Assembly and culture of cellular objects on a contact structure under the action of axial and transverse acoustic radiation forces

Abstract

Device and method for assembling objects (11, 12) by generating an axial acoustic radiation force to position the objects (11, 12) on pressure nodes (N1-N4) and / or antinodes, and generating a transverse acoustic radiation force to move the objects (11, 12) thus positioned on a contact structure (4) of the porous membrane type. Figure for the abstract: Fig. 1

Description

Title of the invention: Assembly and culture of cellular objects on a contact structure under the action of axial and transverse acoustic radiation forces. Technical field

[0001] The invention relates to the field of biotechnology and in particular to the assembly of cells or micrometric particles, for example with a view to reconstructing or modeling living tissues.

[0002] The invention is of particular, but by no means limiting, interest in the fields of cell therapy, pharmacological modeling, agri-food (for example, for the cultivation of meat, microalgae or plants), and aerospace, particularly for cell culture under microgravity conditions. Prior art

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

[0004] The most commonly 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, involves 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 cells to be assembled 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 for satisfactory control of the development of such connections.

[0006] More generally, the assembly techniques known in this field are complex and costly, can require significant amounts of time to assemble and culture cells and, insofar as they are implemented in vitro, can lead to the death of a large number of cells. Description of the invention

[0007] The present invention aims to overcome the aforementioned drawbacks by providing a device for assembling objects, comprising: - a cavity configured to receive a fluid and objects, - a generation system configured to generate in the cavity, along a axial direction, a stationary acoustic wave so as to produce an axial acoustic radiation force capable of positioning objects on pressure nodes and / or antinodes formed by the acoustic wave.

[0008] According to the invention, the device comprises a contact structure forming one or more surfaces extending along the axial direction and the generation system is configured to exert a transverse acoustic radiation force capable of moving objects in the direction of the surface or surfaces of the contact structure.

[0009] The propagation of a stationary acoustic wave in the cavity makes it possible to form in the cavity, along the axial direction, one or more pressure nodes, that is to say places where the fluid pressure is zero, and one or more antinodes or pressure antinodes, that is to say places where this pressure is maximum.

[0010] In a manner known per se, depending on the properties of the objects and in particular their density-compressibility factor, or acoustic contrast with respect to the fluid contained in the cavity, the axial acoustic radiation force moves the objects either towards a pressure node when their acoustic contrast is positive or towards a pressure antinode when it is negative.

[0011] The axial acoustic radiation force thus makes it possible to form in the cavity one or more aggregates of objects which follow one another along the axial direction, this in an extremely rapid manner - typically in a few seconds - and with the help of equipment which is particularly simple to implement and inexpensive.

[0012] The transverse acoustic radiation force, which is preferably generated after such axial positioning of the objects, makes it possible to put each of the aggregates thus formed in contact with the contact structure.

[0013] The invention thus makes it possible to create one or more assemblies of objects and to maintain these assemblies in acoustic levitation in support of the contact structure.

[0014] By maintaining such assemblies in acoustic levitation for the required duration, for example several hours or days, it is possible to promote interactions between objects when these are living, in particular when these objects are biological cells.

[0015] The invention thus makes it possible to carry out a cell culture in acoustic levitation, by controlling, for each of the assemblies, the development of the connections and interactions between the cells of this assembly and / or between these cells and other objects or elements which can be arranged on or in the contact structure (see further below).

[0016] In the present description, an "object" means a living or inert element preferably having a small size compared to the length of the stationary acoustic wave generated in the cavity.

[0017] By way of non-limiting example, the objects may have a micrometer size, by example between 1 pm and 100 pm, typically when the stationary Fonde frequency has a value in the MHz range.

[0018] For another example, in particular when the generation frequency is in the kHz range, the objects can have a size of millimeters, for example between 1 mm and 100 mm.

