A microfluidic chip for structuring cell aggregates by optical exclusion and acoustic levitation

Abstract

The present invention relates to a microfluidic chip, in particular for cell culture, comprising: a block (5) made of a biocompatible material; passage channels (7, 8) made in the block for the passage of cells immersed in a liquid, in particular a nutrient solution; a resonant cavity (6) made in the block, connected to the passage channels and including walls for containing the cells originating from the passage channels; a generator (12) for generating acoustic waves capable of forming at least one cell aggregate in acoustic levitation in the resonant cavity; and at least one light emitter (13, 13') capable of irradiating the cells in the resonant cavity through at least one wall of the resonant cavity and simultaneously with the generation of acoustic waves in such a way as to structure the at least one aggregate, by optical exclusion techniques.

Description

[Technical field]

[0001] The present invention relates to a microfluidic chip for manipulating objects such as microparticles or nanoparticles having a size comprised between 0.1 μm and 300 μm. More specifically, the present invention relates to the levitation of cells by application of acoustic waves. In the following, reference is mainly made to cells, but the present invention applies to any particle that is at least sensitive to acoustic waves.

[0002] In general, the principle of acoustic levitation and the formation of particle (or cell) aggregates during acoustic levitation is summarized in Figure 1. Depending on the frequency, it is possible to generate 1 to N pressure nodes between two opposing walls 1 and 2 of a channel 3. The condition to be fulfilled is h=N.λ ac / 2, where h is the height of the cavity, λ ac is the ultrasonic wavelength (sound wave). A transducer 4 is placed outside the channel in contact with the wall 2 to generate sound waves through said wall 2. The wall 1 is a reflector that reflects the sound waves in the channel 3 to generate standing waves and N pressure nodes distributed over the height of the cavity. These changes in pressure result in the creation of an acoustic radiation force field. The axial component of the acoustic radiation force thus created then promotes the formation of aggregates at each of the pressure nodes that define the acoustic levitation plane. The cells are distributed over a number of aggregates, each of which is located at a pressure node that defines the plane. The lines drawn in FIG. 1 only show the acoustic radiation forces that result in pressure nodes.

[0003] More precisely, under the effect of the acoustic radiation force, the suspended particles (or cells) are forced to move towards the acoustic pressure nodes. Once in the different acoustic levitation planes, the radial component of the acoustic radiation force forces the particles to gather together and form agglomerates at each level. The frequency of the ultrasound is set to obtain several pressure nodes distributed over the height h of the channel.

[0004] In current acoustofluidic applications, the acoustic radiation force is used to separate species as a function of their physical and mechanical properties, i.e., their density and their compressibility as well as their size, since the acoustic force depends directly on these parameters as defined by the following equation: F ac =4π.r 3 .k ac .E0.Φ.sin(2k ac .z)

[0005] where r is the radius of the particle, Φ is the acoustic contrast coefficient, and k ac is the acoustic wave number and E0 is the acoustic energy. It defines the sensitivity of a particle (or cell) to the acoustic radiation force. The denser and the larger the particle, the greater the acoustic force it experiences and the faster it moves towards the pressure node. This force is typically used to separate flowing particles or cells as a function of these characteristics.

[0006] Document WO 2019 / 002551 is known, which describes a method for separating flowing particles by applying sound waves in order to levitate them. This document also describes the principle of optical exclusion. The present invention refers to this document WO 2019 / 002551 for the method for levitating particles and the use of the principle of optical exclusion.

[0007] The object of the present invention is a novel use of the principle of acoustic levitation with optical manipulation. Another object of the invention is a device that allows optimized manipulation of the agglomerates created by acoustic levitation. Yet another object of the present invention is a novel method for producing aggregates.

[0008] At least one of the above objects is a microfluidic chip, comprising: a block made of a biocompatible material; - a passage channel made in said block for the passage of cells immersed in a liquid, in particular a nutrient solution (culture medium); a resonant cavity, for example intended for cell culture, made in the block, connected to the through-channel and including a wall for containing the cells originating from the through-channel; an acoustic wave generator capable of forming at least one cell aggregate in acoustic levitation within said resonant cavity; - Irradiating cells in the resonant cavity through at least one wall of the resonant cavity and simultaneously with the generation of acoustic waves in such a way as to structure the at least one aggregate, by the technique of optical exclusion, is achieved with a microfluidic chip comprising at least one optical emitter capable of irradiating the cells in the resonant cavity by the technique of optical exclusion.

