Acoustic stimulation of cellular objects

Abstract

The invention relates to a technique for stimulating cellular objects (11A, 11B), for example objects in acoustic levitation or supported by a hydrogel matrix, by means of amplitude and / or frequency modulation of an acoustic wave.

Description

DESCRIPTION Title: Acoustic stimulation of cellular objects Technical field

[0001] The present invention relates to a technique for stimulating micrometric cells or particles, also called "objects", or aggregates of such objects, for example for stimulating biological cells with a view to reconstructing or modeling living tissues.

[0002] The invention is of particular interest, in no way limiting, in the sectors of cell therapy, pharmacological modeling, agri-food, for example for the cultivation of meat, microalgae or plants, or even aerospace, in particular for cell culture in microgravity conditions. State of the art

[0003] In the context of research on the reconstruction and modeling of organs-on-a-chip and organoids, a growing number of experimental approaches aim to enable the structuring of cellular assemblies.

[0004] The most widely used techniques for this purpose include the manipulation of cells within microfluidic devices and the formation of tissues by additive manufacturing.

[0005] Another known technique, described in the following document, consists of structuring cell sheets by acoustic levitation in hydrogels: Bouyer et al. A bio-Acoustic Levitational (BAL) Assembly Method for Engineering of Multilayered, 3D Brain-Like Constructs, Using Human Embryonic Stem Cell Derived Neuro-Progenitors, Adv. Mater. 2016, 28, 161- 167. This technique allows assembling cells in a hydrogel in the form of layers or sheets with the aim of establishing connections between cells of different layers. However, this technique does not allow the development of such connections to be controlled satisfactorily. Statement of the invention

[0006] The present invention aims to overcome all or part of the drawbacks of the techniques known in the prior art by providing a method of acoustic stimulation of one or more objects received in a fluid or semi-solid medium within a cavity, comprising: - a step of generating a standing acoustic wave so as to form, in the medium, one or more pressure nodes and / or antinodes at respective axial positions called reference positions, in order to produce an acoustic radiation force which is exerted on the objects, - from a so-called reference configuration in which the objects are positioned on one or more of said pressure nodes and / or anti-nodes, a step of modifying, over time, one or more parameters of the acoustic wave in order to modify the acoustic radiation force and / or to move one or more of said pressure nodes and anti-nodes in an axial direction relative to their reference position.

[0007] The invention thus makes it possible to control over time the mechanical deformation of objects which can typically form tissues manufactured or reconstituted within a three-dimensional culture system and to improve the modeling and / or reproduction of physiological and / or pathological deformations of such tissues.

[0008] The majority of tissues in a living organism are in fact continuously subjected to mechanical deformations and / or pressure variations caused by bodily movements, pulsatile peristaltic activities (e.g. lungs, heart) or contractile activities (e.g. intestines), or even movements of liquids (e.g. pulse waves, lymphatic blood flows) causing a pulsatile deformation of veins and arteries with repercussions on all irrigated tissues and organs.

[0009] The invention makes it possible in particular to implement mechanical deformations and / or controlled pressure variations in a cell culture system so as to mimic cellular responses and accelerate the differentiation of the cells subjected to it, under conditions similar to those of a living organism.

[0010] The method of the invention can be implemented to stimulate objects that are not biological cells, for example droplets of a substance such as silicone oil, or a biomaterial such as a hydrogel.

[0011] The parameters of the acoustic wave are preferably chosen from a list including an amplitude and a frequency.

[0012] According to one embodiment of the method, the step of modifying one or more of said parameters of the acoustic wave comprises a modification of the amplitude of the acoustic wave at fixed frequency over time.

[0013] According to another embodiment of the method, the step of modifying one or more of said parameters of the acoustic wave comprises a modification of the frequency of the acoustic wave with fixed amplitude over time.

[0014] Of course, these different modes can be implemented successively and do not exclude other modes of implementation, for example a mode in which the step of modifying one or more of said parameters of the acoustic wave comprises a simultaneous modification of the frequency and the amplitude of the acoustic wave.

[0015] The step of modifying one or more of said parameters of the acoustic wave may comprise a periodic modification of one or more of said parameters.

[0016] The step of modifying one or more of said parameters of the acoustic wave may in particular comprise a modification of one or more of said parameters at a modulation frequency which is relatively low compared to a relatively high frequency of the acoustic wave.

