Structuring of a set of objects of the cell and micrometric particle type by acoustic force

Abstract

The invention relates to a technique for moving various objects, such as cells (6) and particles (7) of hydrogel or a compressible material, suspended in a fluid (5), to form a layered structure resembling human organ tissue. A standing acoustic wave (4) is propagated through the fluid (5) to position the cells (6) at a pressure node and the particles (7) at a pressure antinode. As a result, the cells (6) have a positive acoustic contrast with the fluid (5), while the particles (7) have a negative acoustic contrast with the fluid (5). Figure for the abstract: Fig. 2

Description

Title of the invention: Structuring of a set of objects of the cell and micrometric particle type by acoustic force technical field

[0001] The invention relates to the field of biotechnology and in particular to the structuring of cellular assemblies, 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, consists of structuring cells by acoustic levitation: 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 makes it possible to structure cells in layers with the aim of establishing connections between cells of different layers, but does not, however, allow for satisfactory control of the development of such connections.

[0006] In general, the techniques known in this field are complex and expensive, may require significant amounts of time to structure and culture cells and, insofar as they are implemented in vitro, may lead to the death of a large number of cells. Description of the invention

[0007] In order to overcome the aforementioned drawbacks, a method for structuring a set of objects is proposed within the scope of the present invention, comprising: - a step of arranging a fluid and said objects suspended in the fluid within a cavity, and - a step of generating a stationary acoustic wave in the cavity of in order to produce an acoustic radiation force causing the objects within the cavity to move, said objects comprising: - the first objects that exhibit a positive acoustic contrast with the fluid, and - second objects that present a negative acoustic contrast with respect to the fluid.

[0008] The propagation of a stationary acoustic wave in the cavity allows the formation in the cavity, along the direction of propagation, of one or more nodes, that is to say places where the pressure of the fluid is zero, and one or more antinodes or antinodes, that is to say places where this pressure is maximum.

[0009] The first objects, exhibiting a positive acoustic contrast, i.e., a positive density-compressibility factor relative to the fluid, will be transported by the acoustic radiation force towards a pressure node. The second objects, exhibiting a negative acoustic contrast, or density-compressibility factor, will be transported by the acoustic radiation force towards a pressure antinode.

[0010] The invention thus makes it possible to form in the cavity one or more aggregates of first objects and one or more aggregates of second objects in the form of layers, or sheets, which follow one another along the direction of propagation, 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.

[0011] Such a layered structure is similar to the structure of human organ tissues, which typically comprise layers of cells separated by layers of an extracellular matrix. For example, epithelia, particularly cardiac, pulmonary, or endothelial epithelia, may comprise differentiated or undifferentiated layers that, in a number of cases, rest on basal laminae of a protein nature, such as epithelial or muscle cells. As another example, the blood-brain barrier or the cerebral parenchyma typically comprises layers of interconnected neurons.

[0012] Moreover, the invention makes it possible to maintain in acoustic levitation the whole of the objects thus structured, under the action of the stationary acoustic wave whose generation can be maintained for the required duration, for example several hours or days, in order to promote interactions between objects when they are alive, in particular when they are formed by biological cells.

[0013] The invention thus makes it possible to carry out cell culture in acoustic levitation, by controlling the permeability and consequently the development of connections and interactions between layers of cells forming the first objects, by a choice of second objects forming one or more layers of chosen porosity.

[0014] This innovative approach also makes it possible to limit the contact of objects with walls or surfaces, thus preserving their mechanical and functional integrity.

[0015] In the present description, an "object" means a living or inert element preferably having a small size compared to the length of the acoustic wave generated in the cavity.

[0016] By way of non-limiting example, the objects can typically have a micrometric size, for example between 1 pm and 100 or several hundred pm.

[0017] Thus, in a preferred but by no means limiting embodiment, said first objects are living elements such as biological cells, for example of the eukaryotic or prokaryotic type.

[0018] These second objects may themselves be inert elements such as particles comprising a hydrogel, for example based on collagen, gelatin, or even fibrin and extracellular matrix protein. As a non-limiting alternative, the second objects may comprise a compressible elastomer, for example polydimethylsiloxane.

[0019] The invention can however be implemented with objects of different sizes, i.e. located outside the aforementioned range.

[0020] Thus, the objects or some of them may have a size of less than 1 pm, for example being formed by bacteria or viruses, and / or have a size of several hundred pm.

[0021] The objects, in particular the first objects, or some of them, may be multicellular elements or artificially formed objects or objects taken from an organ.

[0022] 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.

[0023] The invention thus provides a simple solution for reconstituting artificial tissues for research purposes or in the context of cell therapies.

[0024] 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.