[0019] The invention can, however, be implemented with objects of different sizes. For example, the objects, or some of them, may be smaller than 1 µm, being formed, for example, by bacteria or viruses, and / or be several hundred µm in size. Furthermore, the objects, or some of them, may be multicellular elements, artificially formed objects, or objects taken from an organ.

[0020] The fluid in which the objects are suspended is preferably a liquid which, depending on the intended application, may include water or form a culture medium or more generally an aqueous medium comprising, for example, a hydrogel pre-polymer or colloidal particles.

[0021] The invention also provides a particularly precise solution in terms of positioning objects in space and which allows, where appropriate, the control of the development of intercellular interactions.

[0022] The device of the invention can thus form an acousto-fluidic chip implemented for various applications and exhibit varied structural characteristics, in particular with regard to its contact structure.

[0023] In one embodiment, the contact structure comprises one or more membranes or walls.

[0024] In the present description, a membrane, also called a "wall", is a structure typically comprising two surfaces that are relatively large in relation to a thickness of this structure, that is to say in relation to the distance separating these two surfaces.

[0025] In one embodiment, the cavity comprises several chambers delimited by one or more of said membranes.

[0026] By way of non-limiting example, the cavity may comprise two chambers and the contact structure may comprise a single membrane arranged so that a first surface of the membrane delimits one of these chambers and a second surface of the membrane delimits the other chamber.

[0027] In one embodiment, each of the chambers comprises a respective part of the objects.

[0028] Thus, in the context of the aforementioned example of compartmentalizing the cavity into two chambers, the objects can be divided into two series, the objects of the first series being able to be placed in one of the chambers and the objects of the second series in the other room.

[0029] In this non-limiting example, the transverse acoustic radiation force can be configured so as to move the objects of the first series towards the membrane in order to group these objects against the first surface of the membrane and so as to move the objects of the second series towards the membrane in order to group these objects against the second surface of the membrane.

[0030] Objects of the first series and of the second series arranged at the same position along the axial direction can thus be grouped on either side of the membrane, allowing, for example, the development of interactions, in particular through the membrane when it is porous.

[0031] Thus, in one embodiment, one or more of said membranes are porous.

[0032] In one embodiment, one or more of said surfaces formed by the contact structure are surfaces extending each around a respective direction.

[0033] Preferably, this direction is parallel or oblique with respect to said axial direction.

[0034] For example, one or more of said surfaces formed by the contact structure can each extend around a respective direction so as to form a surface of revolution around that direction, for example so as to present a cylindrical geometry.

[0035] Such a surface of revolution may have a relatively small cross-section compared to the length of stationary acoustic background generated in the cavity and / or compared to the axial dimension of this surface of revolution, so as to allow one or more assemblies of objects around such a surface, for example in the form of spheroid or ovoid assemblies.

[0036] In one embodiment, one or more of said surfaces formed by the contact structure can each form a hollow structure, allowing for example to infuse and / or collect biological or chemical objects, elements or samples, or to encapsulate objects such as biological cells in the contact structure.

[0037] Thus, in one embodiment, the device includes elements, such as biological cells, connected to the contact structure in such a way as to allow interactions between these elements and one or more of said objects when the latter are moved towards the surfaces of the contact structure.

[0038] Of course, these principles can be generalized so that one or more of said surfaces of the contact structure, whether the latter takes the form of one or more membranes and / or hollow structures and / or structures arranged around one or several directions and / or one or more three-dimensional structures of any geometry, can be cellularized or, more generally, include or carry elements containing biological, physico-chemical and / or physical information, for example using molecules that promote or inhibit cell multiplication, nanoparticles or micrometric or nanometric texturing elements.

[0039] In one embodiment, the contact structure comprises or forms one or more electrodes and / or has conductive properties.

[0040] In one embodiment, the contact structure comprises a gas-permeable material, for example polydimethylsiloxane, for example so as to diffuse oxygen into the core of the object assembly or assemblies.

