming the aggregate was completed within 1 h. Chondrocytes levitated in the trap synchronously formed functional gap junctions (as assessed by CMFDA dye transfer assays) in less than 1 h of initiation of cell-cell contact in the trap. It was shown that Cx43 gene expression was retained in isolated chondrocytes in suspension. Preincubation of cells with the protein synthesis inhibitor cycloheximide caused a six-fold decrease in Cx43 accumulation (as assessed by immunofluorescence) at the interfaces of chondrocytes in the aggregate. It is shown that the ultrasound trap provides an approach to studying the early stages of cytoskeletal and gap junction development as cells progress from physical aggregation, through molecular adhesion, to display the intracellular consequences of receptor interactions.

Fluorescein isothiocynate-dextran uptake by chinese hamster ovary cells in a 1.5 MHz ultrasonic standing wave in the presence of contrast agent.

Uptake of fluorescein isothiocynate-dextran (FITC-dextran) by Chinese hamster ovary cells was studied after exposure to ultrasonic standing wave (USW) in presence of Optison, an ultrasound contrast agent. Confluent Chinese hamster ovary cells were harvested and suspended in phosphate-buffered saline + 0.1% bovine serum albumin containing FITC-dextran (10, 40, and 500 kDa) at 10 microM final concentration. The suspension was seeded with contrast agent (75 microL/mL) and exposed to a 1.5 MHz USW system at acoustic pressures ranging from 0.98 to 4.2 MPa. Macromolecular uptake was assessed by fluorescent microscopy and quantified by flow cytometry 10 min after exposure. FITC-dextran positive cells, as assessed by flow cytometry, were 1 +/- 0.05% and 2.58 +/- 0.27% for acoustic pressures of 1.96 and 4.2 MPa, respectively (p = 0.006). Fluorescent microscopy indicated a degree of macromolecular loading at 0.98 MPa with 46% of peripherally FITC-dextran- and/or propidium iodide-stained cells coincident with the appearance of significant frequency (f0/2 and 2 f0) emission signals. At higher pressures, high macromolecular loading with 6% peripherally stained cells at 1.96 MPa was associated with lower order emission signals and white noise. The study conclusively demonstrates macromolecular loading in an USW, a significantly higher macromolecular loading at higher pressures and indicates potential of emission signals for a feedback loop to control the acoustic power outputs and fine-tune the biologic effects associated with sonoporation.

Improvement of immunodetection of bacterial spore antigen by ultrasonic cavitation.

Ultrasonic cavitation was employed to enhance sensitivity of bacterial spore immunoassay detection, specifically, enzyme-linked immunosorbent assay (ELISA) and resonant mirror (RM) sensing. Bacillus spore suspensions were exposed to high-power ultrasound in a tubular sonicator operated at 267 kHz in both batch and flow modes. The sonicator was designed to deliver high output power and is in a form that can be cooled efficiently to avoid thermal denaturation of antigen. The 30-s batch and cooled flow (0.3 mL/min) sonication achieved an approximately 20-fold increase in ELISA sensitivity compared to unsonicated spores by ELISA. RM sensing of sonicated spores achieved detection sensitivity of approximately 10(6) spores/mL, whereas unsonicated spores were undetectable at the highest concentration tested. Improvements in detection were associated with antigen released from the spores. Equilibrium temperature increase in the tubular sonicator was limited to 14 K after 30 min and was maintained for 6 h with cooling and flow (0.3 mL/min). The work described here demonstrates the utility of the tubular sonicator for the improvement in the sensitivity of the detection of spores and its suitability as an in-line component of a rapid detection system.

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.

Optical leaky waveguide sensor for detection of bacteria with ultrasound attractor force.

An integrated, sensitive, and rapid system was developed for the detection of bacteria. The system combined an optical metal-clad leaky waveguide (MCLW) sensor with ultrasound standing waves (USW). The performance of a MCLW sensor for the detection of bacteria has been increased (>100 fold) by using USWs to drive bacteria onto the sensor surface. By forming the USW nodes at or within the surface of the MCLW, the diffusion-limited capture rate has been replaced by fast movement. Immobilized anti-BG antibody on the MCLW sensor surface was used to capture Bacillus subtilis var. niger (BG) bacterial spores driven to the surface. This combination of sensor and attractor force combination has been tested by detecting the evanescent scattering from bacterial spores at the sensor surface. Application of ultrasound for 3 min gave a detection limit for BG bacterial spores of 1 x 10(3) spores/mL.

Molecular adhesion development in a neural cell monolayer forming in an ultrasound trap.

A 2-dimensional aggregate of C6 neural cells was formed rapidly (within 30 s) in suspension in a recently developed 1.5 MHz ultrasound standing wave trap. A typical 1 mm diameter aggregate contained about 3,500 cells. Spreading of membrane occurred between the aggregated cells. The rate of spreading of the tangentially developing intercellular contact area was 0.19 microm/min. The form of the suspended aggregate changed from one of a hexagonal arrangement of cells to one of a cell-monolayer-like continuous sheet of mostly quadrilateral and pentagonal cells as in a cell monolayer on a solid substratum. A range of fluorescent indicators showed that the >99% viability of the cells did not change during 1 h exposures; therefore cell viability was not compromised during the monolayer development. The average integral intensities from stained actin filaments at the spreading cell-cell interfaces after 1, 8 and 30 min were 14, 25 and 46 microm(2) respectively. The cells in this work progressed from physical aggregation, through molecular adhesion, to displaying the intracellular consequences of receptor interactions. The ability to form mechanically strong confluent monolayer structures that can be monitored in situ or harvested from the trap provides a technique with general potential for monitoring the synchronous development of cell responses to receptor-triggered adhesion.