[0017] For information purposes, the frequency of the acoustic wave may be greater than 0.5 MHz and said modulation frequency may be less than 100 kHz, the modulation frequency being able for example to be of the order of a few Hz, or of the order of a few tens of Hz, or even of the order of a few kHz.

[0018] The objects may be maintained in the cavity, in particular in the reference configuration before implementing the step of modifying one or more of said parameters of the acoustic wave and / or in different configurations during this step, in several ways including those described below.

[0019] According to a first variant, the objects are held in the cavity under the action of the acoustic radiation force.

[0020] In the context of this first variant, the objects can in particular be kept in acoustic levitation in the medium, the medium being able to be liquid for this purpose, for example aqueous, at least in the reference configuration.

[0021] According to a second variant, the objects are held in the cavity by being supported by the medium, the medium being able for example to be semi-solid (or semi-liquid) at least in the reference configuration.

[0022] According to a third variant, the objects are held in the cavity by being supported by, and / or in contact with, one or more contact structures which extend into the cavity.

[0023] Without limitation, each of these contact structures can be chosen from a list including a wall, a pillar and an electrode.

[0024] Such contact structures may form one or more surfaces extending along the axial direction and / or one or more surfaces extending along a transverse direction.

[0025] The method can be implemented so that, in an initial configuration, the objects are suspended in the medium which is fluid in this initial configuration.

[0026] The step of generating the acoustic wave can be carried out in such a way as to move the objects under the action of the acoustic radiation force to place them in said reference configuration, for example from the aforementioned initial configuration or another configuration.

[0027] In one embodiment, the objects include objects having positive acoustic contrast relative to the medium and / or objects having negative acoustic contrast relative to the medium.

[0028] In a manner known per se, an acoustic radiation force, in particular an axial component of this force, which is exerted on objects having a positive acoustic contrast makes it possible to move these objects towards pressure nodes formed by the acoustic wave, i.e. places where the pressure of the medium is zero. An acoustic radiation force, in particular an axial component of this force, which is exerted on objects having a negative acoustic contrast makes it possible to move these objects towards pressure anti-nodes formed by the acoustic wave, i.e. places where this pressure is maximum.

[0029] The method may include a step of incubating the objects.

[0030] The incubation step can be carried out for the purpose of differentiation and / or self-organization and / or maturation of aggregates of cellular objects.

[0031] Such an incubation step can be carried out while the objects are subjected to the acoustic radiation stress.

[0032] Such an incubation step can be carried out before and / or during implementation of the step of modifying one or more of said parameters of the acoustic wave.

[0033] According to another aspect, the invention also relates to a device for acoustic stimulation of objects configured to implement a method as described above.

[0034] The device may for this purpose comprise a cavity capable of containing said medium receiving the objects, a transducer configured to generate said acoustic wave and a transducer control member configured to modify one or more of said parameters of the acoustic wave.

[0035] The transducer is preferably an ultrasonic transducer.

[0036] In embodiments, the device may include one or more contact structures as defined above.

[0037] Other advantages and characteristics of the invention will appear on reading the detailed, non-limiting description which follows. Brief description of the figures

[0038] The following detailed description refers to the attached drawings in which: - Figure 1 is a schematic view of a device comprising a cavity which contains a fluid with suspended objects, including objects with positive acoustic contrast and objects with negative acoustic contrast; - Figure 2 is a schematic view of the device of Figure 1 in which the objects with positive acoustic contrast are positioned in the form of aggregates on two pressure nodes formed by a standing acoustic wave and the objects with negative acoustic contrast are positioned in the form of aggregates on an anti-pressure node formed by this standing acoustic wave; - Figure 3 is a schematic view of a device comprising a cavity and a wall extending vertically in the cavity, the cavity containing a fluid and aggregates of objects with positive acoustic contrast which are positioned in the form of aggregates on four pressure nodes formed by a standing acoustic wave, under the action of an acoustic radiation force, and which are supported on the wall to adhere to it; - figure 4 is a schematic view of the device of figure 3 in which the aggregates of objects are, after adhesion to the wall, subjected to a first mechanical stress under the action of a variation in the acoustic radiation force, driving the objects vertically upwards; - Figure 5 is a schematic view of the device of Figure 3 in which the aggregates of objects are subjected to a second mechanical stress under the action of a new variation in the acoustic radiation force, driving the objects vertically downwards; - Figure 6 is a schematic view of a device comprising a cavity and a membrane extending horizontally in the cavity, the cavity containing a fluid and aggregates of objects which are in pressure on the membrane under the action of an acoustic radiation force; - Figure 7 is a schematic view of the device of Figure 6 in which the aggregates of objects and the membrane are moved under the action of a variation in the acoustic radiation force; - Figure 8 is a schematic view of a portion of a cavity of a device similar to that of Figure 1, the cavity containing a fluidic medium receiving a droplet of silicone oil shown in an initial state; - Figure 9 is a schematic view of the part of the cavity of Figure 8 showing the droplet in a deformed state; - Figure 10 shows a diagram illustrating a deformation over time of the droplet of Figures 8 and 9; - Figure 11 is a schematic view of a portion of a cavity of a device similar to that of Figure 1, the cavity containing a fluidic medium receiving an aggregate of cells shown in an initial state; - Figure 12 is a schematic view of the cavity portion of Figure 11 showing the aggregate in a deformed state. Detailed description of embodiments