[0041] In one embodiment, the generation system comprises one or more piezoelectric and / or ultrasonic transducers which may comprise several elements arranged in a matrix manner and / or one or more acoustic holographic lenses.

[0042] In one embodiment, said objects include objects exhibiting a positive acoustic contrast with respect to the fluid such that the axial acoustic radiation force moves these objects towards the pressure nodes and / or objects exhibiting a negative acoustic contrast with respect to the fluid such that the axial acoustic radiation force moves these objects towards the anti-pressure nodes.

[0043] The invention also relates to a method of assembling objects using a device as defined above.

[0044] This method includes generating axial acoustic radiation force so as to position objects on pressure nodes and / or antinodes and generating transverse acoustic radiation force so as to move objects in the direction of the surface or surfaces of the contact structure.

[0045] The transverse acoustic radiation force is preferably generated after positioning the objects on the pressure nodes and / or antinodes, for example with a generation system comprising a matrix of piezoelectric elements.

[0046] Alternatively, axial and transverse acoustic radiation forces can be generated simultaneously, for example with a generation system comprising a single transducer or an acoustic holographic lens.

[0047] The method can implement all steps enabling different combinations of the functional characteristics described above to be obtained, depending in particular on the structural characteristics of the device and in particular on the contact structure.

[0048] In general, the invention does not cover applications in which said objects comprise human embryonic stem cells involving the destruction of a human embryo and applications in which the invention would implementation in such a way as to constitute or develop a human body.

[0049] Other advantages and features of the invention will become apparent from the following detailed, non-limiting description. Brief description of the drawings

[0050] The detailed description that follows refers to the attached drawings on which:

[0051] [Fig-1] is a schematic, axial cross-sectional view of a device conforming to the invention, this device comprising a cavity, a porous membrane arranged in the cavity so as to delimit two chambers, an ultrasonic transducer comprising an array of piezoelectric elements and a reflector, each of the chambers containing cellular objects maintained in acoustic levitation in the form of aggregates at a distance from the membrane;

[0052] [Fig.2] is a schematic view of the device of [Fig.1], the aggregates of cellular objects being arranged against the membrane so as to form assemblies of aggregates which can develop intercellular interactions with each other through pores in the membrane;

[0053] [Fig.3] is a schematic cross-sectional view of a device according to the invention which differs from that of [Fig.1] in that it comprises two porous membranes arranged in the cavity so as to delimit four chambers which each contain cellular objects maintained in acoustic levitation in the form of aggregates at a distance from the membrane;

[0054] [Fig.4] is a schematic view of the device of [Fig.4], the aggregates of cellular objects being arranged against the membranes so as to form assemblies of aggregates which can develop intercellular interactions with each other through pores in the membranes;

[0055] [Fig.5] is a schematic axial cross-sectional view of a device according to the invention, this device comprising a cavity, elongated contact elements arranged in the cavity, an ultrasonic transducer comprising an array of piezoelectric elements and a reflector, the cavity containing cellular objects maintained in acoustic levitation in the form of aggregates at a distance from the contact elements;

[0056] [Fig.6] is a schematic view of the device of [Fig.5], the aggregates of cellular objects being arranged around the contact elements. Detailed description of implementation methods

[0057] Fig. 1 schematically illustrates an example of a device conforming to a first embodiment of the invention.

[0058] This device includes a container which forms a cavity 1 suitable for containing a fluid and / or various substances, in particular in liquid form.

[0059] Generally, the cavity 1 extends along an axial direction Al, which In this example, this corresponds to a vertical direction. Cavity 1 has a dimension B1 along the direction Al, defining a height of cavity 1.

[0060] In this example, the cavity 1 has an overall cylindrical shape, the direction Al forming an axis of symmetry of the cavity 1. In variants not shown, the cavity 1 may have another geometry, for example a rectangular section.

[0061] The device of the invention further comprises an acoustic wave generation system, in this case a transducer 2 equipped with a matrix of piezoelectric elements 2A and 2B and an acoustic reflector 3.

