STRUCTURING A SET OF OBJECTS SUCH AS CELLS AND MICRON-SIZED PARTICLES USING ACOUSTIC FORCE

Abstract

A technique for moving various objects such as cells and particles of hydrogel or a compressible material, suspended in a fluid, so as to form a layered structure akin to human organ tissue. A standing sound wave is propagated through the fluid so as to position the cells on a pressure node and the particles on a pressure antinode. As such, the cells have a positive acoustic contrast relative to the fluid, while the particles have a negative acoustic contrast relative to the fluid.

Claims

1-9. (canceled)

10. A method for structuring a set of objects, comprising: arranging a fluid and said objects suspended in the fluid in a cavity, and generating a stationary acoustic wave in the cavity so as to produce an acoustic radiation force causing the objects to move in the cavity, wherein said objects comprise: first objects which have a positive acoustic contrast with respect to the fluid, and second objects which have a negative acoustic contrast with respect to the fluid, and the stationary acoustic wave is held so as to keep the objects thus moved in acoustic levitation.

11. The method according to claim 10, wherein the stationary acoustic wave generated in the cavity has a wavelength less than twice a dimension of the cavity along a direction of propagation of the stationary acoustic wave, preferably less than or equal to this dimension.

12. The method according to claim 10, comprising, after positioning the objects under the action of the acoustic radiation force, a step of introducing a substance into the cavity so as to form a matrix capable of supporting the first objects.

13. The method according to claim 12, wherein said substance comprises a hydrogel prepolymer.

14. The method according to claim 12, wherein the matrix is porous.

15. The method according to claim 12, wherein said substance comprises a photopolymerisable material, the method comprising, after introducing the substance into the cavity, a step of light stimulating the substance so as to polymerise it.

16. The method according to claim 10, comprising, after positioning the objects under the action of the acoustic radiation force, a step of incubating the objects.

17. The method according to claim 10, comprising, after positioning the objects under the action of the acoustic radiation force, a step of heating the second objects so as to melt them.

18. The method according to claim 10, comprising, after positioning the objects under the action of the acoustic radiation force, a step of encapsulating the first objects, this step comprising introducing third objects having a positive acoustic contrast with respect to the fluid into the cavity.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The following detailed description refers to the attached drawings in which:

[0058] FIG. 1 is a schematic view of a device comprising a cavity and a transducer capable of generating a stationary acoustic wave in the cavity, the cavity containing a fluid with suspended objects which are relatively homogeneously distributed in the cavity before undergoing the effects of the acoustic wave;

[0059] FIG. 2 is a schematic view of the device of FIG. 1, in which the objects have been moved by an acoustic radiation force produced by the acoustic wave so as to be aligned on a node or an antinode of this wave respectively;

[0060] FIG. 3 is a schematic view illustrating a phenomenon of diffusion between layers of cells;

[0061] FIG. 4 is a schematic view illustrating a phenomenon of development of cell extensions;

[0062] FIG. 5 is a schematic view illustrating a phenomenon of cell migration;

[0063] FIG. 6 is a schematic view of the device of FIG. 2, the objects being held in the configuration of FIG. 2 by means of a gel matrix.

DETAILED DESCRIPTION

[0064] FIGS. 1 and 2 represent a device for implementing the invention.

[0065] This device comprises, on the one hand, a container which forms a cavity 1 capable of containing a fluid and/or different substances in the form of a liquid or gel, for example.

[0066] Generally speaking, the cavity 1 extends along a direction A1, which in this example corresponds to a vertical direction. The cavity 1 has a dimension B1 along direction A1, which in this example corresponds to the height of the cavity 1.

[0067] The cavity 1 has an overall cylindrical shape. Of course, cavity 1 may have another geometry, for example a rectangular cross-section.

[0068] On the other hand, the device of FIGS. 1 and 2 comprises an acoustic wave generation system.