[0009] The resonant cavity includes walls for containing the cells, in other words, the cells can enter a region within the resonant cavity where they are no longer subject to the flow in the through channel. As a result, the cells can be subjected to the action of sound and light waves. The cells can also remain immobile with respect to the flow motion in the passing channel, meaning that they are exposed to small movements of the medium but not to the linear displacement motion of the flow.

[0010] The flow rate can be adjusted such that the rate does not induce the cells to form particularly one or more aggregates, in which case the acoustic force is sufficient to oppose the flow.

[0011] In a microfluidic chip according to the invention, acoustic waves and optical beams can be used simultaneously to hold cells in levitation across one or more layers by acoustic waves while the cells are manipulated by optical beams according to the technique of optical exclusion.

[0012] The microfluidic chip according to the invention constitutes a photoacoustic bioreactor in which cells can be cultured in acoustic levitation and manipulated by specific irradiation to structure the aggregates into successive layers of cells, structuring meaning the spatial distribution of the layers of cells that differ from one another. Within the resonant cavity, the cells are immersed in a liquid that does not perturb the acoustic or optical beams used and is suitable for cell culture.

[0013] The resonant cavity is advantageously intended for the cultivation of cells in acoustic levitation. It will be clear to the skilled artisan that any micro- or nanoparticle that is intrinsically or extrinsically sensitive to sound waves and possibly certain optical beams can be used in the present invention.

[0014] It is therefore possible to spatially structure cell aggregates in acoustic levitation using both optical and acoustic properties, and this operation can be repeated several times by successively injecting different types of cells that respond to different wavelengths of light, either marked (fluorescent biological markers) or not.

[0015] It is possible to create particle agglomerates that can be held in acoustic levitation for as long as necessary by the acoustic radiation force of the sound waves, which is dimensioned to hold the agglomerates in acoustic levitation within the resonant cavity that constitutes the acoustic trap.

[0016] According to an advantageous feature of the invention, the microfluidic chip may further comprise at least one second light emitter, the two emitters being arranged to emit respectively through two opposing walls of the resonant cavity.

[0017] Preferably, the two light emitters emit at different wavelengths. Such an arrangement makes it possible to act simultaneously or sequentially on different types of cells by the technique of optical exclusion. It also makes it possible to act symmetrically on the aggregates, since there is an attenuation of the intensity of the beam passing through the thickness of the fluid and the encounter with the aggregates along the axis of the beam's propagation. Thanks to this configuration, the upper aggregates are acted on in the same way as the lower aggregates.

[0018] According to an embodiment of the present invention, the acoustic wave generator can include upper and lower elements on two opposing walls that sandwich at least a portion of a resonant cavity, the upper element being fixed or removable and being an upper transducer or an acoustic wave reflector, and the lower element being a lower transducer, and the upper and lower transducers can emit acoustic waves. Further, the upper and / or lower elements can be configured to reflect light beams provided to illuminate the cells. transparency It is.

[0019] In other words, the generator includes a lower transducer combined with a reflector capable of reflecting sound waves from the lower transducer so as to create an acoustic radiation force within the resonant cavity, except that the reflector can be replaced with an upper transducer that synchronously generates the same sound waves as the lower transducer so as to also generate an acoustic radiation force within the resonant cavity.

[0020] Preferably, at least one of the two transducers is transparency or is otherwise annular to allow optical illumination to pass through its center. The removable nature of the upper element may allow for the draining or recovery of cells that form aggregates.

[0021] According to an embodiment of the invention, the resonant cavity is closed at its lower end by a lower transducer, which thus constitutes the lower wall of the resonant cavity. In such an embodiment, the lower transducer is in direct contact with the medium. This configuration makes it possible to improve the energy efficiency of the resonant cavity.

[0022] According to an advantageous feature of the invention, the lower transducer may be arranged inside the resonant cavity. Thus, a removable lower transducer is provided that can slide inside the resonant cavity. Preferably, the dimensions of the transducer make it possible to ensure that the resonant cavity is sealed. In this case, the usable volume forming the medium is smaller than the total volume of the resonant cavity. It is therefore possible to define different volumes of the resonant cavity. The resonant cavity is thus a cavity with a variable volume and therefore a variable number of levitation planes. Advantageously, the resonant cavity may have at least one stop for blocking the head of the lower transducer once it is inserted into the resonant cavity.

