Acoustic levitation as a tool for cell-driven self-organization of human cell spheroids during long-term 3D culture.

Acoustic levitation, which allows contactless manipulation of micro-objects with ultrasounds, is a promising technique for spheroids formation and culture. This acoustofluidic technique favors cell-cell interactions, away from the walls of the chip, which leads to the spontaneous self-organization of cells. Using this approach, we generated spheroids of mesenchymal stromal cells, hepatic and endothelial cells, and showed that long-term culture of cells in acoustic levitation is feasible. We also demonstrated that this self-organization and its dynamics depended weakly on the acoustic parameters but were strongly dependent on the levitated cell type. Moreover, spheroid organization was modified by actin cytoskeleton inhibitors or calcium-mediated interaction inhibitors. Our results confirmed that acoustic levitation is a rising technique for fundamental research and biotechnological industrial application in the rapidly growing field of microphysiological systems. It allowed easily obtaining spheroids of specific and predictable shape and size, which could be cultivated over several days, without requiring hydrogels or extracellular matrix.

Feasibility of an acoustophoresis-based system for a high-throughput cell washing: application to bioproduction.

BACKGROUND AIMS: These last decades have seen the emergence and development of cell-based therapies, notably those based on mesenchymal stromal cells (MSCs). The advancement of these promising treatments requires increasing the throughput of processed cell for industrialization in order to reduce production costs. Among the various bioproduction challenges, downstream processing, including medium exchange, cell washing, cell harvesting and volume reduction, remains a critical step for which improvements are needed. Typically, these processes are performed by centrifugation. However, this approach limits the automation, especially in small batch productions where it is performed manually in open system. METHODS: An acoustophoresis-based system was developed for cell washing. The cells were transferred from one stream to another via the acoustic forces and were collected in a different medium. The optimal flow rates of the different streams were assessed using red blood cells suspended in an albumin solution. Finally, the impact of acoustic washing on adipose tissue-derived MSCs (AD-MSCs) transcriptome was investigated by RNA-sequencing. RESULTS: With a single passage through the acoustic device at input flow rate of 45 mL/h, the albumin removal was up to 90% while recovering 99% of RBCs. To further increase the protein removal, a loop washing in two steps was performed and has allowed an albumin removal ≥99% and a red blood cell/AD-MSCs recovery of 99%. After loop washing of AD-MSCs, only two genes, HES4 and MIR-3648-1, were differently expressed compared with the input. CONCLUSIONS: In this study, we developed a continuous cell-washing system based on acoustophoresis. The process allows a theoretically high cell throughput while inducing little gene expression changes. These results indicate that cell washing based on acoustophoresis is a relevant and promising solution for numerous applications in cell manufacturing.

Controlling the force and the position of acoustic traps with a tunable acoustofluidic chip: Application to spheroid manipulations.

A multi-node acoustofluidic chip working on a broadband spectrum and beyond the resonance is designed for cell manipulations. A simple one-dimensional (1D) multi-layer model is used to describe the stationary standing waves generated inside a cavity. The transmissions and reflections of the acoustic wave through the different layers and interfaces lead to the creation of pressure nodes away from the resonance condition. A transparent cavity and a broadband ultrasonic transducer allow the measurement of the acoustic energy over a wide frequency range using particle image velocimetry measurements and the relation between acoustic energy and the particles velocity. The automation of the setup allows the acquisition over a large spectrum with a high frequency definition. The results show a wide continuous operating range for the acoustofluidic chip, which compares well with the 1D model. The variation of the acoustic radiation force when varying the frequency can be compensated to ensure a constant amplitude for the ARF. This approach is finally applied to mesenchymal stem cell (MCS) spheroids cultured in acoustic levitation. The MSC spheroids can be moved and merged just by varying the acoustic frequency. This approach opens the path to various acoustic manipulations and to complex 3D tissue engineering in acoustic levitation.

Influence of the temperature on the opto-acoustophoretic effect.