[0039] Figures 1 and 2 schematically represent a device 1 according to the invention.

[0040] The device 1 forms a cavity 2 intended to contain a medium which can generally be fluid, in particular liquid, or semi-solid of the hydrogel type.

[0041] Generally, the cavity 2 extends along an axial direction Al, which in this example extends vertically when using the device 1. The cavity 2 has a dimension B1 along the direction Al, which defines a height of the cavity 2.

[0042] In this example, cavity 2 has a generally cylindrical shape, the direction Al forming an axis of symmetry of cavity 2.

[0043] The device 1 is equipped with an acoustic wave generation system which comprises in this example a transducer 3 arranged at a first end of the cavity 2 in the direction A1, in this case vertically below the cavity 2, as well as an acoustic reflector 4 which delimits a second end of the cavity 2 in the direction A1, the reflector 4 being in this case arranged vertically above the cavity 2.

[0044] With reference to figure 2, this system is configured to be able to generate in the cavity 2, and in particular in the medium which it contains, a standing acoustic wave 6, along the direction Al.

[0045] In the configuration illustrated in Figure 2, wave 6 has a frequency identical to the resonant frequency of cavity 2 which forms a resonator. The height B1 of cavity 2 is a multiple of half the acoustic wavelength.

[0046] This acoustic wave may have a frequency different from the resonance frequency of cavity 2, particularly in certain phases during which aggregates of objects are acoustically stimulated (see further below).

[0047] The system is generally configured to be able to generate, in particular, a wave 6 forming along the direction Al at least one pressure node and / or at least one antinode or pressure antinode.

[0048] In this example, transducer 3 is a broadband piezoelectric transducer used as an ultrasonic source.

[0049] Such a transducer 3 makes it possible in particular to modify the axial position of the node(s) and anti-nodes formed by the wave 6 and / or the distance between these nodes and anti-nodes, by acting on the frequency of this wave 6.

[0050] The device 1 of figures 1 and 2 comprises a member (not shown) for controlling the transducer 3, which is configured to be able to modify one or more parameters of the wave that it generates (see further below).

[0051] A method according to the invention will now be described, making it possible to stimulate objects using the device 1 of figures 1 and 2, or a similar device.

[0052] Figure 1 illustrates an initial configuration in which objects 11 and 12 are suspended in a liquid medium, in this case aqueous, within the cavity 2.

[0053] For information purposes, each of the objects 11 and 12 may have a size of between 0.1 pm and 100 pm, for example between 1 pm and 100 pm, and the height B1 of the cavity 2 may be of the order of λ / 2, with λ being the acoustic wavelength, typically from several millimeters to several centimeters.

[0054] In this non-limiting example, the objects 11 are biological cells having a density p ol greater than the density p f from the middle (acoustic contrast F y > 0), while the objects 12 are polydimethylsiloxane beads having a density p o2 less than the density p f from the middle (acoustic contrast F y < 0).

[0055] The objects 11 are chosen so that the speed c ol propagation of an acoustic wave in these objects 11 is greater than the speed c f of propagation of this acoustic wave in the medium. The objects 12 are chosen so that the speed c o2 of propagation of an acoustic wave in these objects 12 is less than the speed c f of propagation of this acoustic wave in the medium.

