Development of a novel compact sonicator for cell disruption.

Ultrasound microbial cell disrupters operating at around 20 kHz are often physically large and, due to significant heating, can be unsuitable for small sample volumes where biochemical integrity of the extracted product is required. Development of a compact device based on a 63.5-mm diameter, 6.5-mm thick tubular transducer for rapid cell disruption in small-volume samples in a high-intensity acoustic cavitation field with minimal temperature rises is described here. Suspensions of Saccharomyces cerevisiae were exposed to cavitation for various times in the compact device and a 20-kHz probe sonicator. Cell disruption was assessed by protein release and by staining. Yeast cell disruption was greater in the novel 267-kHz sonicator than in the 20-kHz probe sonicator for the same exposure time. A 1-dimensional (1-D) transfer matrix model analysis for piezoelectric resonators was applied to an axial cross-section of the tubular sonicator to predict frequencies of mechanical resonance in the sample volume associated with maximum acoustic pressure. Admittance measurements identified frequencies of electrical resonance. Ultrasonic cavitation noise peaks were detected by a hydrophone at both the mechanical and electrical resonances. Cell breakage efficiency was twice as great in terms of protein released per dissipated watt at the mechanical resonance predicted by the model, compared to those at the electrical resonance frequencies. The results form a basis for rational design of an ultrasound cell disruption technique for small-volume samples.

Ultrasound-induced physiological changes in cultured cells of Petunia hybrida.

Petunia hybrida cell suspension cultures were exposed to ultrasonic standing wave fields at 2.43 MHz for 40 min with mean sound pressures (within homogenous sound fields) varying from 0 (control) to ca. 1.1 MPa. Mean (+/- s.d.; n =6-9) cell viability was reduced to 87+/-10% at 0.6 MPa and to 59 +/- 23% at 1.1 MPa, compared to an initial control value of 92 +/- 6% (P <0.05). Mean (n = 3) cell alkaline phosphatase concentration increased linearly with sound pressure from a control value of 0.006+/-0.001 to 0.02+/-0.01 Sigma-Units microg(-1) protein at 1.1 MPa (P<0.05). Similarly, mean cell catalase activity increased from a control value of 0.020 +/- 0.003 to 0.026 +/- 0.008 arbitrary units at 1.1 MPa. In contrast, mean cellular lactate dehydrogenase concentration was unchanged. These observations indicate that cellular repair processes associated with increased alkaline phosphatase activity might be triggered by physical cell damage caused by ultrasound. The observed increase in catalase activity suggests increasing production of free radicals and other sonochemicals, which warrants further study. The absence of changes in lactate dehydrogenase indicates that there was no major damage to respiratory pathways or to overall cellular integrity.

A study of the spatial organisation of microbial cells in a gel matrix subjected to treatment with ultrasound standing waves.

Retention and manipulation of microbial cells through exploitation of ultrasonic forces has been reported as a novel cell immobilisation technique. The spatial ordering of yeast cells, within suspensions subjected to an ultrasonic standing wave field, was analysed for the first time. A technique, based on 'freezing' the spatial arrangement using polymer gelation was developed. The resultant gel was then sectioned and examined using microscopic techniques. Light Microscopy confirmed the presence of specific regions in the ultrasonic field, where the cells are organised into bands corresponding to the standing waves' pressure nodal planes. Computer Image Analysis measurement of several physical parameters associated with this cell distribution matched the values derived from the theoretical model. The spatial cell-cell re-arrangement within each band and uneven distribution along the nodal planes have been analysed by Scanning Electron Microscopy. These results complement the ongoing study of the process of immobilisation of microbial cells by ultrasound standing waves.

Mechanical culture conditions effect gene expression: gravity-induced changes on the space shuttle.

Three-dimensional suspension culture is a gravity-limited phenomenon. The balancing forces necessary to keep the aggregates in suspension increase directly with aggregate size. This leads to a self-propagating cycle of cell damage by balancing forces. Cell culture in microgravity avoids this trade-off. We determined which genes mediate three-dimensional culture of cell and tissue aggregates in the low-shear stress, low-turbulent environment of actual microgravity. Primary cultures of human renal cortical cells were flown on the space shuttle. Cells grown in microgravity and ground-based controls were grown for 6 days and fixed. RNA was extracted, and automated gene array analysis of the expression of 10, 000 genes was performed. A select group of genes were regulated in microgravity. These 1,632 genes were independent of known shear stress response element-dependent genes and heat shock proteins. Specific transcription factors underwent large changes in microgravity including the Wilms' tumor zinc finger protein, and the vitamin D receptor. A specific group of genes, under the control of defined transcription factors, mediate three-dimensional suspension culture under microgravity conditions.

Viability of plant cell suspensions exposed to homogeneous ultrasonic fields of different energy density and wave type.