[0023] According to an embodiment of the present invention, the resonant cavity is configured to receive at its lower end an acoustic wave originating from a lower transducer disposed outside the resonant cavity. transparency It can be closed by a fixed or removable film. This film can be made from polydimethylsiloxane (PDMS) material or from cyclic olefin copolymer (COC), or it can be an adhesive Polymerase Chain Reaction (PCR) film that is easily removable and allows very good transmission of ultrasound. The removable film allows for the passage or recovery of cell aggregates, for example, at the end of the culture.

[0024] Preferably, the resonant cavity is a cylinder, the side walls of which are constituted by blocks. The upper base can be formed by the reflector or the upper transducer. The lower base can be formed by a film or directly by the lower transducer. According to an embodiment, the through channel can lead to the resonant cavity at the top of the side wall of the cylinder, such an arrangement makes it possible to supply the cells to the cavity through the top of the side wall of the cylinder.

[0025] According to the invention, the resonant cavity can have a stop positioned so that the head of the lower transducer can be inserted to reduce the height of the usable volume in the resonant cavity until its height is equal to the height of the passing channel. Ideally, the available volume allows for the creation of one or more monolayer or multilayer cell aggregates.

[0026] According to an advantageous embodiment of the invention, the passage channels can be made on the upper surface of the block, and a connected or removable strip can cover all of the surface of the block, including the upper end of the resonant cavity, which strip is transparent to an optical beam provided to illuminate the cells of the resonant cavity from the outside. transparency Furthermore, if a transducer is placed on the opposite side of the strip, the strip will reflect sound waves from the transducer inside the resonant cavity.

[0027] Preferably, the through-channel and the cylinder can be arranged perpendicular to each other, so that the block can further comprise two microchannels passing through the block from one side to the other, parallel to the cylinder and connected respectively to the two free ends of the through-channel, the first microchannel intended for the arrival of cells in the through-channel and the second microchannel intended for the discharge of cells from the through-channel.

[0028] As a non-limiting example, the reflector is a strip made of glass, polymethyl methacrylate (PMMA), quartz, silicon, polydimethylsiloxane (PDMS) or cyclic polyolefin copolymer (COC). Such a strip is designed to ensure good transmission on the one hand and good reflection on the other hand. Preferably, the reflector can be designed starting from the same material as the material of the block and with an inner surface treated to reflect sound waves.

[0029] The chip according to the invention can comprise a number of microchannels, made in the thickness of the block and leading to the resonant cavity for the entry and / or exit of cells. Preferably, these microchannels are aligned with the pressure nodes provided in the resonant cavity due to the acoustic radiation force. These microchannels can be used, for example, to inject cells of different nature than those injected through the through channel, to inject nutrients and also to expel cells.

[0030] Advantageously, the height of the resonant cavity can be a function of the number of pressure nodes created and the wavelength of the sound waves generated by the generator. The resonant cavity is designed to accommodate the superposition of several cell layers, while the height of the through channels is preferably adapted to the size of the cells.

[0031] The resonant cavity is higher than the passage channel, for example several times the diameter of the cell, in particular at least ten times the diameter. It is possible to try to place the first pressure node at the level of the passage channel, thus at a channel height of the order of λ / 4.

[0032] Preferably, the through channel can have a linear shape such that the section of the through channel between the inlet end and the resonant cavity is collinear with the section of the through channel between the resonant cavity and the other end of the through channel, the outlet end.

[0033] According to an embodiment of the present invention, the blocks may be made from polydimethylsiloxane (PDMS) or cyclic olefin copolymer (COC). In particular, the block is designed starting from a gas-permeable material to facilitate gas exchange between the culture medium, the contents of the resonant cavity and, if necessary, the outside. Channels are etched or molded into the block and can be placed in an incubator to ensure optimal culture conditions in terms of gas and temperature.

