Acoustic streaming induced by MHz-frequency ultrasound extends the volume limit of cell suspension culture.

Large-scale cell suspension culture technology opens up opportunities for numerous medical and bioengineering applications. For these purposes, scale-up of the culture system is paramount. For initial small-scale culture, a simple static suspension culture (SSC) is generally employed. However, cell sedimentation due to the lack of agitation limits the culture volume feasible for SSC. Thus, when scaling up, cell suspensions must be manually transferred from the culture flask to another vessel suitable for agitation, which increases the risk of contamination and human error. Ideally, the number of culture transfer steps should be kept to a minimum. The present study describes the fabrication of an ultrasonic suspension culture system that stirs cell suspensions with the use of acoustic streaming generated by ultrasound irradiation at a MHz frequency. This system was applied to 100-mL suspension cultures of Chinese hamster ovary cells-a volume ten-fold larger than that generally used. The cell proliferation rate in this system was 1.88/day when applying an input voltage of 40 V to the ultrasonic transducer, while that of the SSC was 1.14/day. Hence, the proposed method can extend the volume limit of static cell suspension cultures, thereby reducing the number of cell culture transfer steps.

The fast dynamics of cavitation bubbles within water confined in elastic solids.

Many applications such as ultrasonic cleaning or sonochemistry use the ability of bubbles to oscillate and drive liquid flow. But bubbles have also received attention in porous media, where drying may cause cavitation, a phenomenon occurring in plant tissues. Here we explore the dynamics of cavitation bubbles when the liquid is fully entrapped in an elastic solid, using light scattering, laser strobe photography and high speed camera recordings. Our experiments show unexpectedly fast bubble oscillations in volume. They depend on the confinement size and elasticity, which we explain with a simple model where liquid compressibility is a key parameter. We also observe rich non-spherical dynamics, with ejection away from the walls and bubble fragmentation, which reveal extreme fluid motion at short timescales.

Homogeneous nucleation in water in microfluidic channels.

It has been an experimental challenge to test the rupture of liquids with homogeneous nucleation of vapor bubbles. Many prior studies suffered from the ubiquitous presence of impurities in liquids or at container surfaces that spontaneously nucleate and grow under tension. Here, we propose a microfluidic approach to eliminate such impurities and obtain homogeneous bubble nucleation. We stretch the liquid dynamically via the interaction between a laser-induced shock and an air-liquid interface in a microchannel. Reproducible observations of the nucleation of vapor bubbles are obtained, supporting our claim of homogeneous nucleation. From comparisons of the distribution of vapor cavities with Euler flow simulations, the nucleation threshold for water at room temperature is predicted to be -60 MPa.

Elaborate color patterns of individual chicken feathers may be formed by the agouti signaling protein.

Hair and feather pigmentation is mainly determined by the distribution of two kinds of melanin, eumelanin and pheomelanin, which produce brown to black and yellow to red colorations, respectively. The agouti signaling protein (ASIP) acts as an antagonist or an inverse agonist of the melanocortin 1 receptor (MC1R), a G protein-coupled receptor for α-melanocyte-stimulating hormone (α-MSH). This antagonism of the MC1R by ASIP on melanocytes initiates a switch of melanin synthesis from eumelanogenesis to pheomelanogenesis in mammals. In the present study, we isolated multiple ASIP mRNA variants generated by alternative splicing and promoters in chicken feather follicles. The mRNA variants showed a discrete tissue distribution. However, mRNAs were expressed predominantly in the feather pulp of follicles. Paralleling mRNA distribution, ASIP immunoreactivity was observed in feather pulp. Interestingly, ASIP was stained with pheomelanin but not eumelanin in pulp areas that face developing barbs. We suggest that the elaborate color pattern of individual feathers is formed in part by the antagonistic action of ASIP that is produced by multiple mRNA variants in chicken feather follicles.

Quantitative Analysis of Acoustic Pressure for Sonophoresis and Its Effect on Transdermal Penetration.

