Suspension culture in a T-flask with acoustic flow induced by ultrasonic irradiation

Suspension culture is an essential large-scale cell culture technique for biopharmaceutical development and regenerative medicine. To transition from monolayer culture on the culture surface of a flask to suspension culture in a bioreactor, a pre-specified cell number must first be reached. During this period of preparation for suspension culture, static suspension culture in a flask is generally performed because the medium volume is not large enough to use a paddle to circulate the medium. However, drawbacks to this static method include cell sedimentation, leading to high cell density near the bottom and resulting in oxygen and nutrient deficiencies. Here, we propose a suspension culture method with acoustic streaming induced by ultrasonic waves in a T-flask to create a more homogeneous distribution of oxygen, nutrients, and waste products during the preparation period preceding large-scale suspension culture in a bioreactor. To demonstrate the performance of the ultrasonic method, Chinese hamster ovary cells were cultured for 72 h. Results showed that, on average, the cell proliferation was improved by 40% compared with the static method. Thus, the culture time required to achieve a 1000-fold increase could be reduced by 32 h (a 14% reduction) compared with the static method. Furthermore, the ultrasonic irradiation did not compromise the metabolic activity of the cells cultured using the ultrasonic method. These results demonstrate the effectiveness of the ultrasonic method for accelerating the transition to large-scale suspension culture.     

Ultrasonic cavitation at liquid/solid interface in a thin Ga–In liquid layer with free surface

Cavitation in thin layer of liquid metal has potential applications in chemical reaction, soldering, extraction, and therapeutic equipment. In this work, the cavitation characteristics and acoustic pressure of a thin liquid Ga–In alloy were studied by high speed photography, numerical simulation, and bubble dynamics calculation. A self-made ultrasonic system with a TC4 sonotrode, was operated at a frequency of 20 kHz and a max output power of 1000 W during the cavitation recording experiment. The pressure field characteristic inside the thin liquid layer and its influence on the intensity, types, dimensions, and life cycles of cavitation bubbles and on the cavitation evolution process against experimental parameters were systematically studied. The results showed that acoustic pressure inside the thin liquid layer presented alternating positive and negative characteristics within 1 acoustic period (T). Cavitation bubbles nucleated and grew during the negative-pressure stage and shrank and collapsed during the positive-pressure stage. A high bubble growth speed of 16.8 m/s was obtained and evidenced by bubble dynamics calculation. The maximum absolute pressure was obtained at the bottom of the thin liquid layer and resulted in the strongest cavitation. Cavitation was divided into violent and weak stages. The violent cavitation stage lasted several hundreds of acoustic periods and had higher bubble intensity than the weak cavitation stage. Cavitation cloud preferentially appeared during the violent cavitation stage and had a life of several acoustic periods. Tiny cavitation bubbles with life cycles shorter than 1 T dominated the cavitation field. High cavitation intensities were observed at high ultrasonication power and when Q235B alloy was used because such conditions lead to high amplitudes on the substrate and further high acoustic pressure inside the liquid.     

High-speed imaging of ultrasonic emulsification using a water-gallium system

Aiming at elucidating ultrasonic emulsification mechanisms, the interaction between a single or multiple acoustic cavitation bubbles and gallium droplet interface was investigated using an high-speed imaging technique. To our best knowledge, the moment of emulsification and formation of fine droplets during ultrasound irradiation were observed for the first time. It was found that the detachment of fine gallium droplets occurs from the water-gallium interface during collapse of big cavitation bubbles. The results suggest that the maximum size of cavitation bubble before collapsing is of prime importance for emulsification phenomena. Previous numerical simulation revealed that the collapse of big cavitation bubble is followed by generation of high-velocity liquid jet directed toward the water-gallium interface. Such a jet is assumed to be the prime cause of liquid emulsification. The distance between cavitation bubbles and water-gallium interface was found to slightly affect the emulsification onset. The droplet fragmentation conditions are also discussed in terms of the balance between (1) interfacial and kinetic energies and (2) dynamic and Laplace pressure during droplet formation.     

Acoustic resonance and atomization for gas-liquid systems in microreactors

It is shown that a liquid slug in gas–liquid segmented flow in microchannels can act as an acoustic resonator to disperse large amounts of small liquid droplets, commonly referred to as atomization, into the gas phase. We investigate the principles of acoustic resonance within a liquid slug through experimental analysis and numerical simulation. A mechanism of atomization in the confined channels and a hypothesis based on high-speed image analysis that links acoustic resonance within a liquid slug with the observed atomization is proposed. The observed phenomenon provides a novel source of confined micro sprays and could be an avenue, amongst others, to overcome mass transfer limitations for gas–liquid processes in flow.     