[0062] With reference to [Fig. 1], the transducer 2 is arranged at one end of the cavity 1 in the direction Al, in this case vertically above the cavity 1. The reflector 3 delimits a second end of the cavity 1 in the direction Al, in this case being arranged vertically below the cavity 1.

[0063] The piezoelectric elements 2A and 2B are distributed transversely, i.e., across the width of the cavity 1, such that each of these elements is located at a respective position radially with respect to the AL direction

[0064] This system is configured to generate in cavity 1 and propagate in the fluid it contains a standing acoustic wave, along an axial propagation direction corresponding to the AL direction

[0065] The standing wave can be generated by one or more of the piezoelectric elements of the transducer 2, with a frequency identical to the resonant frequency of the cavity 1, which in this case forms a resonator. Alternatively, the standing wave can have a frequency different from the resonant frequency of the cavity 1.

[0066] In all cases, the system is configured to be able to generate, in particular, a wave having a wavelength 2 less than or equal to twice the height B1 of the cavity 1, in order to form along the direction Al at least one pressure node and at least one pressure antinode.

[0067] In this example, transducer 2 is a broadband transducer equipped with an ultrasonic source. Such a transducer 2 makes it possible to modify the position of the nodes of the standing wave along the direction Al and / or the distance between these nodes, by changing the frequency of the wave.

[0068] In the embodiment of [Fig.1], the device includes a wall 4, also called a "membrane", which extends into the cavity 1 so as to separate the cavity 1 into two chambers 5 and 6.

[0069] The membrane 4 comprises a first surface 7 which delimits the chamber 5 and a second surface 8 which delimits the chamber 6.

[0070] The membrane 4 and its surfaces 7 and 8 extend along the direction Al, in this case parallel to this direction.

[0071] In this example, the membrane 4 is made of nitrocellulose and includes pores 9 forming openings which pass through the membrane 4 from one side to the other, so as to open onto the surfaces 7 and 8.

[0072] By way of example, the membrane 4 may have a thickness, defined as the distance between surfaces 7 and 8, of 150 pm and pores having a diameter of 3 pm. Without limitation, the size of such pores may range from the nanometer scale to the micrometer scale.

[0073] In this example, chambers 5 and 6 are filled with a fluid containing objects 11 and 12, each having a size between 1 pm and 100 pm.

[0074] In this non-limiting example, objects 11 are biological cells of a first type which are received in chamber 5 while objects 12 are biological cells of a second type received in chamber 6. The fluid forms a culture medium for the cellular objects 11 and 12.

[0075] Without limitation, objects 11 and 12 may be neurons or glial, tumor, endothelial, epithelial, bone or immune cells.

[0076] In this specific example, each of the objects 11 and 12 has a density Po greater than the density Pf of the fluid. The objects 11 and 12 are further chosen such that the propagation speed of an acoustic wave in these objects 11 and 12 is greater than the propagation speed cf of this acoustic wave in the fluid.

[0077] The device in [Fig.1] is implemented in this example so as to be able to structure objects 11 and 12 in the manner described below.

[0078] In an initial state, not shown, objects 11 and 12 are suspended in the fluid, respectively in chambers 5 and 6.

[0079] The transducer 2 is implemented so as to generate a standing acoustic wave in the cavity 1, along the direction Al, in this example with a wavelength forming four pressure nodes N1-N4. In this case, the wave is generated via the piezoelectric elements 2A, identified by rectangles with a cross on [Fig.1], the piezoelectric elements 2B, identified by rectangles without a cross on [Fig.1], being inactivated.

[0080] This standing wave produces an axial acoustic radiation force which is exerted on objects 11 and 12.

[0081] This axial acoustic radiation force FRAa can in particular be described according to the following model, known per se, of K. Yosioka and Y. Kawasima:

[0082] [Math.l] FRAa = p? vg k d3 Fy sin^Æ z)

[0083] where vo is the Fonde velocity, k the wave number, Fy the contrast factor acoustic, or density-compressibility, and z the axial position of the object 11 or 12 considered along the direction Al, that is to say along the direction of wave propagation.