Ultrasound facilitates the penetration of macromolecular compounds through the skin and offers a promising non-invasive technique for transdermal delivery. However, technical difficulties in quantifying ultrasound-related parameters have restricted further analysis of the sonophoresis mechanism. In this study, we devise a bolt-clamped Langevin transducer-based sonophoresis device that enables us to measure with a thin lead zirconate titanate (PZT) sensor. One-dimensional acoustic theory accounting for wave interaction at the skin interface indicates that the acoustic pressure and cavitation onset on the skin during sonophoresis are sensitive to the subcutaneous support, meaning that there is a strong need to perform the pressure measurement in an experimental environment replacing the human body. From a series of the experiments with our new device, the transdermal penetration of polystyrene, silica and gold nanoparticles is found to depend on the size and material of the particles, as well as the hardness of the subcutaneous support material. We speculate from the acoustic pressure measurement that the particles' penetration results from the mechanical action of cavitation.

Chemically controlled megasonic cleaning of patterned structures using solutions with dissolved gas and surfactant.

Acoustic cavitation is used for megasonic cleaning in the semiconductor industry, especially of wafers with fragile pattern structures. Control of transient cavitation is necessary to achieve high particle removal efficiency (PRE) and low pattern damage (PD). In this study, the cleaning performance of solutions with different concentrations of dissolved gas (H2) and anionic surfactant (sodium dodecyl sulfate, SDS) in DIW (DI water) on silicon (Si) wafers was evaluated in terms of PRE and PD. When only DIW was used, PRE was low and PD was high. An increase in dissolved H2 gas concentration in DIW increased PRE; however, PD also increased accordingly. Thus, we investigated the megasonic cleaning performance of DIW and H2-DIW solutions with various concentrations of the anionic surfactant, SDS. At 20 ppm SDS in DIW, PRE reached a maximum value and then decreased with increasing concentration of SDS. PRE decreased slightly with increasing concentrations of SDS surfactant when dissolved in H2-DIW. Furthermore, PD decreased significantly with increasing concentrations of SDS surfactant in both DIW and H2-DIW cases. A high-speed camera setup was introduced to analyze bubble dynamics under a 0.96 MHz ultrasonic field. Coalescence, agglomeration, and the population of multi-bubbles affected the PRE and PD of silicon wafers differently in the presence of SDS surfactant. We developed a hypothesis to explain the change in bubble characteristics under different chemical environmental conditions.

Improvement of acoustic theory of ultrasonic waves in dilute bubbly liquids.

The theory of the acoustics of dilute bubbly liquids is reviewed, and the dispersion relation is modified by including the effect of liquid compressibility on the natural frequency of the bubbles. The modified theory is shown to more accurately predict the trend in measured attenuation of ultrasonic waves. The model limitations associated with such high-frequency waves are discussed.

Low-intensity ultrasound induced cavitation and streaming in oxygen-supersaturated water: Role of cavitation bubbles as physical cleaning agents.

A number of acoustic and fluid-dynamic phenomena appear in ultrasonic cleaning baths and contribute to physical cleaning of immersed surfaces. Propagation and repeated reflection of ultrasound within cleaning baths build standing-wave-like acoustic fields; when an ultrasound intensity gradient appears in the acoustic fields, it can in principle induce steady streaming flow. When the ultrasound intensity is sufficiently large, cavitation occurs and oscillating cavitation bubbles are either trapped in the acoustic fields or advected in the flow. These phenomena are believed to produce mechanical action to remove contaminant particles attached at material surfaces. Recent studies suggest that the mechanical action of cavitation bubbles is the dominant factor of particle removal in ultrasonic cleaning, but the bubble collapse resulting from high-intensity ultrasound may be violent enough to give rise to surface erosion. In this paper, we aim to carefully examine the role of cavitation bubbles from ultrasonic cleaning tests with varying dissolved gas concentration in water. In our cleaning tests using 28-kHz ultrasound, oxygen-supersaturated water is produced by oxygen-microbubble aeration and used as a cleaning solution, and glass slides spin-coated with silica particles of micron/submicron sizes are used to define cleaning efficiency. High-speed camera recordings and Particle Image Velocimetry analysis with a pressure oscillation amplitude of 1.4 atm at the pressure antinode show that the population of cavitation bubbles increases and streaming flow inside the bath is promoted, as the dissolved oxygen supersaturation increases. The particle removal is found to be achieved mainly by the action of cavitation bubbles, but there exists optimal gas supersaturation to maximize the removal efficiency. Our finding suggests that low-intensity ultrasound irradiation under the optimal gas supersaturation in cleaning solutions allows for having mild bubble dynamics without violent collapse and thus cleaning surfaces without cavitation erosion. Finally, observations of individual bubble dynamics and the resulting particle removal are reported to further support the role of cavitation bubbles as cleaning agents.