[0025] The simplicity and precision of this technique are due in particular to the limited number of control parameters, namely the respective density and speed of sound wave propagation of the fluid and the objects, as well as the speed and frequency of the acoustic wave.

[0026] Of course, many variations can be implemented on the basis of the principle described above.

[0027] For example, other objects can be injected into the fluid after positioning the first and second objects, so as to form, for example, additional or complementary aggregates.

[0028] In a particular embodiment, the standing acoustic wave generated in the cavity has a wavelength less than twice a dimension of the cavity along a direction of propagation of the standing acoustic wave.

[0029] Preferably, this wavelength is less than or equal to this dimension.

[0030] It is preferred that the standing acoustic wave has at least one antinode and at least one or two nodes.

[0031] This allows, in the context of a preferred embodiment variant, the formation of two layers of first objects separated by a layer of second objects.

[0032] In particular, in the context of such a variant, the use of hydrogel or compressible elastomer to form the second objects makes it possible to constitute a porous intermediate layer, allowing the development of interactions between the layers of first objects extending on either side of this intermediate layer, when the first objects include living cells.

[0033] The invention not only makes it possible to carry out cell culture in acoustic levitation but also, alternatively or complementaryly, to initiate or continue such a process by maintaining the objects in position using a matrix.

[0034] In particular, the method may include, 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.

[0035] This substance is preferably a biocompatible active substance promoting the phase change of the medium constituted by the fluid.

[0036] This substance may include a hydrogel prepolymer or another element suitable for forming a matrix in the form of a gel.

[0037] This substance may include a catalyst and / or a photoinitiator.

[0038] A gel-like matrix allows objects to be adequately held in position in space while being elastically deformable.

[0039] Furthermore, it is preferred that the matrix be porous, whether in the form of a gel or in another form.

[0040] The porosity of the matrix makes it permeable and perfusable, so as to allow the development of cellular connections.

[0041] In one embodiment, said substance comprises a photopolymerizable material, the process comprising, after introduction of the substance into the cavity, a step of light stimulation of the substance so as to polymerize it.

[0042] The invention thus makes it possible to sculpt a support matrix for the structured set of objects, in particular the first objects.

[0043] In the context of the different embodiment variants that have just been described, the process may include, after positioning the objects under the action of the acoustic radiation force, an incubation step of the objects.

[0044] For example, the cavity and its contents can be placed in an incubator for this purpose.

[0045] Incubation promotes the differentiation, self-organization and maturation of cell layers.

[0046] In one embodiment, the process includes, after positioning the objects under the action of the acoustic radiation force, a step of heating the second objects so as to fuse them.

[0047] The heating step can be carried out using a laser sheet.

[0048] When a support matrix such as described above is used, such a heating step is preferably carried out before the formation of this matrix.

[0049] In one embodiment, the process includes, after positioning the objects under the action of the acoustic radiation force, a step of encapsulating the first objects.

[0050] Preferably, this encapsulation step includes an introduction into the cavity of third objects exhibiting a positive acoustic contrast with respect to the fluid.

[0051] The third objects can thus be transported by the force of acoustic radiation to a pressure node to form a protective shell around the first objects that are there.

[0052] By way of example, the third objects may include hydrogel beads or another material enabling the formation of a porous protective shell.

[0053] The encapsulation step is preferably implemented, but not necessarily, when no use is made of a support matrix.

[0054] The invention can also be implemented for cell therapy purposes, for example by in vivo injection of a culture or proto-culture carried out using the principles described in this document.

[0055] Other advantages and features of the invention will become apparent from the following detailed, non-limiting description. Brief description of the drawings

[0056] The following detailed description refers to the attached drawings on which:

[0057] [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 that are distributed relatively homogeneously in the cavity before undergoing the effects of the acoustic wave;

[0058] [Fig.2] is a schematic view of the device of [Fig.1], in which the objects were displaced by an acoustic radiation force produced by the acoustic wave so as to be respectively aligned on a node or an antinode of this wave;

[0059] [Fig.3] is a schematic view illustrating a diffusion phenomenon between cell layers;

[0060] [Fig.4] is a schematic view illustrating a phenomenon of development of cell extensions;

[0061] [Fig.5] is a schematic view illustrating a cell migration phenomenon;

[0062] [Fig.6] is a schematic view of the device in [Fig.2], the objects being held in the configuration of [Fig.2] using a gel matrix. Detailed description of implementation methods

[0063] Figures 1 and 2 show a device for implementing the invention.

[0064] This device includes on the one hand a container which forms a cavity 1 suitable for containing a fluid and / or different substances in the form of, for example, liquid or gel.