[0084] The acoustic contrast factor, or density-compressibility, Fy can be defined as follows:

[0085] [Math.2]

[0086] where Po is the density of the object 11 or 12 considered and the propagation speed of Fonde within the object 11 or 12 considered.

[0087] Considering the density and the speed of propagation of the acoustic wave of objects 11 and 12 relative to the fluid, objects 11 and 12 exhibit a positive density-compressibility factor, or acoustic contrast.

[0088] Taking into account the aforementioned respective properties of the fluid and of the objects 11 and 12, from said initial state in which the objects 11 and 12 are distributed relatively homogeneously throughout the cavity 1, the axial acoustic radiation force causes a displacement of the objects 11 and 12 towards the nodes of the standing acoustic wave, so as to reach a configuration as illustrated in [Fig.1].

[0089] The axial acoustic radiation force thus causes objects 11 and 12 to be positioned in groups, also called "aggregates", in the manner illustrated in [Fig.1], which are located radially at the level of the activated piezoelectric elements 2A.

[0090] Thus, a group 21 of objects 11 is formed axially at the pressure node NI and radially at the piezoelectric elements 2A located opposite the chamber 5. Three other groups 22, 23 and 24 of objects 11 are formed axially at the pressure nodes N2, N3 and N4, respectively, and radially at the same level as group 21.

[0091] Symmetrically, a group 25 of objects 12 is formed axially at the pressure node NI and radially at the piezoelectric elements 2A located opposite the chamber 6. Three other groups 26, 27 and 28 of objects 12 are formed axially at the pressure nodes N2, N3 and N4, respectively, and radially at the same level as group 25.

[0092] The actuated piezoelectric elements 2A can maintain the objects 11 and 12 in acoustic levitation for the time necessary to obtain sufficient self-organization of the aggregates 21 to 28, which in this example constitute substantially ovoid three-dimensional structures.

[0093] The transducer 2 can then be controlled to produce a transverse acoustic radiation force in order to move objects 11 and 12 in the direction of the membrane 4, in particular so that aggregates 21 to 24, located in chamber 5, are pressed against surface 7 of membrane 4 and aggregates 25 to 28, located in chamber 6, are pressed against surface 8 of membrane 4.

[0094] In this example, such a transverse acoustic radiation force is achieved by changing the activation state of the piezoelectric elements 2A and 2B, i.e. by progressively activating the piezoelectric elements radially in the direction of the Al direction and by deactivating, as new piezoelectric elements are activated, the previously activated piezoelectric elements.

[0095] The transducer 2 thus allows spatial control of the acoustic field in the cavity 1 so as to exert a transverse acoustic radiation force capable of translating the aggregates 21 to 28 towards the membrane 4, until reaching a configuration such as illustrated in [Fig.2].

[0096] In a manner known per se, the transverse acoustic radiation force FRAT can be expressed as a function of the acoustic energy density gradient:

[0097] [Math.3] FRA^x, y, {E^x, y, z)}

[0098] where d corresponds to the diameter of objects 11 and 12 and V ( Eac(x, y, z)} to the acoustic energy density gradient.

[0099] In the configuration of [Fig.2], the piezoelectric elements 2A located radially at the level of the membrane 4 can remain activated so as to maintain the aggregates 21 to 28 in contact with the membrane 4, by acoustic levitation.

[0100] The invention thus makes it possible to assemble structures of objects, in this example aggregates 21-24 of objects 11 of a first type with aggregates 25-28 of objects 12 of a second type, without the different types of objects located in the same plane of acoustic levitation coming into direct contact with each other and so that contact can in this example be established via the porous membrane 4.