Exposure of Petunia hybrida cell suspensions to ultrasound at a frequency of 2.43 MHz in a standing wave field at an energy density of 70 Jm-3 (pressure amplitude of 0.78 MPa) decreased their mean viability to 35% after 20 min of sonication. A comparison of propagating wave and standing wave treatments at equal frequency (2.15 MHz) and energy density (8.5 Jm-3) showed, in the first case, a rapid decline in mean viability of cells (to 30% after 10 min of sonication) and, in the second case, a retaining of the initial viability (95%), respectively. Cells sonicated 4 days after subculture were more sensitive than cells sonicated 2 or 6 days after transfer to new culture medium. It was concluded that cellular viability depends primarily on the acoustic energy density, the exposure time, and the mechanical properties of the cells determined by age. As a consequence of the trapping of cells in the anti-node planes of the standing wave, propagating wave fields reduced cellular viability compared with standing wave fields at equal energy density.

Viability of yeast cells in well controlled propagating and standing ultrasonic plane waves.

Recent studies have shown that there is no loss of cell viability when the cells are subjected to ultrasonic standing wave fields in acoustic cell retention systems. These systems are characterised by waves that spatially vary in pressure amplitude in the direction of sound propagation. In this work an anechoic 'one-dimensional' sonication chamber has been developed that produces propagating waves, which differ from standing waves in that the pressure amplitude remains constant as the wave travels in a medium with negligible attenuation. The viability of yeast cell suspensions as a function of treatment time was investigated during exposure to both standing and propagating wave fields with frequencies slightly above 2 MHz. The influence of 12% (vol/vol) of ethanol in water on the spatial arrangement of the cells in suspension was also studied. Changes in yeast cell morphology caused by the different types of suspension media and the ultrasonic treatment were examined by transmission electron microscopy (TEM). The agglomeration of yeast cells within the pressure nodal planes appears to minimise damaging effects due to ultrasonic fields.

Nuclear factor kappa B mediates lipopolysaccharide-induced inflammation in the urinary bladder.

PURPOSE: The proteins which constitute the final common pathway linking receptors on cell surfaces to the inflammatory cascade have recently been identified and cloned. Central to activation of this inflammatory cascade is translocation from cytosol to nucleus of the nuclear transcription factor known as nuclear factor-kappa B (NF-kappaB). The purpose of this study was to determine whether NF-kappaB cascade plays a role in lipopolysaccharide (LPS)-induced inflammation of the mouse urinary bladder. MATERIALS AND METHODS: Bladder inflammation was induced in anesthetized mice by intravesical instillation of lipopolysaccharide (LPS) and quantified by morphometric analysis. The NK-1 receptors for substance P were quantified by flow cytometry. LPS-induced degradation of inhibitory IkappaB subunit was quantified by Western blotting analysis and translocation of NF-kappaB protein from cytosol to the nucleus was determined by confocal microscopy and Western blotting analysis. In addition, we determine the effect of lactacystin, a proteosome inhibitor, on LPS-induced IkappaB degradation and NF-kappaB translocation, NK-1 receptor fluorescence intensity, and bladder inflammation. RESULTS: LPS instillation into the mouse bladder resulted in time dependent loss of the inhibitory IkappaB subunit of the NF-kappaB protein complex. IkappaB cleavage was followed by translocation of NF-kappaB from the cytosol to the nucleus. This was associated with increased expression of an NF-kappaB dependent inflammatory component, the NK-1 receptor. Pretreatment of mouse bladders with the NF-kappaB inhibitor, lactacystin, prevented cleavage of IkappaB in a dose-dependent manner. Lactacystin prevented increases in the NF-kappaB dependent inflammatory cascade components such as the NK-1 receptor. At the whole tissue level, the marked inflammatory infiltrate and mucosal breakdown associated with LPS administration was completely abolished by lactacystin. CONCLUSION: NF-kappaB mediates many features of urinary bladder inflammation induced by LPS. The NF-kappaB cascade is an important target for anti-inflammatory management of cystitis.

Breakdown of immobilisation/separation and morphology changes of yeast suspended in water-rich ethanol mixtures exposed to ultrasonic plane standing waves.

Some physiological/morphological changes have been reported before, when suspended yeasts have been irradiated with well-defined ultrasonic standing, as well as propagating, plane waves around 2.2 MHz, as used in ultrasonic coagulation, e.g., for cell filtering. Thus we used yeast as a biological model to explore the reasons for both those morphology changes and some unusual macroscopic behaviour in the case of water-rich ethanol mixtures when used as carrier liquid. When the cells were suspended in 12% (v/v) ethanol-water mixture separation was greatly reduced; the yeast cells were not retained in the pressure nodal planes of the standing wave, but mixed turbulently through the separation system. How this behaviour alters the efficiency of retention/immobilisation was measured. As the viability of the yeast was decreased as well the morphology of the cells was examined using transmission electron microscopy. Two effects, according to the type of assessment, were evident; a disruption of the cells vacuole and also damage to the cell wall/membrane complex. The extent of the alterations in vacuole structure with sonication time, utilising a fluorescent vacuole membrane dye, was measured. Transient cavitation was not detected and thus could be excluded as being responsible for the observed effects. Other possible reasons for the disruption of the intracellular compartments may be acoustic pressure, displacement or other, secondary effects like (sub) harmonic cavitation. The investigations contribute to a better understanding of the physical conditions experienced when a cell is stressed in a high-frequency ultrasonic wave in the MHz range.