[0065] Generally, the cavity 1 extends along a direction Al, which in this example corresponds to a vertical direction. The cavity 1 has a dimension B1 along the direction Al, which in this example corresponds to a height of the cavity 1.

[0066] The cavity 1 here has an overall cylindrical shape. Of course, the cavity 1 can have another geometry, for example a rectangular cross-section.

[0067] On the other hand, the device in Figures 1 and 2 includes an acoustic wave generation system.

[0068] In this example, this system includes a piezoelectric transducer 2 disposed at a first end of the cavity 1 in the direction Al, in this case vertically below the cavity 1, and an acoustic reflector 3 which delimits a second end of the cavity 1 in the direction Al, in this case disposed vertically above the cavity 1.

[0069] This system is configured to be able to generate in the cavity 1 and propagate in the fluid it contains a standing acoustic wave 4, along a propagation direction that corresponds to the AL direction

[0070] The standing wave 4 thus generated can have a frequency identical to the resonance frequency of the cavity 1, which consequently forms a resonator.

[0071] Alternatively, this standing wave 4 may have a different frequency from the resonance frequency of cavity 1.

[0072] In all cases, the system is configured to be able to generate, in particular, a wave 4 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 belly.

[0073] In this example, transducer 2 is a broadband transducer equipped with an ultrasonic source.

[0074] Such a transducer 2 makes it possible to modify the position of the node(s) of the wave 4 along the direction Al and / or the distance between different nodes of the wave 4, by playing on the frequency of this wave 4.

[0075] In the context of the invention, the device just described, or any similar device, is implemented to position small objects, typically of micrometric size, within the cavity 1 according to a spatial organization determined by one or more parameters of Fonde 4, in particular its frequency.

[0076] For this purpose, the cavity 1 is filled with a fluid 5 and with objects 6 and 7 suspended in this fluid 5.

[0077] 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 polydi-methylsiloxane beads.

[0078] For the sake of indication, each of the objects 6 and 7 has a size between 1 pm and 100 pm and the height B1 of the cavity 1 is several centimeters.

[0079] In this example, each of the objects 6 has a density P^ greater than the density Pf of the fluid 5. Conversely, each of the objects 7 has a density Po\ less than the density Pf of the fluid 5.

[0080] The objects 6 are further chosen such that the speed Fa of propagation of an acoustic wave in these objects 6 is greater than the speed Cf of propagation of this acoustic wave in the fluid 5. Conversely, the objects 7 are chosen such that the speed c<>2 of propagation of the acoustic wave in these objects 7 is less than the speed Cf of propagation of this acoustic wave in the fluid 5.

[0081] After arranging the fluid 5 in the cavity 1 and the objects 6 and 7 suspended in the fluid 5 in the manner illustrated in [Fig.1], the transducer 2 is actuated so as to generate a standing acoustic wave 4 in the cavity 1.

[0082] The generation of this wave 4 makes it possible to produce an acoustic radiation force which is exerted on objects 6 and 7.

[0083] This acoustic radiation force FRA can in particular be described according to the following model, known per se, of K. Yosioka and Y. Kawasima:

[0084] [Math.l] FRA = fpf yg k d3 Fy sin( / c

[0085] where vo is the velocity of Fonde 4, & the wave number, F y a density-compressibility factor and z the position of the object 6 or 7 considered along the direction Al, that is, along the direction of wave propagation 4.

[0086] The density-compressibility factor Fy can be defined as follows:

[0087] [Math.2]

[0088] where Pox is the density P»] or Po2 of the object 6, or respectively 7, considered, and is the propagation speed or Fa of the wave 4 within the object 6, or respectively 7, considered.

[0089] Considering the respective density and the respective propagation speed of acoustic background of objects 6 and 7 with respect to the fluid 5, objects 6 exhibit a positive density-compressibility factor, or acoustic contrast, while objects 7 exhibit a negative density-compressibility factor, or acoustic contrast.

[0090] In the example of figures 1 and 2, the wave 4 has a wavelength 2 equal to the height B1 of the cavity 1, forming respectively along the direction Al a first node at a coordinate Cl, an antinode at a coordinate C2 and a second node at a coordinate C3.

[0091] Given the aforementioned respective properties of the fluid 5 and the objects 6 and 7, from the configuration of [Fig.1] in which the objects 6 and 7 are distributed relatively homogeneously throughout the cavity 1, the acoustic radiation force produced by the wave 4 thus causes a displacement of the objects 6 towards the nodes of the wave 4 and a displacement of the objects 7 towards the antinode of the wave 4, so as to reach a configuration such as that illustrated in [Fig.2].

[0092] The invention thus makes it possible to spatially organize the objects 6 and 7 in the form of spaced layers along the direction Al and to keep them thus positioned in acoustic levitation, under the action of the wave 4.