[0056] From the initial configuration of figure 1, the transducer 3 is actuated so as to generate in the cavity 2 a standing acoustic wave 6 (see figure 2).

[0057] The wave 6 thus generated produces an acoustic radiation force which is exerted on objects 11 and 12.

[0058] This acoustic radiation force FRA can be described in particular by the following model, known in itself, by K. Yosioka and Y. Kawasima: 7T9_ FRA = —pf Vg kd 3 F y sin (kz) where v0 is the speed of wave 6, k the wave number, F y the acoustic contrast factor and z the position of the object 11 or 12 considered along the direction Al.

[0059] The acoustic contrast factor, or density-compressibility factor, F y can be defined as follows: 1 + + - fl — — j 2 3 p o > P f cj y 2 + — 3 Po Co Po where p0 is the density of the object 11 or 12 considered and c0 the propagation speed of the wave within the object 11 or 12 considered.

[0060] Taking into account the respective speed of propagation of the acoustic wave and the respective density of the objects 11 and 12 relative to the medium, the objects 11 have a positive density-compressibility factor, or acoustic contrast, relative to the medium, while the objects 12 have a negative density-compressibility factor, or acoustic contrast, relative to the medium.

[0061] In the example of figure 2, the wave 6 has a wavelength λ equal to the height B1 of the cavity 2, forming along the direction Al a first pressure node NI, an anti-pressure node AN1 and a second pressure node N2, at respective axial positions called reference positions.

[0062] Taking into account the aforementioned respective properties of the medium and of the objects 11 and 12, from the initial configuration of Figure 1 in which the objects 11 and 12 are distributed relatively homogeneously in the cavity 2, the acoustic radiation force produced by the wave 6 causes a displacement of the objects 11 towards the nodes NI and N2 and a displacement of the objects 12 towards the anti-node AN1, so as to reach the configuration illustrated in Figure 2, also called the reference configuration.

[0063] The method thus comprises a step of generating the acoustic wave 6 which, in this non-limiting example, is carried out so as to move the objects 11 and 12 under the action of the acoustic radiation force to place them in said reference configuration.

[0064] In the reference configuration of Figure 2, objects 11 and 12 are spatially organized in the form of layers, or sheets, spaced along the direction Al.

[0065] In this example, the wave generation step 6 is continued to keep the objects 11 and 12 thus positioned, that is to say to keep them in the reference configuration, in acoustic levitation.

[0066] In the configuration of Figure 2, the objects 12 form an intermediate layer, located halfway up the cavity 2, while the objects 11 form two layers extending on either side of the intermediate layer.

[0067] The objects 12 here being polydimethylsiloxane beads, their aggregation or grouping in the form of a layer makes it possible to constitute a porous barrier which allows the development of interactions between the layers of cells 11, this without contact with the walls of the cavity 2, the medium forming in this example a culture medium for these cells 11.

[0068] According to the invention, the method is implemented to stimulate the objects from such a reference configuration, in this example after the process of developing interactions between the layers of cells 11.

[0069] To do this, an operation called actuation or stimulation is implemented, which comprises at least one step of modification, over time, of one or more parameters of the acoustic wave 6 in order to modify the acoustic radiation force and / or to move the nodes NI and N2 and the anti-node AN1 in the direction Al relative to their reference position.

[0070] The parameters in this example are chosen from a list including the amplitude and frequency of wave 6.

[0071] According to a first variant, the actuation operation comprises a modification of the amplitude of wave 6 at fixed frequency over time, in other words an amplitude modulation of wave 6.

[0072] For example, the signal supplied to the transducer 3 may comprise a periodic change in amplitude, varying the amplitude of the wave 6 between a minimum value and a maximum value according to a function predetermined periodic at a lower frequency, for example a sinusoidal, square, triangular or sawtooth function.

[0073] For information purposes, the minimum and maximum values ​​of the amplitude applied to the transducer 3 may be 5 V and 10 V, respectively, and the function may be a square or sinusoidal function having a period or cycle of one second. Such modulation of the amplitude of the wave 6 makes it possible to exert on the cells 11 an acoustic radiation force having an axial component that may vary in a range from 5 pN to 20 pN every second, typically mimicking the effect of mechanical stresses resulting from a heartbeat of an individual.

[0074] According to a second variant, the actuation operation comprises a modification of the frequency of the acoustic wave 6 with fixed amplitude over time, in other words a frequency modulation of the wave 6.