Opto-acoustophoretic mobility has been demonstrated recently for fluorescent and colored particles acoustically levitated in a stationary ultrasonic field when illuminated with the appropriate optical wavelength [Dumy, Hoyos, and Aider, J. Acoust. Soc. Am. 146, 4557-4568 (2019); Zhou, Gao, Yang, Li, Shao, Zhang, Li, and Li, Adv. Sci. 5, 1800122 (2018)]. It is a repeatable phenomenon, needing both acoustic trapping and specific optic excitation to occur. However, the physical origin of the phenomenon is still debated. In this study, we provide more insights into the probable origin of this phenomenon by confronting numerical simulations with temperature controlled experiments. The phenomenon properties are well reproduced by our model, relying on a thermofluidic instability, hinting at the potential thermally induced fluid density gradient as a drag source for the observed ejection of particles. Thermostated experiments exhibit a surprising threshold above which the phenomenon is not observed anymore no matter how large the optic or acoustic energies used. This exciting observation differs from the initial interpretation of the phenomenon, altering its potential application without removing its interest because it suggests the possible contactless generation of customized flows by acoustically trapped particles.

Acoustic radiation force and torque on spheroidal particles in an ideal cylindrical chamber.

In this article, the acoustic radiation force and torque exerted on a small spheroidal particle immersed in a nonviscous fluid inside an ideal cylindrical chamber is theoretically investigated. The ideal chamber comprises a hard top and bottom (rigid boundary condition) and a soft or hard lateral wall. By assuming that the particle is much smaller than the acoustic wavelength, analytical expressions of the radiation force and torque caused by an acoustic wave of arbitrary shape are presented. Unlike previous results, these expressions are given relative to a fixed laboratory frame. The model is showcased for analyzing the behavior of an elongated metallic microspheroid (with a 10:1 aspect ratio) in a half-wavelength acoustofluidic chamber with a diameter of a few millimeters. The results show that the radiation torque aligns the microspheroid along the nodal plane, and the radiation force causes a translational motion with a speed of up to one body length per second. Finally, the implications of this study on propelled nanorods by ultrasound are discussed.

Observation of selective optical manipulation of particles in acoustic levitation.

Acoustic Radiation Force is commonly used to create stable large-scale aggregates of particles in levitation (so-called "acoustic levitation") in a micro-cavity. The authors show in the following work that this well-known and well-controlled aggregation process can be reversed without contact or external flow if the aggregated particles are enlightened with the proper optical wavelength. This coupled optics and acoustics effect has been observed with various kinds of particles and different optic wavelengths, showing high reproducibility. The phenomenon is studied using fluorescent micro-metric polystyrene particles without flow, and the effects of acoustic energy and illumination power have been quantitatively assessed. It is then exploited to separate a mix of particles with identical mechanical properties based on their different optic absorption. If the phenomenon is not well understood, some possible mechanisms are proposed and discussed that could be responsible for the rapid ejection of the objects in levitation from the illuminated area. Since it is a tag free phenomenon that does not need high energies to happen and since it works with biological objects such as algae, red blood cells, and bacteria, it may open the way to a broad range of applications.

Turbulent spots in channel flow: an experimental study: large-scale flow, inner structure and low-order model.

We present new experimental results on the development of turbulent spots in channel flow. The internal structure of a turbulent spot is measured, with Time Resolved Stereoscopic Particle Image Velocimetry. We report the observation of travelling-wave-like structures at the trailing edge of the turbulent spot. Special attention is paid to the large-scale flow surrounding the spot. We show that this large-scale flow is an asymmetric quadrupole centred on the spot. We measure the time evolution of the turbulent fluctuations and the mean flow distortions and compare these with the predictions of a nonlinear reduced order model predicting the main features of subcritical transition to turbulence.

Influence of microgravity on spontaneous calcium activity of primary hippocampal neurons grown in microfluidic chips.

The influence of variations of gravity, either hypergravity or microgravity, on the brain of astronauts is a major concern for long journeys in space, to the Moon or to Mars, or simply long-duration missions on the ISS (International Space Station). Monitoring brain activity, before and after ISS missions already demonstrated important and long term effects on the brains of astronauts. In this study, we focus on the influence of gravity variations at the cellular level on primary hippocampal neurons. A dedicated setup has been designed and built to perform live calcium imaging during parabolic flights. During a CNES (Centre National d'Etudes Spatiales) parabolic flight campaign, we were able to observe and monitor the calcium activity of 2D networks of neurons inside microfluidic devices during gravity changes over different parabolas. Our preliminary results clearly indicate a modification of the calcium activity associated to variations of gravity.