Agglomeration of particles by a converging ultrasound field and their quantitative assessments

The acoustic radiation force resulting from acoustic waves have been extensively studied for the contact-free generation of organized patterning arrays. The precise arrangement of microscopic objects clustered at the pressure nodes is critical to the development of functional structures and patterned surfaces. However, the size of the clusters is restricted by the saturation limit of the acoustic nodes. Here, we present a bulk acoustic wave (BAW) platform, which employs a two-dimensional acoustic wave to propel particles of various sizes. Experimentally, when particles are large, significant acoustic energy is scattered and partly absorbed by the matched layers in front of the sensors. The acoustic radiation force from a convergent acoustic pressure field agglomerates the large polystyrene (PS) particles towards the central region instead of the pressure nodes. The parametric analysis has been performed to assess the transition in the particles from clustering at the organized nodal arrays to agglomerating in the central region, which is a function of particle size, particle concentration, and load voltage. Statistically, the particles can agglomerate with a cluster ratio greater than 70%, and this ratio can be improved by increasing the load power/voltage supplied to the transducers. With its ability to perform biocompatible, label-free, and contact-free self-assembly, this concept offers a new possibility in the fabrication of colloidal layers, the recreation of tissue microstructure, the development of organoid spheroid cultures, the migration of microorganisms, and the assembly of bioprinting materials.     

Control of the dynamics of a boiling vapour bubble using pressure-modulated high intensity focused ultrasound without the shock scattering effect: A first proof-of-concept study

Boiling histotripsy is a promising High-Intensity Focused Ultrasound (HIFU) technique that can be used to induce mechanical tissue fractionation at the HIFU focus via cavitation. Two different types of cavitation produced during boiling histotripsy exposure can contribute towards mechanical tissue destruction: (1) a boiling vapour bubble at the HIFU focus and (2) cavitation clouds in between the boiling bubble and the HIFU source. Control of the extent and degree of mechanical damage produced by boiling histotripsy is necessary when treating a solid tumour adjacent to normal tissue or major blood vessels. This is, however, difficult to achieve with boiling histotripsy due to the stochastic formation of the shock scattering-induced inertial cavitation clouds. In the present study, a new histotripsy method termed pressure-modulated shockwave histotripsy is proposed as an alternative to or in addition to boiling histotripsy without inducing the shock scattering effect. The proposed concept is (a) to generate a boiling vapour bubble via localised shockwave heating and (b) subsequently control its extent and lifetime through manipulating peak pressure magnitudes and a HIFU pulse length. To demonstrate the feasibility of the proposed method, bubble dynamics induced at the HIFU focus in an optically transparent liver tissue phantom were investigated using a high speed camera and a passive cavitation detection systems under a single 10, 50 or 100 ms-long 2, 3.5 or 5 MHz pressure-modulated HIFU pulse with varying peak positive and negative pressure amplitudes from 5 to 89 MPa and −3.7 to −14.6 MPa at the focus. Furthermore, a numerical simulation of 2D nonlinear wave propagation with the presence of a boiling bubble at the focus of a HIFU field was conducted by numerically solving the generalised Westervelt equation. The high speed camera experimental results showed that, with the proposed pressure-modulated shockwave histotripsy, boiling bubbles generated by shockwave heating merged together, forming a larger bubble (of the order of a few hundred micron) at the HIFU focus. This coalesced boiling bubble then persisted and maintained within the HIFU focal zone until the end of the exposure (10, 50, or 100 ms). Furthermore, and most importantly, no violent cavitation clouds which typically appear in boiling histotripsy occurred during the proposed histotripsy excitation (i.e. no shock scattering effect). This was likely because that the peak negative pressure magnitude of the backscattered acoustic field by the boiling bubble was below the cavitation cloud intrinsic threshold. The size of the coalesced boiling bubble gradually increased with the peak pressure magnitudes. In addition, with the proposed method, an oval shaped lesion with a length of 0.6 mm and a width of 0.1 mm appeared at the HIFU focus in the tissue phantom, whereas a larger lesion in the form of a tadpole (length: 2.7 mm, width: 0.3 mm) was produced by boiling histotripsy. Taken together, these results suggest that the proposed pressure-modulated shockwave histotripsy could potentially be used to induce a more spatially localised tissue destruction with a desired degree of mechanical damage through controlling the size and lifetime of a boiling bubble without the shock scattering effect.     