[0093] 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.

[0094] Since objects 7 are polydimethylsiloxane beads, their aggregation or grouping in the form of a layer allows the formation of a porous barrier which permits the development of interactions between the layers of cells 6, without contact with the walls of the cavity 1.

[0095] The invention thus makes it possible to carry out a cell culture in acoustic levitation.

[0096] The invention also makes it possible to control the interactions between cell layers 6 since it is possible to choose different materials, geometries and sizes for objects 7, these parameters have a direct impact on the porosity of the barrier that they constitute under the action of the acoustic radiation force.

[0097] As an example, it is thus possible to trigger or allow the diffusion of solutes or secretions 10 from cells ([Fig.3]), the development of cellular extensions 11 of the neuronal axon type ([Fig.4]), or the migration of cells 6 ([Fig.5]).

[0098] In one embodiment, the objects 7 comprise hydrogel particles which, after positioning under the action of the acoustic radiation force as described below, are fused by local heating, for example using a laser sheet.

[0099] It is thus possible to constitute a continuous layer of hydrogel interposed between two layers of cells 6.

[0100] The invention also allows cell culture to continue, or to be initiated after positioning of objects 6 and 7 in the manner described above, by producing in cavity 1 a support matrix.

[0101] To do this, once objects 6 and 7 are positioned according to the configuration of [Fig.2] or according to a similar configuration, a hydrogel prepolymer-based substance can be introduced into cavity 1.

[0102] Such a substance makes it possible to constitute a porous matrix 20 in the form of a gel, making it possible to support the layers of objects 6 and 7 ([Fig.6]).

[0103] In one embodiment, this substance also includes a photopolymerizable material which, after introduction into the cavity 1, is subjected to light stimulation leading to polymerization of the matrix.

[0104] 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.

[0105] In an alternative embodiment, starting from the configuration of [Fig.2], other objects (not shown) such as hydrogel beads with positive acoustic contrast are introduced into cavity 1.

[0106] Under the action of the acoustic radiation force resulting from wave 4, these balls or any other objects with positive acoustic contrast will move towards the pressure nodes so as to envelop the layers formed by the objects 6.

[0107] It is thus possible to encapsulate the layers of objects 6 using a shell with porosity controlled by the properties of the objects that form it, for example for in vivo cell therapy applications.

[0108] It follows from the preceding non-limiting description that the invention makes it possible to reconstruct and stimulate complex architectures comprising different layers of cells separated by a variety of objects allowing the interactions between the cell layers to be controlled, using a particularly efficient method and device simple to implement.

Claims

Demands

1. A method for structuring a set of objects (6,7), comprising: - a step of arranging a fluid (5) and said objects (6, 7) suspended in the fluid (5) in a cavity (1), and - a step of generating a standing acoustic wave (4) in the cavity (1) so as to produce an acoustic radiation force causing a displacement of the objects (6, 7) in the cavity (1), characterized in that said objects (6, 7) comprise: - first objects (6) which have a positive acoustic contrast with respect to the fluid (5), and - second objects (7) which have a negative acoustic contrast with respect to the fluid (5), and in that the standing acoustic wave (4) is maintained so as to keep the objects (6, 7) thus displaced in acoustic levitation.

2. Method according to claim 1, wherein Stationary acoustic background (4) generated in cavity (1) has a wavelength less than twice a dimension (Bl) of cavity (1) along a propagation direction (Al) of Stationary acoustic background (4), preferably less than or equal to that dimension (Bl).

3. Method according to claim 1 or 2, comprising, after positioning the objects (6, 7) under Faction of the acoustic radiation force, a step of introducing into the cavity (1) a substance so as to form a matrix (20) suitable for supporting the first objects (6).

4. A method according to claim 3, wherein said substance comprises a hydrogel prepolymer.

5. Method according to claim 3 or 4, wherein the matrix (20) is porous.

6. A method according to any one of claims 3 to 5, wherein said substance comprises a photopolymerizable material, the method comprising, after introduction of the substance into the cavity (1), a step of light stimulation of the substance so as to polymerize it.

7. A method according to any one of claims 1 to 6, comprising, after positioning the objects (6, 7) under Faction of the acoustic radiation force, an incubation step of the objects (6, 7).

8. A method according to any one of claims 1 to 7, comprising, after positioning the objects (6, 7) under the action of the acoustic radiation force, a step of heating the second objects (7) so as to fuse them.

9. A method according to any one of claims 1 to 8, comprising, after positioning the objects (6, 7) under the action of the acoustic radiation force, a step of encapsulating the first objects (6), this step comprising an introduction into the cavity (1) of third objects having a positive acoustic contrast with the fluid (5).

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