[0034] In particular, a resonant cavity can be dimensioned in height greater than the diameter of the through channel leading to it, the through channel being thus distinct from the resonant cavity, where the through channel is a small diameter conduit typically suitable for carrying cells only in single file, and the resonant cavity has a larger dimension capable of accommodating cell aggregates in one plane and in one volume. As a non-limiting example, the resonant cavity can have a diameter of 1 to 50 mm, the height of the resonant cavity is comprised between 5 and 15 mm, and the height of the through channel is equal to 450 μm.

[0035] The microfluidic chip according to the invention may further comprise at least one additional microchannel made in the block in the same plane as the through-channel: this is a simplified arrangement allowing to feed and discharge cells to and from the resonant cavity, where they can form aggregates in the same plane as the cells originating from the through-channel, which is in fact the main microchannel. This additional microchannel can also be used to inject other cells, biomarkers, or alternatively to wash out the medium or to withdraw product cells during their culture.

[0036] According to another aspect of the invention, a method is proposed for manipulating cells in acoustic levitation in a microfluidic chip as defined above, said method comprising the following steps: - injecting cells into the resonant cavity via the inlet of the passage channel; - generating acoustic waves for acoustically levitating the injected cells so as to form cell aggregates in at least one layer; - at least one phase step in which the cells are irradiated with a specific light wavelength whilst simultaneously maintaining the acoustic waves so as to manipulate the cells according to the optical exclusion principle.

[0037] According to the present invention, the selective photoacoustic fluidic exclusion principle is used, which explains the fact that some particles or cells respond to certain light wavelengths, while other particles or cells do not respond at all.

[0038] According to an advantageous embodiment of the invention, the injected cells have different properties and the step of generating and irradiating acoustic waves can be carried out as follows. - generating sound waves to acoustically levitate the injected cells and at the same time applying a light beam at a specific wavelength, allowing the principle of elimination of cells sensitive to this wavelength, so that only cells that are not sensitive to this light wavelength form aggregates in one layer; - Obtaining radially structured aggregates by maintaining the acoustic waves and stopping the light beam so that cells sensitive to the wavelength of the light beam now form aggregates around the already formed aggregates.

[0039] The invention thus makes it possible to create radially structured monolayer cell aggregates, which may therefore consist of continuous rings of different cells, thus allowing the co-culture of cells, an essential step in the formation of organoids. By cell aggregate is meant a layer of cells that meets all of the following characteristics: at least two cells contained in the layer, in particular at least 10%, better 25%, preferably 50% of the cells contained in the layer are in contact, and the layer has a continuum of cells over at least a portion of its length and between displacements in at least one of its lateral dimensions.

[0040] According to an advantageous embodiment of the invention, a three-dimensional structure formed of several layers of aggregates is produced by carrying out the following steps: - the injecting step includes injecting cells into the resonant cavity via one or more inlets; The step of generating acoustic waves further includes generating acoustic waves for acoustically levitating multiple aggregates of injected cells at multiple levels, the levels being acoustic pressure nodes, the number of acoustic pressure nodes depending on the wavelength of the acoustic waves and the height of the resonant cavity. The invention allows the creation of several monolayers (cell sheets) one above the other, which results in considerable time savings for cell culture, since it is no longer necessary to trypsinize the sheet of cells to separate it from a possible solid substrate with the aim of later depositing it on another. This process is more particularly suitable for Mesenchymal Stromal Cell (MSC) type stem cell culture, in which the cells are cultured to produce cell sheets that are then superimposed, but it is relevant for any type of cell culture with the aim of forming cell sheets. With the present invention it is possible to envisage the production in acoustic levitation of spheroids, organoids or tuberoids which can be maintained and cultured in acoustic levitation.

[0041] The present invention allows cell culture to be carried out while the resulting aggregates are kept immobile in acoustic levitation for the duration of the culture.