Viscosity sensor utilizing a piezoelectric thickness shear sandwich resonator.

This paper describes a novel quartz crystal sensor for measurement of the density-viscosity product of Newtonian liquids. The sensor element consists of two piano-convex AT-cut quartz crystals vibrating in a thickness-shear mode with the liquid sample in between. This special set-up allows suppression of disturbing resonances in the liquid layer. Such resonances are generated in the common single-plate arrangements due to compressional waves caused by spurious out-of-plane displacements of the shear vibrating finite plate. The primary measurands of the sensor are the fundamental resonance frequency and the associated resonance Q-value, which are influenced by the viscously entrained liquid contacting the quartz surface. The sensor allows the measurement of samples with viscosities from almost zero (air!) up to 200 cP with a sample volume of 130 microl.

Comparison between BAW and SAW sensor principles.

A comparison is given between piezoelectrically excited bulk acoustic wave (BAW) and surface acoustic wave (SAW) elements with respect to their primary sensitivity functions and principal capabilities for sensor applications. The importance of mode purity for high dynamic range sensors is emphasized. Characteristic sensor examples are reviewed, and the special demands on the electronics for BAW and SAW elements in the sensor field are described (e.g., cable problem, wireless SAW sensors). For a fair evaluation, a performance figure, SQ, defined as the product of reduced sensitivity S and resonator Q-value, is introduced. The potential of alternative piezoelectric materials for future sensor developments is discussed briefly.

Selective retention of viable cells in ultrasonic resonance field devices.

A double-chamber ultrasonic resonance field device was used for the separation and retention of animal cells. By controlling operational parameters such as flow and power input, the device can retain viable cells more efficiently, allowing for selective removal of nonviable cells and cell debris. A simple model describing the forces acting on spherical particles in a sound field (primary radiation force, Bernoulli force, secondary radiation force) is presented. Field stability increases with decreasing average flow rates and increasing power input. At very high field stability, as achieved with low flow rates and high power input, the selectivity for viable cells is reduced, due to the efficient retention of all types of particles. At high flow rates and resulting low field stability, selectivity is also reduced, due to poor separation efficiency, resulting in equally low retention of viable cells, nonviable cells, and cell debris.

A novel ultrasonic resonance field device for the retention of animal cells.

This article describes two types of flow-through cell retention devices based on the concept of layered piezoelectric resonators. A single-chamber device is compared to a novel optimized steam-sterilizable prototype ultrasonic cell separator with improved acoustic design and an integrated cooling circuit, eliminating the problem of local temperature increase caused by the high amplitudes necessary to achieve the separation of animal cells with low acoustic contrast. This setup yields highly reproducible results and is ideal for studying the long-term effects of ultrasonic sound fields and separation efficiency. The novel two-chamber system has the potential for scaleability due to the reduction in thermal and acoustic flow, increased field stability, and separation efficiency. Finally, the effect of power input on separation and cell viability is reported. Such flow-through cell retention systems could be used as systems to retain biomass within the fermentor or as a substitute for centrifugation, with the major advantage of eliminating high-speed rotational motion.

Rapid agglutination testing in an ultrasonic standing wave.

The time taken to perform diagnostic agglutination tests can be significantly reduced by applying an ultrasonic standing wave field to a droplet of reactants held in a capillary tube. Avian erythrocytes, bacteria and latex particles from commercially available test kits were agglutinated in 15 s, 5 min, and 1 min respectively. These times compare favourably with the times of 30 min, 4 h, and 8 min required for agglutination by the methods prescribed for the respective kits. No loss in sensitivity or specificity was observed with the ultrasonic method. A multi-test procedure is also described whereby a series of five droplets loaded in a single capillary can be tested in less than 4 min by drawing the capillary along the axis of the ultrasonic field of a ring transducer.

Motional capacitance of layered piezoelectric thickness-mode resonators.

The Butterworth-Van Dyke equivalent circuit for description of the electrical behavior of piezoelectric bulk resonators is considered. The motional capacitance, C(1), in the circuit characterizes the strength of piezoelectric excitability of a vibration mode. For layered one-dimensional (1-D) structures this parameter can be calculated from the admittance given by the transfer matrix description of H. Nowotny and E. Benes (1987). Introducing the equivalent area of a vibration mode, the calculation is generalized for the three-dimensional (3-D) case of thickness-mode vibration amplitudes varying only slowly in the lateral directions. Detailed formulae are given for the case of singly rotated quartz crystals or ultrasonic transducers with additional layers on one or two sides. Good agreement of the calculated C (1) with experimental data is shown for mass-loaded planoconvex AT-cut quartz crystals.

Laser surgery and immunotherapy in the management of laryngeal papilloma.

Twenty-three cases of laryngeal papilloma have been treated and followed for four years utilizing laser excision and laser excision/immunotherapy. Fourteen cases responded well to laser excision alone, nine did not and immunotherapy was instituted as adjunctive treatment. To date, immunotherapy has contributed little to the relief in this group of refractory cases.