[0075] For example, the signal supplied to the transducer 3 may comprise a periodic modification of frequency, varying the frequency of the wave 6 between a minimum value and a maximum value according to a predetermined periodic function, for example a sawtooth function varying within each cycle the frequency linearly or according to another function, for example logarithmically.

[0076] For information purposes, the minimum and maximum values ​​of the frequency may vary by ±5% or ±10% around the resonance frequency, the minimum and maximum values ​​of the frequency being, for example, 1.9 MHz and 2.0 MHz, respectively, and the function may be a sawtooth function having a period or cycle of four seconds. Such a modification of the frequency of the wave 6 makes it possible to exert on the cells 11 a force which may vary in a range from 3 pN to 9 pN every four seconds, typically mimicking the effect of mechanical constraints resulting from a respiratory activity of an individual.

[0077] A change in the frequency of wave 6 produces a displacement of the pressure nodes and antinodes along the direction Al relative to their reference position, resulting in a corresponding displacement of the objects 11 and 12 along the direction Al which thus undergo a stress depending on both the variation of the acoustic radiation force and the viscosity of the medium.

[0078] It is of course possible to modify both the amplitude and the frequency of wave 6, for example to move the pressure nodes and antinodes relative to their reference position while subjecting the objects to a substantially constant acoustic radiation force over time.

[0079] The invention thus makes it possible to acoustically stimulate objects which are in particular grouped in the form of aggregates within the cavity 2. In the non-limiting examples described above, this stimulation aims to mimic physiological processes by carrying out periodic actuation. In an alternative example, the stimulation may be intended to mimic a trauma, for example by modifying one or more parameters of the wave 6 so as to exceed a corresponding critical threshold and / or by carrying out this modification over a duration less than a predetermined duration.

[0080] The device 1 of the invention can thus be used to form an active acoustic bioreactor.

[0081] In the above description, the acoustic stimulation of objects is carried out from a reference configuration in which the objects are grouped into aggregates and maintained in this configuration by acoustic levitation.

[0082] The acoustic stimulation according to the invention can generally be triggered after formation of such aggregates, or during their formation.

[0083] In an alternative embodiment, the acoustic stimulation is carried out on objects which are maintained in the reference configuration, for example in the configuration illustrated in figure 2, not in acoustic levitation in a liquid medium but by being supported by a semi-solid (or semi-liquid) medium.

[0084] This variant is described below only in terms of its differences from the preceding description, which applies by analogy.

[0085] In the context of this variant, the objects 12 comprise hydrogel particles and the method comprises, after positioning the objects 11 and 12 according to the configuration of figure 2, a step of heating / polymerization of the hydrogel particles, for example using a laser sheet, so as to fuse them to constitute a support matrix for the objects 11.

[0086] In other words, the method may comprise a step of transforming the liquid medium into a semi-solid medium. Maintaining the objects 11 in the reference configuration - and in configurations resulting from their acoustic stimulation - may thus be ensured by the medium which may comprise a hydrogel matrix and / or another type of support matrix.

[0087] Many variations can be envisaged on this basis. For example, the support matrix can be formed by introducing a photopolymerizable substance into the cavity 2 and light stimulation of all or part of this substance in order to cause polymerization.

[0088] Acoustic stimulation can also be carried out on objects supported by such a semi-solid medium, for example during an incubation phase allowing the development of phenomena of differentiation, self-organization and maturation of aggregates of the objects.

[0089] For example, modulation of the frequency of the acoustic wave, particularly around the resonance frequency, makes it possible to mechanically deform or constrain aggregates of objects, in particular to mimic developmental or pathological physiological processes such as those mentioned above.

[0090] Figures 3 to 5 schematically represent an embodiment in which the device 1 differs from that of Figures 1 and 2 in that it further comprises a wall 20 arranged in the cavity 2.

[0091] The embodiment of Figures 3 to 5 is described only in terms of its differences from that of Figures 1 and 2, the preceding description applying by analogy.

[0092] With reference to Figure 3, the wall 20 extends into the cavity 2 so as to separate it into two chambers 21 and 22.

[0093] The wall 20 comprises a first surface 25 which delimits the chamber 21 and a second surface 26 which delimits the chamber 22.

[0094] The wall 20 and its surfaces 25 and 26 extend along the direction A1, in this case parallel to this direction.