[0042] Such a system thus made it possible to culture MSCs in acoustic levitation for 18 days. At the end of this culture, the cells were alive and could grow normally when placed on a substrate. Thus, the system has been validated for short-term (hours) and long-term (weeks) cultures. This is possible thanks to the porosity of the material used, which allows the flow of nutrients and the exchange of gases inside the incubator. To allow the culture of cells, the acoustic bioreactor was placed inside the incubator. The compactness of the system makes it possible to place several chips inside the incubator. It is therefore possible to carry out the culture of several sheets or spheroids in parallel in acoustic levitation (several dozens per well and several wells in the incubator). [Brief description of the drawings]

[0043] Other characteristics and advantages of the invention will become apparent upon reading the detailed description of realizations and embodiments, which are by no means limiting, in the light of the attached figures, in which: [Figure 1] FIG. 1 is a schematic diagram of the creation of a pressure node in a channel by acoustic levitation according to the prior art. [Diagram 2] 1 is a schematic exploded view of an example of a device according to the invention; [Diagram 3] FIG. 2 shows an example of a device according to the invention viewed from below. [Figure 4] FIG. 2 is a schematic cross-sectional view along the longitudinal axis of a passing channel with an optical emitter and an acoustic transducer disposed outside the resonant cavity according to the present invention. [Diagram 5]1 is a schematic cross-sectional view along the longitudinal axis of a passage channel with an optical emitter and an acoustic transducer closing a resonant cavity at its base according to the invention; [Figure 6] FIG. 2 is a schematic cross-sectional view along the longitudinal axis of a passing channel with an optical emitter and an acoustic transducer disposed inside a resonant cavity according to the present invention. [Figure 7] 1 is a schematic cross-sectional view along the longitudinal axis of a passing channel with a light emitter and an acoustic transducer arranged inside the resonant cavity at the top stop, according to the invention; FIG. [Figure 8] A schematic cross-sectional view along the longitudinal axis of a passage channel arranged within the thickness of a block, with an optical emitter and an acoustic transducer closing a resonant cavity at its base, according to the present invention. [Figure 9] FIG. 9 is a diagram of the device from FIG. 8 with two light emitters and an acoustic transducer according to the invention. [Figure 10] 1 is a schematic diagram of a transducer designed starting from a material transparent to an optical beam, according to the present invention, which is provided to realize the exclusion principle. [Figure 11] 1 is a schematic diagram of a transducer having a ring shape according to the present invention; [Figure 12] 1 is a schematic cross-sectional view along the longitudinal axis of a passing channel with two light emitters and a transducer having the shape of a hollow cylinder arranged around a resonant cavity according to the invention; [Figure 13] FIG. 13 is a schematic cross-sectional view of a block adapted for the device of FIGS. 2-12 with multiple microchannels etched into the block leading to a resonant cavity, in accordance with the present invention. [Figure 14] FIG. 1 is a schematic cross-sectional view of a radially structured cell aggregate according to the present invention. [Figure 15] FIG. 1 is a schematic cross-sectional view of several radially and laterally structured cell aggregates according to the present invention. [Figure 16]1 is a curve showing the emission rate of two types of cells as a function of wavelength. [Figure 17] 1 is a curve showing the release rate of several different types of cells having different sizes as a function of wavelength. [Figure 18] 1 is a curve showing the release rate of red blood cells as a function of wavelength. [Figure 19] 1 is a curve showing the release rate of red blood cells as a function of wavelength. [Figure 20] 1 is a curve showing the release rate of leukocytes as a function of wavelength. [Figure 21] 2 shows two photographs showing the formation of white particle agglomerates starting from a mixture of white polystyrene 10 μm particles and 3 μm red particles. [Figure 22] Four photographs taken at different times during the formation of "purified" aggregates during acoustic levitation by optical exclusion of red blood cells. [Diagram 23] 13A-13C are three photographs showing the optical exclusion effect applied to a multi-node cavity. [Figure 24] Shown is a layered ring structure containing 15 μm diameter particles of red polystyrene and 40 μm particles of white polystyrene. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] The embodiments described below are in no way limiting. Variations of the present invention can be realized, in particular, including only a selection of features described below in isolation from other described features (if this selection of features is sufficient to provide a technical advantage or to differentiate the present invention over the state of the art). This selection includes at least one preferably functional feature without structural details or with only a part of structural details (if this part is sufficient to provide a technical advantage or to differentiate the present invention over the state of the art). In particular, all variants and all embodiments described are offered to be combined together in all combinations which are not objectionable from a technical point of view. In the figures, elements that are common to several figures retain the same references.