Improved functionality of hepatic spheroids cultured in acoustic levitation compared to existing 2D and 3D models.

Hepatic spheroids are of high interest in basic research, drug discovery and cell therapy. Existing methods for spheroid culture present advantages and drawbacks. An alternative technology is explored: the hepatic spheroid formation and culture in an acoustofluidic chip, using HepaRG cell line. Spheroid formation and morphology, cell viability, genetic stability, and hepatic functions are analyzed after 6 days of culture in acoustic levitation. They are compared to 2D culture and non-levitated 3D cultures. Sizes of the 25 spheroids created in a single acoustofluidic microphysiological system are homogeneous. The acoustic parameters in our system do not induce cell mortality nor DNA damage. Spheroids are cohesive and dense. From a functional point of view, hepatic spheroids obtained by acoustic levitation exhibit polarity markers, secrete albumin and express hepatic genes at higher levels compared to 2D and low attachment 3D cultures. In conclusion, this microphysiological system proves not only to be suitable for long-term culture of hepatic spheroids, but also to favor differentiation and functionality within 6 days of culture.

Self-organization and culture of Mesenchymal Stem Cell spheroids in acoustic levitation.

In recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.

Self-sustaining process through streak generation in a flat-plate boundary layer.

The existence of a self-sustaining process between streamwise vortices and streaks has been suggested at moderate Reynolds numbers. Such a mechanism has never been demonstrated experimentally. Using small cylinders as vortex generators to create streamwise counterrotating vortices, we show, through the characterization of the spatial transient growth of the energy of the longitudinal and spanwise velocity perturbations, that such a mechanism exists above a given Reynolds number. From instantaneous particle image velocimetry fields in a horizontal plane, we show that the self-sustaining process can also be associated with the longitudinal destabilization of streamwise streaks.

Transitions in the wake of a flapping foil.

We study experimentally the vortex streets produced by a flapping foil in a hydrodynamic tunnel, using two-dimensional particle image velocimetry. An analysis in terms of a flapping frequency-amplitude phase space allows the identification of (i) the transition from the well-known Bénard-von Kármán (BvK) wake to the reverse BvK vortex street that characterizes propulsive wakes, and (ii) the symmetry breaking of this reverse BvK pattern giving rise to an asymmetric wake. We also show that the transition from a BvK wake to a reverse BvK wake precedes the actual drag-thrust transition and we discuss the significance of the present results in the analysis of flapping systems in nature.

Varying the agglomeration position of particles in a micro-channel using Acoustic Radiation Force beyond the resonance condition.

It is well-known that particles can be focused at mid-height of a micro-channel using Acoustic Radiation Force (ARF) tuned at the resonance frequency (h=λ/2). The resonance condition is a strong limitation to the use of acoustophoresis (particles manipulation using acoustic force) in many applications. In this study we show that it is possible to focus the particles anywhere along the height of a micro-channel just by varying the acoustic frequency, in contradiction with the resonance condition. This result has been thoroughly checked experimentally. The different physical properties as well as wall materials have been changed. The wall materials is finally the only critical parameters. One of the specificity of the micro-channel is the thickness of the carrier and reflector layer. A preliminary analysis of the experimental results suggests that the acoustic focusing beyond the classic resonance condition can be explained in the framework of the multilayered resonator proposed by Hill [1]. Nevertheless, further numerical studies are needed in order to confirm and fully understand how the acoustic pressure node can be moved over the entire height of the micro channel by varying the acoustic frequency. Despite some uncertainties about the origin of the phenomenon, it is robust and can be used for improved acoustic sorting or manipulation of particles or biological cells in confined set-ups.

Experimental scaling law for the subcritical transition to turbulence in plane Poiseuille flow.

We present an experimental study of the transition to turbulence in a plane Poiseuille flow. Using a well-controlled perturbation, we analyze the flow by using extensive particle image velocimetry and flow visualization (using laser-induced fluorescence) measurements, and use the deformation of the mean velocity profile as a criterion to characterize the state of the flow. From a large parametric study, four different states are defined, depending on the values of the Reynolds number and the amplitude of the perturbation. We discuss the role of coherent structures, such as hairpin vortices, in the transition. We find that the minimal amplitude of the perturbation triggering transition scales asymptotically as Re(-1).