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.     

New insights into the mechanisms of ultrasonic emulsification in the oil–water system and the role of gas bubbles

Ultrasonic emulsification (USE) assisted by cavitation is an effective method to produce emulsion droplets. However, the role of gas bubbles in the USE process still remains unclear. Hence, in the present paper, high-speed camera observations of bubble evolution and emulsion droplets formation in oil and water were used to capture in real-time the emulsification process, while experiments with different gas concentrations were carried out to investigate the effect of gas bubbles on droplet size. The results show that at the interface of oil and water, gas bubbles with a radius larger than the resonance radius collapse and sink into the water phase, inducing (oil–water) blended liquid jets across bubbles to generate oil-in-water-in-oil (O/W/O) and water-in-oil (W/O) droplets in the oil phase and oil-in-water (O/W) droplets in the water phase, respectively. Gas bubbles with a radius smaller than the resonance radius at the interface always move towards the oil phase, accompanied with the generation of water droplets in the oil phase. In the oil phase, gas bubbles, which can attract bubbles nearby the interface, migrate to the interface of oil and water due to acoustic streaming, and generate numerous droplets. As for the gas bubbles in the water phase, those can break neighboring droplets into numerous finer ones during bubble oscillation. With the increase in gas content, more bubbles undergo chaotic oscillation, leading to smaller and more stable emulsion droplets, which explains the beneficial role of gas bubbles in USE. Violently oscillating microbubbles are, therefore, found to be the governing cavitation regime for emulsification process. These results provide new insights to the mechanisms of gas bubbles in oil–water emulsions, which may be useful towards the optimization of USE process in industry.     

Ultrasound-assisted solution crystallization of fotagliptin benzoate: Process intensification and crystal product optimization

The ultrasound-assisted crystallization process has promising potentials for improving process efficiency and modifying crystalline product properties. In this work, the crystallization process of fotagliptin benzoate methanol solvate (FBMS) was investigated to improve powder properties and downstream desolvation/drying performance. The direct cooling/antisolvent crystallization process was conducted and then optimized with the assistance of ultrasonic irradiation and seeding strategy. Direct cooling/antisolvent crystallization and seeding crystallization processes resulted in needle-like crystals which are undesirable for downstream processing. In contrast, the ultrasound-assisted crystallization process produced rod-like crystals and reduced the crystal size to facilitate the desolvation of FBMS. The metastable zone width (MSZW), induction time, crystal size, morphology, and process yield were studied comprehensively. The results showed that both the seeding and ultrasound-assisted crystallization process (without seeds) can improve the process yield and the ultrasound could effectively reduce the crystal size, narrow the MSZW, and shorten the induction time. Through comparing the drying dynamics of the FBMS, the small rod-shaped crystals with a mean size of 9.6 μm produced by ultrasonic irradiation can be completely desolvated within 20 h, while the desolvation time of long needle crystals with an average size of about 157 μm obtained by direct cooling/antisolvent crystallization and seeding crystallization processes is more than 80 h. Thus the crystal size and morphology were found to be the key factors affecting the desolvation kinetics and the smaller size produced by using ultrasound can benefit the intensification of the drying process. Overall, the ultrasound-assisted crystallization showed a full improvement including crystal properties and process efficiency during the preparation of fotagliptin benzoate desolvated crystals.     

Ultrasound-assisted production and optimization of mini-emulsions in a microfluidic chip in continuous-flow

The use of ultrasound to generate mini-emulsions (50 nm to 1 μm in diameter) and nanoemulsions (mean droplet diameter < 200 nm) is of great relevance in drug delivery, particle synthesis and cosmetic and food industries. Therefore, it is desirable to develop new strategies to obtain new formulations faster and with less reagent consumption. Here, we present a polydimethylsiloxane (PDMS)-based microfluidic device that generates oil-in-water or water-in-oil mini-emulsions in continuous flow employing ultrasound as the driving force. A Langevin piezoelectric attached to the same glass slide as the microdevice provides enough power to create mini-emulsions in a single cycle and without reagents pre-homogenization. By introducing independently four different fluids into the microfluidic platform, it is possible to gradually modify the composition of oil, water and two different surfactants, to determine the most favorable formulation for minimizing droplet diameter and polydispersity, employing less than 500 µL of reagents. It was found that cavitation bubbles are the most important mechanism underlying emulsions formation in the microchannels and that degassing of the aqueous phase before its introduction to the device can be an important factor for reduction of droplet polydispersity. This idea is demonstrated by synthetizing solid polymeric particles with a narrow size distribution starting from a mini-emulsion produced by the device.     