[0101] Such a membrane 4 makes it possible in particular to control the interactions between objects 11 and 12, through the pores 9 which can constitute passages for axon-type connections when objects 11 and 12 are primary neurons.

[0102] Numerous variations can be made to the device just described and to its implementation. For example, the height B1 of the cavity 1 and / or the frequency of the standing acoustic wave can be modified in order to increase the number of pressure nodes, for example so as to assemble several dozen aggregates simultaneously.

[0103] The size and shape of objects 11 and 12 can also be modified, as well as the concentration of objects in cavity 1, the volume of cavity 1, or the frequency of the wave, in order, for example, to determine a number of objects per aggregate or assembly, as well as the size of the assembly(ies) thus obtained. The choice of the acoustic background amplitude and frequency, which determines the magnitude of the acoustic radiation force, can also be controlled to modify the lateral and / or axial dimensions of the assembly(ies). The amplitude of the acoustic pressure applied to the object sets can also be controlled, for example, to force a given spatial organization or to stimulate the objects and thus force certain spatial arrangements and / or functionalities.

[0104] For another example, the membrane 4 can be a glass wall or more generally a non-porous structure, allowing in particular the use of different fluids in each of the chambers 5 and 6 of the cavity 1. Such a wall can of course be functionalized and / or cellularized and / or be permeable to gases.

[0105] Other, non-limiting embodiments of the invention are described below, it being understood that the preceding description applies by analogy to these different embodiments and their variants. In the following description, these embodiments are therefore essentially described according to their differences from the embodiment shown in Figures 1 and 2.

[0106] In the embodiment of figures 3 and 4, the contact structure 4 comprises not one membrane but two porous membranes 4A and 4B separating the cavity 1 into four chambers 5A, 5B, 6A and 6B. Each of the chambers 5A, 5B, 6A and 6B comprises objects 31, 32, 33 and 34, respectively, of a different nature.

[0107] Such a device makes it possible to form complex assemblies, in this case by doubling the types of objects compared to the device in [Fig.1].

[0108] More generally, the contact structure 4 can therefore be configured to compartmentalize the acoustic levitation cavity into several independent chambers, with different culture media, as needed. This makes it possible, in particular, to maintain acoustic levitation and cultivate in three dimensions one cell type per chamber, all within a single acousto-fluidic chip.

[0109] By controlling the frequency of the emitted acoustic waves and / or the shape of the reflector, and consequently the spatial pattern of the acoustic field generated in the cavity, levitating cellular objects can be brought into contact with a contact structure such as a membrane, either at the beginning of the culture or during the culture.

[0110] In these different examples, different symmetrical or asymmetrical shapes of reflector 3 can be implemented in order to promote a given spatial organization of aggregates of acoustically levitating objects, for example an organization in sheets, in spheroid or ovoid structures, in rings, or even in independent lobes.

[0111] The membrane(s) of the contact structure may be of various physicochemical nature, for example be formed of a hydrogel, an elastomer or even of an inorganic material, and / or may include textures of micrometric or nanometric size.

[0112] The use of membranes with controlled porosity makes it possible to control, on the one hand, the self-assembly of different aggregates, particularly spheroids, and thus to reconstruct complex multicellular assemblies, and on the other hand, the nature of the interactions between aggregates of various types. For example, it is possible to control the exchange of solutes, particularly with membrane pores smaller than one micrometer, cellular extensions including neuronal axons, particularly with a porosity between 1 pm and 5 pm, or even the exchange of cells or objects, particularly with a porosity greater than 5 pm.

[0113] Furthermore, the chamber(s) delimited by one or more membranes, or more generally by a contact structure that may be otherwise constituted, may be used solely for initial structuring stages, i.e., assembling objects, these assemblies then being able to be cultured conventionally, for example in a liquid medium or in a hydrogel. Alternatively, the device of the invention may be used to perform long-term culture by acoustic levitation.