[0095] In this example, the wall 20 is a nitrocellulose membrane which comprises pores forming openings passing through the membrane 20 from one side to the other, so as to open onto the surfaces 25 and 26.

[0096] For information purposes, the wall 20 may have a thickness, defined as the distance between the surfaces 25 and 26, of 150 μm and pores having a diameter of 3 μm. In a non-limiting manner, the size of such pores may range from the nanometric scale to the micrometric scale.

[0097] In this example, chambers 21 and 22 are filled with a fluid containing objects 11A and 11B each having a size between 1 pm and 100 pm and exhibiting positive acoustic contrast.

[0098] In this non-limiting example, the objects 11A are biological cells of a first type which are received in the chamber 21 while the objects 11B are biological cells of a second type received in the chamber 22. The fluid forms a culture medium for the cellular objects 11A and 11B. In a non-limiting manner, the objects 11A and 11B may be neurons or glial, tumor, endothelial, epithelial, bone or even immune cells.

[0099] In this example, each of the objects 11A and 11B has a density greater than the density of the fluid and these objects are chosen so that the propagation speed of an acoustic wave in these objects is greater than the propagation speed of this acoustic wave in the fluid.

[0100] Starting from an initial configuration (not shown) in which the objects 11A and 11B are suspended in the fluid, respectively in the chambers 21 and 22, the transducer 3 is implemented so as to generate a standing acoustic wave in the cavity 2, along the direction Al, in this example with a wavelength forming four pressure nodes Nil, N12, N13 and N14, producing a radiation force acoustic having an axial component which is exerted on the objects 11A and 11B so as to position them in aggregates on the nodes Nil, N12, N13 and N14.

[0101] In this example, the transducer 3 is configured to maintain the objects 11A and 11B in acoustic levitation for the time necessary to obtain sufficient self-organization of the aggregates thus formed, which in this example constitute substantially ovoid three-dimensional structures, then to produce an acoustic radiation force having a transverse component in order to move the objects 11A and 11B in the direction of the wall 20 according to the reference configuration illustrated in FIG. 3.

[0102] To do this, the transducer 3 may comprise piezoelectric elements arranged side by side transversely, which can be gradually activated in the direction of the wall 20.

[0103] In this example, in the reference configuration, the objects 11A form aggregates plated on the surface 25 of the membrane 20 and the objects 11B form aggregates plated on the surface 26 of the membrane 20.

[0104] The wall 20 makes it possible in particular to control the interactions between the objects 11A and 11B, through its pores which can constitute passages for axon-type connections when the objects 11A and 11B are primary neurons.

[0105] In this example, the acoustic stimulation is carried out from the reference configuration of Figure 3, in which the object aggregates are fixed to the wall 20 by cellular adhesion in acoustic levitation.

[0106] Acoustic stimulation may include frequency modulation around the resonant frequency, with Figures 4 and 5 showing pressure nodes N11-N14 at the resonant frequency and relative displacement of object aggregates under positive and negative frequency variation, respectively.

[0107] More generally, the modification of one or more parameters of the wave generated in cavity 2 makes it possible to stimulate the aggregates of objects in contact with the wall 20 and to exert on them controlled tensile and / or compressive and / or torsional forces.

[0108] The principles described above with reference to Figures 3 to 5 can be implemented with other types of contact structures than the wall 20.

[0109] For example, the wall 20 of the device 1 of figures 3 to 5 may be a glass wall or more generally a non-porous structure, allowing in particular the use of different fluids in each of the chambers 21 and 22 of the cavity 2. Such a wall may of course be functionalized and / or cellularized and / or be permeable to gases.

[0110] For another example, the device 1 may comprise a contact structure comprising not one but two vertical walls perpendicular to each other, forming four chambers in the cavity 2, in order to produce assemblies with a greater number of types of objects.

[0111] The wall(s) forming such a contact structure may be of varied physicochemical nature, for example being formed from a hydrogel, an elastomer or even an inorganic material, and / or may comprise textures of micrometric or nanometric size.

[0112] For another example of a contact structure, the cavity 2 may comprise one or more pillars, or one or more hollow contact elements, also called “electrodes”, in order to be able to inject and trap in such contact elements and perfuse into the aggregates of objects solutes such as biological and physical compounds or agents, cells or even viruses.