Complexation of maltodextrin-based inulin and green tea polyphenols via different ultrasonic pretreatment

Ultrasound has been applied in food processing for various purpose, showing potential to advance the physical and chemical modification of natural compounds. In order to explore the effect of ultrasonic pretreatment on the complexation of inulin and tea polyphenols (TPP), different frequencies (25, 40, 80 kHz) and output power (40, 80, 120 W) were carried out. According to the comparison in particle size distribution and phenolic content of different inulin-TPP complexes, it was indicated that high-intensity ultrasonic (HIU) treatment (25 kHz, 40 W, 10 min) could accelerate the interaction of polysaccharides and polyphenols. Moreover, a series of spectral analysis including UV–Vis, FT-IR and NMR jointly evidenced the formation of hydrogen bond between saccharides and phenols. However, the primary structure of inulin and the polysaccharide skeleton were not altered by the combination. Referring to field emission scanning electron microscopy (FESEM), the morphology of ultrasound treated-complex presented a slight agglomeration in the form of bent sheets, compared to non-treated sample. The inulin-TPP complex also revealed better stability based on thermogravimetric analysis (TGA). Thus, it can be speculated from the identifications that proper ultrasonic treatment is promising to promote the complexation of some food components during processing.     

Ultrasound-assisted soil washing processes for the remediation of heavy metals contaminated soils: The mechanism of the ultrasonic desorption

Ultrasound-assisted soil washing processes were investigated for the removal of heavy metals (Cu, Pb, and Zn) in real contaminated soils using HCl and EDTA. The ultrasound-assisted soil washing (US/Mixing) process was compared with the conventional soil washing (Mixing) process based on the mechanical mixing. High removal efficiency (44.8% for HCl and 43.2% for EDTA) for the metals was obtained for the most extreme conditions (HCl 1.0 M or EDTA 0.1 M and L:S = 10:1) in the Mixing process. With the aide of ultrasound, higher removal efficiency (57.9% for HCl and 50.0% for EDTA) was obtained in the same extreme conditions and similar or higher removal efficiency (e.g., 54.7% for HCl 0.5 M and L:S = 10:1 and 50.5% for EDTA 0.05 M and L:S = 5:1) was achieved even in less extreme conditions (lower HCl or EDTA concentration and L:S ratio). Therefore, it was revealed that the US/Mixing was advantageous over the conventional Mixing processes in terms of metal removal efficiency, consumption of chemicals, amount of generated washing leachate, and volume/size of washing reactor. In addition, the heavy metals removal was enhanced for the smaller soil particles in the US/Mixing process. It was due to more violent movement of smaller particles in slurry phase and more violent sonophysical effects. In order to understand the mechanism of ultrasonic desorption, the desorption test was conducted using the paint-coated beads with three sizes (1, 2, and 4 mm) for the free and attached conditions. It was found that no significant desorption/removal of paint from the beads was observed without the movement of beads in the water including floatation, collision, and scrubbing. Thus, it was suggested that the simultaneous application of the ultrasound and mechanical mixing could enhance the physical movement of the particles significantly and the very high removal/desorption could be attained.     

Analysis of ultrasound-assisted convective heating/cooling process: Development and application of a Nusselt equation

In this study, the convective heating/cooling process assisted by US irradiation is analyzed with the aims of developing a new convective heat transfer correlation. Heat transfer experiments were conducted with different copper machined geometries (cube, sphere and cylinder), fluid velocities (0.93–5.00 × 10⁻³ m/s), temperatures (5–60 °C), and US intensities (0–6913 W/m²) using water as heat transfer fluid. The Nusselt (Nu) equation was obtained by assuming an apparent Nu number in the US-assisted process, expressed as the sum of contributions of the forced convection and cavitation-acoustic streaming effects. The Nu equation was validated with two sets of experiments conducted with a mixture of ethylene glycol and water (1:1 V/V) or a CaCl₂ aqueous solution (30 g/L) as immersion media, achieving a satisfactory reproduction of experimental data, with mean relative deviations of 17.6 and 17.8%, respectively. In addition, a conduction model with source term and the proposed correlation were applied to the analysis of US-accelerated heating kinetics of dry-cured ham reported in literature. Results demonstrated that US improves heating of ham slices because of the increased heat transfer coefficients and the direct absorption of US power by the foodstuff.     

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.     

Effect of ultrasound on the preparation of soy protein isolate-maltodextrin embedded hemp seed oil microcapsules and the establishment of oxidation kinetics models

In this study, microcapsules were prepared by spray drying and embedding hemp seed oil (HSO) with soy protein isolate (SPI) and maltodextrin (MD) as wall materials. The effect of ultrasonic power on the microstructure and characteristics of the composite emulsion and microcapsules was studied. Studies have shown that ultrasonic power has a significant impact on the stability of composite emulsions. The particle size of the composite emulsion after 450 W ultrasonic treatment was significantly lower than the particle size of the emulsion without the ultrasonic treatment. Through fluorescence microscopy observation, HSO was found to be successfully embedded in the wall materials to form an oil/water (O/W) composite emulsion. The spray-dried microcapsules showed a smooth spherical structure through scanning electron microscopy (SEM), and the particle size was 10.7 μm at 450 W. Fourier transform infrared (FTIR) spectroscopy analysis found that ultrasonic treatment would increase the degree of covalent bonding of the SPI-MD complex to a certain extent, thereby improving the stability and embedding effect of the microcapsules. Finally, oxidation kinetics models of HSO and HSO microcapsules were constructed and verified. The zero-order model of HSO microcapsules was found to have a higher degree of fit; after verification, the model can better reflect the quality changes of HSO microcapsules during storage.