[0114] In the embodiment of Figures 1 and 2, the transverse acoustic radiation force is generated by selective activation of piezoelectric elements of the transducer 2. This force can, of course, be generated using another type of acoustic field control device, for example, with an acoustic holographic lens. An acoustic holographic lens can be encoded on several frequencies, as is known per se. Thus, a first frequency can be used to organize the objects into aggregates as illustrated in [Fig. 1], and a second frequency to bring and hold these aggregates against the contact structure 4 and form assemblies as illustrated in [Fig. 2].More generally, the wave generation system producing axial and transverse acoustic radiation forces may include one or more transducers or combinations of transducers of different types and / or positioned axially and / or transversely.

[0115] Another type of contact structure is illustrated in Figures 5 and 6.

[0116] With reference to [Fig. 5], the device comprises a contact structure comprising a network of contact elements 40 which each have an elongated geometry along the axial direction Al of the cavity 1.

[0117] In this non-limiting example, each of the contact elements 40 has a cylindrical geometry so as to form an external surface 41 extending around a direction A2 parallel to the axial direction Al of the cavity 1.

[0118] In a manner analogous to the embodiment of Figures 1 and 2, the ge system The acoustic wave generation of the device in [Fig.5] also includes an ultrasonic transducer 2 equipped with piezoelectric elements 2C arranged in a matrix and a reflector 3, each extending in a transverse plane of the cavity 1 so as to be able to move objects 42 present in the cavity 1.

[0119] From an initial state, not shown, in which the objects 42 are suspended in the fluid, the axial acoustic radiation force resulting from the standing wave generated by the transducer 2 causes the objects 42 to be positioned as aggregates as illustrated in [Fig.5].

[0120] The aggregates of objects 42 are formed in this particular example on three pressure nodes formed by the standing wave, the objects 42 exhibiting a positive acoustic contrast with respect to the fluid, transversely with respect to activated piezoelectric elements 2C, in this configuration at a distance from the contact elements 40.

[0121] In a manner analogous to the embodiment of Figures 1 and 2, the activation of the piezoelectric elements 2C is then modified to produce a transverse acoustic radiation force causing a displacement of the aggregates around the contact elements 40 as illustrated in [Fig.6], so as to form in this example spheroid assemblies which can thus be cultured.

[0122] The contact elements 40 can be of different physico-chemical and geometric natures. They can, for example, be solid or hollow, permeable or non-permeable, electroactive or non-electroactive, which can allow, in particular, the controlled perfusion, stimulation, monitoring and / or sampling of the assemblies.

[0123] Thus, in one embodiment, the contact elements 40 can form electrodes designed to measure or impose electrical activity. Electroactive and / or conductive contact elements 40 make it possible, in particular, to model a vascularization phenomenon, with electrical stimulation and recording of the electrical activity of the assemblies of objects 42.

[0124] In one embodiment, the contact elements 40 may be hollow and / or porous, for example, to allow the injection and trapping of solutes such as biological and physical compounds or agents, cells, or viruses into the contact elements 40 and their perfusion into the object assemblies 42. The nature of the diffusion / migration of such solutes can be controlled through the porosity of the contact elements 40.

[0125] Such variants can be implemented or combined with other types of contact structures, for example one or more membranes 4 as described above with reference to Figures 1 to 4.

[0126] More generally, the contact structure of the device of the invention can be functionalized so as to modify the interaction between cellular objects assembled on the contact structure.

[0127] Furthermore, the functionalization of the contact structure may include grafting objects onto one or more surfaces of the contact structure and / or within the contact structure, for example within contact elements 40 as illustrated in Figures 5 and 6. The grafted objects may be particles conferring new properties to the contact structure, for example in terms of electrical, magnetic or acoustic conductivity, bioactive substances influencing the development of cellular objects, or cells, for example endothelial or astrocytic, or other living organisms.