[0069] In this example, this system comprises a piezoelectric transducer 2 arranged at a first end of the cavity 1 along direction A1, in this case vertically below the cavity 1, as well as an acoustic reflector 3 which delimits a second end of the cavity 1 along direction A1, in this case being arranged vertically above the cavity 1.

[0070] This system is configured so as to be able to generate in cavity 1 and propagate in the fluid it contains a stationary acoustic wave 4, along a direction of propagation corresponding to direction A1.

[0071] The stationary wave 4 generated in this way can have a frequency identical to the resonant frequency of the cavity 1, which consequently forms a resonator.

[0072] Alternatively, this stationary wave 4 may have a frequency different from the resonant frequency of the cavity 1.

[0073] In any case, the system is configured to be able to generate, especially, a wave 4 having a wavelength A less than or equal to twice the height B1 of the cavity 1, in order to form at least one pressure node and at least one pressure antinode along direction A1.

[0074] In this example, the transducer 2 is a broadband transducer equipped with an ultrasound source.

[0075] Such a transducer 2 makes it possible to modify position of the node or nodes of the wave 4 along direction A1 and/or the distance between different nodes of the wave 4, by varying the frequency of this wave 4.

[0076] In the scope of the invention, the device just described, or any similar device, is implemented to position small-sized, typically micro-sized, objects within the cavity 1 in a spatial organisation determined by one or more parameters of the wave 4, in particular its frequency.

[0077] Indeed, the cavity 1 is filled with a fluid 5 and objects 6 and 7 suspended in this fluid 5.

[0078] In this non-limiting example, the objects 6 are biological cells, the fluid 5 forms a culture medium for these cells 6 and the objects 7 are polydimethylsiloxane beads.

[0079] By way of indicating purposes, each of the objects 6 and 7 is between 1 m and 100 m in size and the height B1 of the cavity 1 is several centimetres.

[0080] In this example, each of the objects 6 has a density .sub.o1 greater than the density .sub.f of the fluid 5. Conversely, each of the objects 7 has a density .sub.o1 less than the density pf of the fluid 5.

[0081] The objects 6 are also chosen so that the velocity c.sub.o1 of propagation of an acoustic wave in these objects 6 is greater than the velocity c.sub.f of propagation of this acoustic wave in the fluid 5. Conversely, the objects 7 are chosen so that the velocity c.sub.o2 of propagation of the acoustic wave in these objects 7 is less than the velocity c.sub.f of propagation of this acoustic wave in the fluid 5.

[0082] After arranging the fluid 5 in the cavity 1 and the objects 6 and 7 suspended in the fluid 5 as illustrated in FIG. 1, the transducer 2 is actuated so as to generate a stationary acoustic wave 4 in the cavity 1.

[0083] The generation of this wave 4 makes it possible to produce an acoustic radiation force which is exerted on the objects 6 and 7.

[0084] This acoustic radiation force FRA can be described in particular according to the following model, known per se, by K. Yosioka and Y. Kawasima:

[00001]$\mathrm{FRA}=\frac{}{8}{}_f{v}_0^2{\mathrm{kd}}^3{F}_y\sin \left(\mathrm{kz}\right)$ [0085] where v.sub.0 is the velocity of wave 4, k the wavenumber, F.sub.y a density-compressibility factor and z the position of the object 6 or 7 under consideration along direction A1, that is along the direction of propagation of the wave 4.

[0086] The density-compressibility factor F.sub.y can be defined as follows:

[00002]$F_y=\frac{1+\frac{2}{3}\left(1-\frac{{}_f}{{}_{\mathrm{ox}}}\right)}{2+\frac{{}_f}{{}_{\mathrm{ox}}}}-\frac{{}_f{c}_f^2}{3{}_{\mathrm{ox}}{c}_{\mathrm{ox}}^2}$ [0087] where .sub.ox is the density .sub.o1 or .sub.o2 of the object 6, or respectively 7, under consideration, and cox is the velocity c.sub.o1 or c.sub.o2 of propagation of the wave 4 within the object 6, or respectively 7, under consideration.