[0045] Although the invention is not limited thereto, a microfluidic chip suitable for culturing cell aggregates in acoustic levitation is described herein. An example set of components of a microfluidic chip according to the invention is represented in figure 2. A block 5 can be seen, made for example from PDMS material, with a parallelepiped shape and with an upper and lower surface. A cylindrical resonant cavity 6 is made in the centre of this block, spanning the entire thickness of the block 5 between the two surfaces. The purpose of this resonant cavity 6 is to trap cells by acoustic forces.

[0046] For the supply of the resonant cavity 6, through channels 7, 8 are etched into the upper surface of the block 5 so that the through channels and the inside of the cavity are accessible. The through channels have a first part 7 intended for the entry of cells from an inlet microchannel 9 towards the resonant cavity. The through channels also have a second part 8 intended for the exit of cells from the resonant cavity 6 towards an outlet microchannel 10. The two inlet and outlet microchannels are made in the thickness of the block as for the resonant cavity and lead to the lower surface of the block 6, making the back of this block more accessible to various devices for the supply of the microfluidic chip according to the invention and for the management of the microfluidic chip according to the invention.

[0047] Preferably, this block is designed starting from a biocompatible material that can ensure gas exchange between the resonant cavity and the outside (incubator) as required. The block is designed so that the microfluidic chip according to the invention can ensure nutrient and medium flow in the resonant cavity as required. Of course, the block makes it possible to inject cells and to eject them, and to create aggregates of large dimensions in the resonant cavity. The microfluidic chip is mounted inside an incubator to ensure optimal culture conditions (gas and temperature).

[0048] One can also see a glass strip 11 intended to cover the upper surface of the block 5, in particular by plasma bonding, which may be a strip manufactured together with the block 5. This glass strip 11 has at least its inner wall, facing the resonant cavity, and on the opposite side a transducer 12, the inner surface of which is capable of reflecting the sound waves from the transducer 12, on the side of the lower surface of the block 6. Advantageously, the glass strip is transparent to the optical beam from an optical emitter 13 arranged above the strip 11. transparency It is.

[0049] The strip 11 can be designed starting from one or a combination of the following materials to ensure good transmission on the one hand and good reflection on the other hand: glass, PMMA, quartz, silicon, COC, PDMS. The transducer 12 consists of a stainless steel cylinder containing a piezoelectric element, whose operating frequency can be selected as a function of the height of the resonant cavity: this frequency can be chosen between 0.1 MHz and 10 MHz for resonant cavities whose thickness (height) can vary from a few mm to a few tens of μm.

[0050] Figure 3 shows the block 5 seen from above. The ends of the microchannels 9 and 10 can be seen at each end of the passing channels 7, 8. This passing channel has a shape that widens from the microchannel towards the resonant cavity so as to ensure good progression of the cells when entering and when exiting. The passing channel can have, for example, a (circular, rectangular, square or other) cross section with a height of 1 to 30 mm. As will be seen later, stops 14 and 15 are placed on the inner wall of the resonant cavity and make it possible to define a diameter at the upper level of the resonant cavity of about 5 to 30 mm. Other inlets / outlets (not shown) of microchannels types 9, 10 and through-channels types 7, 8 can be made to supply the same or different cells, biomarkers to the resonant cavity, or alternatively to wash the medium or to harvest product cells during their cultivation.

[0051] An embodiment is shown in FIG. 4 in which the lower base of the resonant cavity is closed by an impermeable film 16. Such a configuration makes it possible to prevent any contamination of the medium by the transducer 12 placed outside the cavity. This film 16 may be removable so as to make it possible to drain any agglomerates formed in the resonant cavity. Ideally, this film is impermeable to the sound waves provided to generate an acoustic radiation force in the cavity. transparency The film can be made from PDMS, COC or adhesive PCR film. The flow of cells 17 and the formation of aggregates 18 from cells trapped in the resonant cavity 6 under the action of acoustic waves emitted by the transducer 12 are shown in FIG.

[0052] In an embodiment such as that seen in Fig. 5, for example, the packaged transducer 12 can be in direct contact with the medium in the resonant cavity. In this case, there is no transparent wall, only a glass strip 11, which is reflective, is placed on the opposite side of the transducer 12. This configuration makes it possible to improve the energy efficiency of the resonant cavity by omitting a transparent layer, such as the film 16 in Fig. 4, through which energy can be dissipated.