Growth control of leaf lettuce with exposure to underwater ultrasound and dissolved oxygen supersaturation.

The growth rate of vegetables in plant factories can be regulated by environmental factors including light, temperature, and chemicals, which might give rise to mutation in leaf health. Here, we aim to devise a new way that allows for controlling the growth rate of plants in hydroponics as well as maintaining the product quality; we apply underwater ultrasound and dissolved oxygen supersaturation as external stimuli to plants. As an example, we examine the growth of leaf lettuce in hydroponics with exposure to 28-kHz ultrasound and dissolved oxygen supersaturation up to 36 mg/L at 20 °C. Our results show that exposure to the ultrasound of peak-to-peak pressure at 20 kPa or larger works as the growth inhibitor of the leaves and the roots, while the oxygen supersaturation as the growth promoter, without any degradation of chlorophyll in the leaves. This suggests that these external stimuli can be used in the growth control system of plant factories.

Experiment and modeling of translational dynamics of an oscillating bubble cluster in a stationary sound field.

Translational motion of an oscillating bubble cluster under sound irradiation is studied experimentally and is modeled in the framework of the classical approach of Bjerknes. An experimental technique is proposed to observe bubble cluster formation and its translational dynamics interacting with wall boundaries due to the secondary Bjerknes force. The translational motion observed in the experiment is modeled by extending the classical theory of Bjerknes on a single bubble; a bubble cluster is treated as a single bubble. The extended Bjerknes theory is shown to allow us to predict the overall trajectory of the cluster translating toward a wall of finite acoustic impedance by tuning acoustic energy loss at the wall. The drag force turns out to be unimportant for the translation of a millimeter-sized cluster that we observed.

Statistical equilibrium of bubble oscillations in dilute bubbly flows.

The problem of predicting the moments of the distribution of bubble radius in bubbly flows is considered. The particular case where bubble oscillations occur due to a rapid (impulsive or step change) change in pressure is analyzed, and it is mathematically shown that in this case, inviscid bubble oscillations reach a stationary statistical equilibrium, whereby phase cancellations among bubbles with different sizes lead to time-invariant values of the statistics. It is also shown that at statistical equilibrium, moments of the bubble radius may be computed using the period-averaged bubble radius in place of the instantaneous one. For sufficiently broad distributions of bubble equilibrium (or initial) radius, it is demonstrated that bubble statistics reach equilibrium on a time scale that is fast compared to physical damping of bubble oscillations due to viscosity, heat transfer, and liquid compressibility. The period-averaged bubble radius may then be used to predict the slow changes in the moments caused by the damping. A benefit is that period averaging gives a much smoother integrand, and accurate statistics can be obtained by tracking as few as five bubbles from the broad distribution. The period-averaged formula may therefore prove useful in reducing computational effort in models of dilute bubbly flow wherein bubbles are forced by shock waves or other rapid pressure changes, for which, at present, the strong effects caused by a distribution in bubble size can only be accurately predicted by tracking thousands of bubbles. Some challenges associated with extending the results to more general (nonimpulsive) forcing and strong two-way coupled bubbly flows are briefly discussed.