[0088] Given the respective density and the respective velocity of propagation of the acoustic wave of objects 6 and 7 relative to the fluid 5, objects 6 have a positive density-compressibility factor, or acoustic contrast, while objects 7 have a negative density-compressibility factor, or acoustic contrast.

[0089] In the example of FIGS. 1 and 2, the wave 4 has a wavelength equal to height B1 of the cavity 1, forming respectively along direction A1, a first node at a coordinate C1, an antinode at a coordinate C2 and a second node at a coordinate C3.

[0090] Given the aforementioned respective properties of the fluid 5 and the objects 6 and 7, starting from the configuration of FIG. 1 in which the objects 6 and 7 are relatively homogeneously distributed throughout the cavity 1, the acoustic radiation force produced by the wave 4 thus causes the objects 6 to move towards the nodes of the wave 4 and the objects 7 to move towards the antinode of the wave 4, so as to achieve a configuration such as that illustrated in FIG. 2.

[0091] The invention thus makes it possible to spatially organise the objects 6 and 7 as layers spaced along direction A1 and to keep them thus positioned in acoustic levitation, under the action of the wave 4.

[0092] In this example, the objects 7 form an intermediate layer, located halfway up the cavity 1, while the objects 6 form two layers extending on either side of the intermediate layer.

[0093] As the objects 7 are polydimethylsiloxane beads, their aggregation or pooling as a layer makes it possible to constitute a porous barrier which affords interactions to develop between the layers of cells 6, without any contact with the walls of the cavity 1.

[0094] The invention thus makes it possible to produce a cell culture with acoustic levitation.

[0095] The invention also makes it possible to control interactions between layers of cells 6, since it is especially possible to choose different materials, geometries and sizes for the objects 7, these parameters having a direct effect on the porosity of the barrier they constitute under the action of the acoustic radiation force.

[0096] By way of example, it is thus possible to trigger or afford diffusion of solutes or cell secretion product 10 (FIG. 3), the development of cellular extensions 11 of the neuronal axon type (FIG. 4), or even the migration of cells 6 (FIG. 5).

[0097] In one alternative embodiment, the objects 7 comprise hydrogel particles which, after positioning under the action of the acoustic radiation force as described below, are molten by local heating, for example using a laser sheet.

[0098] It is thus possible to constitute a continuous hydrogel layer interposed between two layers of cells 6.

[0099] The invention also makes it possible to continue cell culture, or to initiate it after positioning the objects 6 and 7 as described above, by producing a support matrix in the cavity 1.

[0100] To do this, once the objects 6 and 7 have been positioned in the configuration of FIG. 2 or in a similar configuration, a hydrogel prepolymer-based substance can be introduced into cavity 1.

[0101] Such a substance makes it possible to constitute a porous matrix 20 as a gel, making it possible to support the layers of objects 6 and 7 (FIG. 6).

[0102] In one embodiment, this substance also comprises a photopolymerisable material which, after introduction into cavity 1, is subjected to light stimulation leading to polymerisation of the matrix.

[0103] The acoustic wave 4 can then be interrupted so that cell culture occurs within such a matrix, for example by placing the container in an incubator.

[0104] In one alternative embodiment, starting from the configuration of FIG. 2, other objects (not represented) such as positive acoustic contrast hydrogel beads are introduced into the cavity 1.

[0105] Under the action of the acoustic radiation force resulting from wave 4, these beads or any other objects with positive acoustic contrast will move towards the pressure nodes so as to envelop layers formed by the objects 6.

[0106] It is thus possible to encapsulate the layers of objects 6 using a shell with a porosity controlled by the properties of the objects that form it, for example for in vivo cell therapy applications.

[0107] It follows from the above non-limiting description that the invention makes it possible to reconstruct and stimulate complex architectures including different layers of cells separated by a variety of objects making it possible to control interactions between the cell layers, using a method and a device that are particularly simple to implement.