[0113] The device may include one or more contact structures that may generally form one or more surfaces extending along the axial direction Al and / or one or more surfaces extending along a transverse direction.

[0114] Figures 6 and 7 illustrate in this regard an embodiment in which the device 1 comprises a membrane 30, made for example of polydimethylsiloxane, extending transversely, in this case perpendicular to the direction Al when the device 1 is in the reference configuration illustrated in figure 6.

[0115] In this example, objects 11C which have a positive acoustic contrast are held in acoustic levitation and pressed against the membrane 30 under the action of the acoustic radiation force which is generated in the reference configuration so as to form a pressure node N21 axially at the level of the membrane 30.

[0116] The objects 11C thus pressed against the membrane 30 can attach to it, for example by cellular adhesion.

[0117] Figure 7 shows the device 1 in a stimulation configuration in which the frequency is modulated so as to move the objects 11C under the action of an axial displacement of said pressure node N21, the membrane 30 being deformed under the action of the displacement of the objects 11C.

[0118] The preceding description naturally applies by analogy to the embodiment of Figures 6 and 7.

[0119] Of course, the invention is not limited to the examples which have just been described. For example, the cavity may have a geometry different from that described above. In particular, in an alternative embodiment, the cavity may be configured to excite different modes at distinct frequencies, in order to modulate the acoustic field, for example using an acoustic lens or multi-element transducer, and the distribution of the acoustic radiation force which is exerted on the objects when the acoustic wave passes from one frequency to another frequency.

[0120] For another example, the acoustic stimulation can be carried out on objects of different natures and / or forming different types of aggregates. For example, the method can be implemented to stimulate an aggregate comprising a group of first objects forming a core encapsulated by a group of second objects forming a porous protective shell, said second objects being able for example to be introduced into the cavity after aggregation of said first objects in acoustic levitation.

[0121] In an alternative embodiment described below with reference to figures 8 to 10, the acoustic stimulation is carried out on one or more objects of the silicone oil droplet type.

[0122] Such an implementation of the invention can in particular make it possible to carry out acoustic pressure measurements from the deformation of such a droplet whose properties are known.

[0123] Figures 8 and 9 schematically show a part of a cavity 2 of a device 1 similar to that described above, the preceding description applying by analogy to this embodiment.

[0124] Cavity 2 in this example contains a liquid medium.

[0125] In this example, the medium includes a surfactant such as sodium dodecyl sulfate with a concentration in the medium of 10 mM, in order to stabilize the droplets.

[0126] Figure 8 more specifically illustrates a droplet 13 of silicone oil received in the medium, the droplet 13 being placed in a reference configuration under the action of an acoustic radiation force produced by the acoustic wave generation system of the device 1.

[0127] In the reference configuration, droplet 13 is positioned at a pressure antinode.

[0128] In this example, the acoustic wave generated by the system is 2 MHz.

[0129] In the reference configuration, the droplet 13, also called an object or cell, has an initial state in which it generally forms a sphere having a diameter which can typically be between 10% and 100% of the wavelength used, for example a diameter of the order of 400 pm.

[0130] In this non-limiting example, the amplitude applied to the transducer of the generation system is modulated between 5 V and 10 V, causing a deformation of the droplet 13 between the initial state illustrated in FIG. 8 and another state illustrated in FIG. 9. This actuation operation results in this case in a modification of the dimension of the droplet 13 along the direction A1, under the effect of an increase in the acoustic force.

[0131] Figure 10 shows a spatio-temporal diagram illustrating such a deformation of the droplet 13 under the effect of a variation in the acoustic force. The diagram comprises an X1 axis of the abscissas representing time and an X2 axis of the ordinates representing a spatial position of parts 13A and 13B of the droplet 13 along the direction A1. The parts 13A and 13B of the droplet 13 correspond to the extrema of the droplet 13 along the direction A1 (see Figures 8 and 9).

[0132] The diagram in Figure 10 shows an experimental result of an actuation cycle carried out over a period from time t1 to time t4 of the order of 16 seconds. During this cycle, the amplitude applied to the transducer is 5 V between time t1 and time t2, 10 V between time t2 and time t3, then again 5 V between times t3 and t4. The dimension of the droplet 13 between times t1 and t2, and between times t3 and t4, has a value DI greater than its value D2 between times t2 and t3. For information, the ratio D1 / D2 is here of the order of 1.15.