[0128] In one embodiment, the contact elements 40 of the device in Figures 5 and 6 can be formed from a hardened hydrogel, for example by confocal photopolymerization. These contact elements 40 may include cellular or functionalizing objects, such as those described above, trapped in the hydrogel forming these contact elements 40. Of course, such contact elements can have any more or less complex three-dimensional geometry, forming, for example, a lattice structure or the like.

[0129] The invention enables numerous applications, including the reconstruction of complex tissue elements, the modeling of interactions between several organs in series, for example, interactions between different brain regions, the serial or parallel arrangement of organ models, etc. Among other examples of applications, the invention allows for the three-dimensional reconstruction of neuroanatomical pathways or three-dimensional models of tissue barriers such as the blood-brain or placental barrier. In the context of neuroscience, the anisotropy of porosity, for example, of conical holes in membranes, would allow for the control of the growth direction of axons and / or nerves from one organoid to another. As another example, complex biological interfaces can be modeled by cellularizing membranes, whether porous or not, particularly by pre-seeding the membranes with cells of interest.

Claims

Demands

1. A device for assembling objects (11, 12, 31-34, 42), comprising: - a cavity (1) configured to receive a fluid and the objects (11, 12, 31-34, 42), - a generation system (2) configured to generate in the cavity (1), along an axial direction (A1), a stationary acoustic wave so as to produce an axial acoustic radiation force capable of positioning the objects (11, 12, 31-34, 42) on pressure nodes (N1-N4) and / or antinodes formed by the acoustic wave, characterized in that it comprises a contact structure (4, 40) forming one or more surfaces (7, 8, 41) extending along the axial direction (A1) and in that the generation system (2) is configured to exert a transverse acoustic radiation force capable of moving the objects (11, 12, 31-34, 42) in the direction of the surface(s) (7, 8, 41) of the contact structure (4, 40).

2. Device according to claim 1, wherein the contact structure comprises one or more membranes (4, 4A, 4B).

3. Device according to claim 2, wherein the cavity (1) comprises several chambers (5, 6, 5A, 5B, 6A, 6B) delimited by one or more of said membranes (4, 4A, 4B), each of the chambers (5, 6, 5A, 5B, 6A, 6B) preferably comprising a respective part of the objects (11, 12, 31-34).

4. Device according to claim 2 or 3, wherein one or more of said membranes (4, 4A, 4B) are porous.

5. Device according to any one of claims 1 to 4, wherein one or more of said surfaces (41) formed by the contact structure (40) are surfaces extending each around a respective direction (A2), this direction (A2) preferably being parallel or oblique with respect to said axial direction (Al).

6. Device according to any one of claims 1 to 5, wherein the contact structure (4, 40) comprises or forms one or more electrodes.

7. A device according to any one of claims 1 to 6, comprising elements, such as biological cells, connected to the contact structure (4, 40) so as to permit interactions between these elements and one or more of said objects (11, 12, 31-34, 42) when the latter are displaced towards the surfaces (7, 8, 41) of the contact structure (4, 40).

8. Device according to any one of claims 1 to 7, wherein the generation system (2) comprises one or more piezoelectric and / or ultrasonic transducers which may comprise several elements (2A, 2B, 2C) arranged in a matrix and / or one or more acoustic holographic lenses.

9. Device according to any one of claims 1 to 8, wherein said objects (11, 12, 31-34, 42) comprise objects exhibiting a positive acoustic contrast with respect to the fluid such that the axial acoustic radiation force moves these objects towards the pressure nodes (N1-N4) and / or objects exhibiting a negative acoustic contrast with respect to the fluid such that the axial acoustic radiation force moves these objects towards the anti-pressure nodes.

10. Method of assembling objects (11, 12, 31-34, 42) using a device according to any one of claims 1 to 9, comprising generating axial acoustic radiation force so as to position the objects (11, 12, 31-34, 42) on the pressure nodes (N1-N4) and / or antinodes and generating transverse acoustic radiation force so as to move the objects (11, 12, 31-34, 42) towards the surface(s) (7, 8, 41) of the contact structure (4, 40).

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