Effects of gas sparging and mechanical mixing on sonochemical oxidation activity

The effects of air sparging (0–16 L min⁻¹) and mechanical mixing (0–400 rpm) on enhancing the sonochemical degradation of rhodamine B (RhB) was investigated using a 28 kHz sonoreactor. The degradation of RhB followed pseudo first-order kinetics, where sparging or mixing induced a large sonochemical enhancement. The kinetic constant varied in three stages (gradually increased → increased exponentially → decreased slightly) as the rate of sparging or mixing increased, where the stages were similar for both processes. The highest sonochemical activity was obtained with sparging at 8 L min⁻¹ or mixing at 200 rpm, where the standing wave field was significantly deformed by sparging and mixing, respectively. The cavitational oxidation activity was concentrated at the bottom of the sonicator when higher sparging or mixing rates were employed. Therefore, the large enhancement in the sonochemical oxidation was attributed mainly to the direct disturbance of the ultrasound transmission and the resulting change in the cavitation-active zone in this study. The effect of the position of air sparging and mixing was investigated. The indirect inhibition of the ultrasound transmission resulted in less enhancement of the sonochemical activity. Moreover, the effect of various sparging gases including air, N₂, O₂, Ar, CO₂, and an Ar/O₂ (8:2) mixture was compared, where all gases except CO₂ induced an enhancement in the sonochemical activity, irrespective of the concentration of dissolved oxygen. The highest activity was obtained with the Ar/O₂ (8:2) mixture. Therefore, it was revealed that the sonochemical oxidation activity could be further enhanced by applying gas sparging using the optimal gas.     

Particle separation in microfluidics using different modal ultrasonic standing waves

Microfluidic technology has great advantages in the precise manipulation of micro and nano particles, and the separation of micro and nano particles based on ultrasonic standing waves has attracted much attention for its high efficiency and simplicity of structure. This paper proposes a device that uses three modes of ultrasonic standing waves to continuously separate particles with positive acoustic contrast factor in microfluidics. Three modes of acoustic standing waves are used simultaneously in different parts of the microchannel. According to the different acoustic radiation force received by the particles, the particles are finally separated to the pressure node lines on both sides and the center of the microchannel. In this separation method, initial hydrodynamic focusing and satisfying various equilibrium constraints during the separation process are the key. Through numerical simulation, the resonance frequency of the interdigital transducer, the distribution of sound pressure in the liquid, and the relationship between the interdigital electrode voltage and the output sound pressure are obtained. Finally, the entire separation process in the microchannel was simulated, and the separation of the two particles was successfully achieved. This work has laid a certain theoretical foundation for the rapid diagnosis of diseases in practical applications.     

Setup of a dipole trap for all-optical trapping

Micromotion induced by the radio-frequency field contributes greatly to the systematic frequency shifts of optical frequency standards. Although different strategies for mitigating this effect have been proposed, trapping ions optically has the potential to provide a generic solution to the elimination of micromotion. This could be achieved by trapping a single ion in the dipole trap composed of a highpower laser field. Here, we present the setup of the dipole trap composed of a 532 nm laser at a power of 10 W aiming to optically trap a single ~(40)Ca~+ and we observe an AC-Stark shift of the fluorescence spectrum line of ～22 MHz caused by the 532 nm dipole beam. The beam waist of the dipole laser is several microns, which would provide a dipole potential strong enough for all-optical trapping of a single ~(40)Ca~+ ion.     

Geometrical structure and nonlinear response variations of metal (M = Ni2+, Pd2+, Pt2+) octaphyrin complex derivatives: A DFT study

Nonlinear optical response of designed organometallic complexes of Ni²⁺, Pd²⁺, and Pt²⁺ metal ions with octaphyrin (OP) as ligand were explored by using DFT at CAM-B3LYP/6–311G++(d, p)/LANL2DZ/DEF2SV level of theory. The geometries of these organometallic complexes were studied in terms of effect on molecular framework by metal ion and substituent groups. The optimized geometry of free ligand displays that one of the four pyrrole rings orients out of plane to reduce the steric hindrance. The effect of the substituents on the geometry was found more prominent in the Ni²⁺-OP complexes. The calculations reveal enhancement in the values of dipole moment and hyperpolarizability on introducing electron withdrawing and electron donating groups in ligand framework with maximum enhancement in case of Pt²⁺-OP derivatives. In this study no regular trend was observed for the HOMO and LUMO energies with the second-order hyperpolarizability of M²⁺-OP complexes. However, we have observed that the excited-state properties calculated by using TD-DFT correlate well with the second-order hyperpolarizability values and the dependence was rationalized in terms of two-level model. Thus, from overall calculations we have observed that the designed organometallic complexes display higher polarizability and hyperpolarizability values and can be effective candidates for nonlinear response.