[0133] This example illustrates an example of deformation of a droplet-like object by modulation of the acoustic radiation force.

[0134] The above description applies by analogy to actuation operations of different types of objects, which can be deformed and / or moved individually or in groups by modulation of an acoustic radiation force.

[0135] Thus, in the context of another variant embodiment described with reference to figures 11 and 12, the device 1 is implemented to stimulate not a droplet but an aggregate of objects 14 which may, in a non-limiting manner, comprise biological cells.

[0136] The embodiment of Figures 11 and 12 is described only in terms of its differences from that of Figures 8 to 10, the preceding description applying by analogy.

[0137] In the reference configuration illustrated in figure 11, the aggregate 14 is in an initial state in which its dimension along Al has a first value, figures 11 and 12 showing a line 40 whose length corresponds to a dimension of the order of 300 pm.

[0138] In the reference configuration, aggregate 14 is positioned at a pressure node.

[0139] In this non-limiting example, the amplitude applied to the transducer of the generation system is modulated between 5 V and 10 V so as to cause the aggregate 14 to pass from the initial state illustrated in figure 11 and a deformed state illustrated in figure 12, under the effect of an increase in the acoustic force.

[0140] Figures 11 and 12 thus illustrate the possibility of modifying the dimension of the aggregate 14 along the direction A1, in particular of compressing it, by acoustic stimulation using a method according to the invention.

Claims

CLAIMS 1. Method for acoustic stimulation of one or more objects (11, 11A, 11B, 11C, 12, 13, 14) received in a fluid or semi-solid medium within a cavity (2), comprising: - a step of generating a standing acoustic wave (6) so as to form, in the medium, one or more pressure nodes (NI, N2, N11-N14, N21) and / or anti-nodes (AN1) at respective axial positions called reference positions, in order to produce an acoustic radiation force which is exerted on the objects, - from a so-called reference configuration in which the objects are positioned on one or more of said pressure nodes and / or anti-nodes, a step of modifying, over time, one or more parameters of the acoustic wave in order to modify the acoustic radiation force and / or to move one or more of said pressure nodes and anti-nodes in an axial direction relative to their reference position.

2. Method according to claim 1, in which the parameters of the acoustic wave are chosen from a list including an amplitude and a frequency.

3. The method of claim 1 or 2, wherein the step of modifying one or more of said parameters of the acoustic wave comprises modifying the amplitude of the fixed frequency acoustic wave over time.

4. A method according to any one of claims 1 to 3, wherein the step of modifying one or more of said parameters of the acoustic wave comprises modifying the frequency of the fixed amplitude acoustic wave over time.

5. A method according to any one of claims 1 to 4, wherein the step of modifying one or more of said parameters of the acoustic wave comprises periodically modifying one or more of said parameters, preferably at a relatively low modulation frequency relative to a relatively high frequency of the acoustic wave.

6. Method according to any one of claims 1 to 5, in which, in particular during the step of modifying one or more of said parameters of the acoustic wave, the objects are held in the cavity (2): - under the action of the acoustic radiation force, for example in acoustic levitation in the medium which may be liquid in the reference configuration, and / or - by being supported by the medium, the medium being able for example to be semi-solid in the reference configuration, and / or - being supported by, and / or in contact with, one or more contact structures (20, 30) which extend into the cavity (2), each of these contact structures being able to be chosen from a list including a wall, a pillar and an electrode.

7. Method according to any one of claims 1 to 6, in which, in an initial configuration, the objects are suspended in the medium which is fluid in this initial configuration, the step of generating the acoustic wave being carried out so as to move the objects under the action of the acoustic radiation force to place them in said reference configuration.

8. A method according to any one of claims 1 to 7, wherein the objects comprise objects (11, 11A, 11B, 11C) having a positive acoustic contrast with respect to the medium and / or objects (12) having a negative acoustic contrast with respect to the medium.

9. Method according to any one of claims 1 to 8, comprising a step of incubating the objects while they are subjected to the acoustic radiation force.

10. Device (1) for acoustic stimulation of objects (11, 11A, 11B, 11C, 12, 13, 14) configured to implement a method according to any one of claims 1 to 9, the device (1) comprising a cavity (2) capable of containing said medium receiving the objects, a transducer (3) configured to generate said acoustic wave (6) and a control member of the transducer (3) configured to modify one or more of said parameters of the acoustic wave.

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