[0053] Figure 6 shows an embodiment in which a transducer 12 is inserted into the resonant cavity 6. Such an embodiment makes it possible to define different volumes as a function of the intended application. The effective volume of the resonant cavity is then variable. Stops 14 and 15 can be placed at different places on the inner wall of the resonant cavity to position the head of the transducer in a given position. In figure 7 these stops are placed at the top of the resonant cavity.

[0054] An embodiment is shown in Fig. 8, in which the through channels 7, 8 are made entirely in the thickness of the block 5 and lead to the resonant cavity at a mid-level, rather than at its top. Of course, this embodiment is compatible with the various configurations presented above, i.e., for example, with the transducer inside or outside the cavity. However, if the transducer is inside, it must remain below the entrance of the channel 7 to the resonant cavity. In addition to cell culture, the production of spatially structured organoids or spheroids with distinct layers of cells is advantageously provided. The microfluidic chip according to the invention makes it possible in particular to inject particles or cells into the resonant cavity and thus to create cell aggregates within the cavity, which allows cell culture over long periods of time by providing the necessary medium for the aggregated cells during acoustic levitation. The present invention also makes it possible to create complex structured layers of cells, which may be useful from the point of view of tissue engineering. To do this, emitter 13 is used to irradiate the cells with specific wavelengths to perform the technique of optical exclusion.

[0055] A mixture of the two types of particles or cells can be injected, which under the action of sound waves form aggregates that mix the two species. When it is desired to spatially organize the aggregates, in particular when it is desired to structure the aggregates into circular and concentric successive layers, it is possible to use the optical exclusion principle.

[0056] The optical exclusion principle makes it possible to release particles or cells in acoustic levitation under the effect of optical illumination at a given wavelength suitable for the cells or particles that one wishes to exclude. This effect depends on the optical absorption properties of the particles / cells. Cells marked with a fluorescent marker also respond to illumination at the absorption wavelength of the fluorescent marker, see in particular Figures 16-20.

[0057] The invention allows the formation of two-dimensional aggregates structured in a plane by successive bands at the periphery of the aggregate. Radially structured aggregates 25 as shown in Figure 14 are thus obtained. This allows the production of sheets composed of circular bands of cells of various nature, which may prove useful for the production of complex organoids. To do this, a mixture of two cells C1 and C2 is injected, one of which receives a given light wavelength λ opt1 The other absorbs light at wavelength λ and the other does not. In this case, the aggregates are easily structured. In fact, the aggregated regions are opt1 When the laser is irradiated at wavelength λ, this prevents C1 from agglomerating under the effect of the acoustic force. Thus, the C2 species forms the first aggregate. Then, the laser is irradiated at wavelength λ so that the C1 species forms aggregates around the first aggregate. opt1 It is sufficient to stop irradiation at

[0058] It is therefore possible to spatially structure cell aggregates in acoustic levitation using both optical and acoustic properties, and this operation can be repeated several times by successively injecting different types of cells, marked or not, that respond to different light wavelengths.

[0059] In Fig. 9, two optical emitters 13 and 13' are positioned to illuminate and / or observe cells simultaneously from either side of the resonant cavity, thus allowing for the simultaneous exclusion of two species from the acoustic concentration region.

[0060] To do this, as can be seen in Figure 10, transparency In practice, a piezoelectric transducer (PZT) 19 is used that actually passes the provided optical wavelengths. Permeability It is possible to fabricate PZT and thus not be limited to single illumination from only one side. An (unpackaged) annular transducer (PZT) can also be used, in which case it is possible to illuminate through a ring and thus couple two light sources simultaneously.

[0061] Double illumination can also be carried out in the embodiment from figure 12, in which a transducer 24 having the shape of a hollow cylinder is arranged around the resonant cavity. In this case, a film 21 is provided, which forms a seal at the base of the resonant cavity and which is directed against the optical beam provided. transparency In this case, the two emitters are free to irradiate the cells inside the resonant cavity.

[0062] Another advantage of culturing in levitation is that it is possible to create multiple pressure nodes in the cavity, and thus to form levitation cell aggregates above and below. As shown in FIG. 13, in a cavity with a height of a few mm, for example 6 mm, several nodes can be created, for example about 15 (or several tens). To do this, multiple inlet microchannels 22 can be made, opposite multiple outlet microchannels 23. Each pair of inlet / outlet microchannels can be provided in the plane of a pressure node. However, the placement of cells in levitation across multiple pressure nodes can also be performed with a single inlet channel, such as a through channel.