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.

A new immobilisation method to arrange particles in a gel matrix by ultrasound standing waves.

Ultrasonic forces may be used to manipulate particles in suspension. For example, a standing wave ultrasound (US) field applied to a suspension moves the particles toward areas of minimal acoustic pressure, where they are orderly retained creating a predictable heterogeneous distribution. This principle of ultrasonic retention of particles or cells has been applied in numerous biotechnological applications, such as mammalian cell filtering and red blood cell sedimentation. Here, a new US-based cell immobilisation technique is described that allows manipulation and positioning of cells/particles within various nontoxic gel matrices before polymerisation. Specifically, gel immobilisation was used to directly demonstrate that the viability of yeast cells arranged by an US standing wave is maintained up to 4 days after treatment. The versatility of this immobilisation method was validated using a wide range of acoustic devices. Finally, the potential biotechnological advantages of this US-controlled particle positioning method combined with gel immobilisation/encapsulation technology are discussed.

Continuous cell washing and mixing driven by an ultrasound standing wave within a microfluidic channel.

Ultrasound standing wave radiation force and laminar flow have been used to transfer yeast cells from one liquid medium to another (washing) by a continuous field-flow fractionation (FFF) approach. Two co-flowing streams, a cell-free suspending phase (flow rate > 50% of the total flow-through volume) and a yeast suspension, were introduced parallel to the nodal plane of a 3 MHz standing wave resonator. The resonator was fabricated to have a single pressure nodal plane at the centre line of the chamber. Laminar flow ensured a stable interface was maintained as the two suspending phases flowed through the sound field. Initiation of the ultrasound transferred cells to the cell-free phase within 0.5 s. This particle transfer procedure circumvents the pellet formation and re-suspension steps of centrifuge based washing procedures. In addition, fluid mixing was demonstrated in the same chamber at higher sound pressures. The channel operates under negligible back-pressure (cross-section, 0.25 [times] 10 mm) and with only one flow convergence and one flow division step, the channel cannot be easily blocked. The force acting on the cells is small; less than that experienced in a centrifuge generating 100g. The acoustically-driven cell transfer and mixing procedures described may be particularly appropriate for the increasingly complex operations required in molecular biology and microbiology and especially for their conversion to continuous flow processes.

Ultrasonic deposition of cells on a surface.

Bacteria in water have been driven to a glass surface by an ultrasonic standing wave. On an antibody coated surface capture of Bacillus subtilis var niger (BG) spores (6.6 x 10(6) ml(-1)) was increased more than 200-fold over above the efficiency in the absence of ultrasound. In microfluidic (non-turbulent) systems detection of particles by sensors operating at a surface is diffusion limited. This results in very low detection abilities particularly for particles with diameters greater than 1 microm. Ultrasound is used here to drive bacterial spores to a wall and overcome this limitation. The results confirm: (1) pressure nodes can be formed close to the water-glass interface when the glass thickness is near half the ultrasonic wavelength; (2) the antibody used was able to capture spores in the presence of an ultrasonic standing wave.

Contrast agent bubble and erythrocyte behavior in a 1.5-MHz standing ultrasound wave.

Human erythrocytes and Optison contrast agent have been exposed to ultrasound, both alone and in combination, in a single-half-wavelength chamber driven at its resonance frequency (fo) of 1.5 MHz. Cell movements were recorded by video microscopy at speeds up to 500 frames/s. The hypothesis that cells near a standing wave pressure node might be stressed by the microbubble products of sonicated contrast agent was examined. In the absence of contrast agent, cells moved rapidly to form an aggregate in the standing wave pressure node plane. First subharmonic and second harmonic emissions were detected from cell-contrast agent suspensions immediately on exposure to a threshold peak pressure amplitude of 0.98 MPa. Emissions at 3fo/2 occurred at 1.47 MPa, whereas white noise and lower-order subharmonic emissions coincided with the appearance of visible bubbles at a threshold of approximately 1.96 MPa. Cells exposed together with contrast agent at a pressure of 0.98 MPa precessed very rapidly about the pressure node plane. This behavior was discussed in the context of a recent analysis predicting that, in contrast to the situation for lower-pressure amplitudes, subresonant size bubbles translate about pressure node plane if the driving pressure amplitude is sufficiently high. Many precessing erythrocytes were clearly spiculated and this morphology persisted after the cells had left the area of precession. Hemoglobin release was significant under conditions inducing precession with first subharmonic and first harmonic emissions. Protein release increased discontinuously near the pressure thresholds, where more complex categories of frequency emission were detected. The potential of this system, which induces erythrocyte morphology changes and some protein release at the first emission threshold, to provide some control on the membrane-permeabilizing stress experienced by cells in a cavitation field is discussed.