[0063] It is possible to use the optical exclusion effect on some cell aggregates in acoustic levitation and thus structure objects within the volume of the resonant cavity. The aggregation area is therefore centered on the axis of the transducer. It is then possible to structure several superimposed aggregates 26 in the volume, as shown in Figure 15, each aggregate being itself composed of several circular bands (annular structuring) of different cells. This makes it possible to envisage the production in acoustic levitation of spheroids, organoids or tuberoids that can be kept and cultured in acoustic levitation. Creating several monolayers above and below represents a considerable time saving for cell culture.

[0064] The present invention therefore proposes a new means for cell culture, suitable to replace the conventional techniques of cell culture on solid substrates. Indeed, in the case of conventional cell culture, cells, such as stem cells for example, grow on a solid substrate, but also migrate in order to return and come into contact with other cells. The culture is considered to be finished when the cells reach "confluency", i.e. when they form a layer of cells (single layer of cells) that are in contact with each other and thus are a "monolayer". The present invention relates to the design of a photoacoustic bioreactor in which cells can be cultured in acoustic levitation and cell aggregates can be manipulated and structured by specific irradiation to form structured aggregates.

[0065] The inventors have determined that the photoacoustofluid effect increases the emission velocity V ej It has been shown that the levitational velocity of the objects according to the invention can be quantified by the wavelength of the levitational signal, which includes showing that the objects leave the illuminated area at a speed that is in particular a function of the wavelength of the levitational signal. These objects are microparticles or nanoparticles sensitive to the wavelength used, with a size comprised between 0.1 μm and 300 μm. In general, the release rate can be measured for various types of particles as a function of the light wavelength, the intensity of the irradiation, and the magnification of the microscope objective. These parameters make it possible to control the power of the irradiation. It can be particularly seen, for example in FIG. 18, that the size of the particles also plays an important role: the smaller the particles, the faster the release rate.

[0066] Figure 16 shows the emission rate of a sample or particle as a function of wavelength. Two populations of particles are formed, one red and one blue, with a diameter of 15 μm. The curves show that the emission rate depends on the light wavelength, but also on the color of the particles. Red particles are emitted from the illuminated area for several light wavelengths from 365 nm to 770 nm, while blue particles are not emitted. In this example, the acoustic frequency is f for an amplitude A = 13 Vpp. ac =0.650MHz.

[0067] In FIG. 17, the following samples were analyzed under the same conditions as in FIG. -3μm green fluorescent particles, -3μm red fluorescent particles, -10μm green fluorescent particles, -10μm red fluorescent particles, -15μm red particles. It is observed that the size of the sample affects the release rate. It is also observed that the red particles have a much higher release rate than the fluorescent particles.

[0068] The curve in Figure 19 relates to the variation of the release rate of red blood cells (RBC) (at a hemodilution of 1:1000) as a function of wavelength. All experiments were carried out at a frequency of 751 kHz and an amplitude of 8 V, and the analyzed blood aggregates were placed in the center of the cavity. It is observed that the emission rate of RBCs is very large from 365 nm to about 490 nm, then from 515 nm to 600 nm, above which the red blood cells remain clumped.

[0069] FIG. 20 relates to a curve showing the release rate of leukocytes isolated from a whole blood sample derived from a healthy patient and marked with DAPI and CD45 (λexc=415 nm, λemission=500 nm) and with acoustic parameters of 751 kHz and 8 V. The maximum release rate is λ opt = 385 nm. The other two wavelengths to which the marked GB responds are λ opt = 405 and 460 nm. GB does not respond to other wavelengths.

[0070] The table below shows the photoacoustofluidic response of various cells. [Table 1]

[0071] Figure 21 shows the formation of white particle aggregates starting from a mixture of 10 μm white polystyrene particles and 3 μm red particles. The mixture is subjected to an acoustic force and irradiated at 460 nm (the wavelength to which the red particles respond) with 60% of the maximum power and a magnification of 10x. The acoustic frequency is 1.91 MHz with an amplitude of 9 V. The flow rate is 0.15 ml / h. The formation of aggregates of white particles is observed, accompanied by few trapped red particles. The left image corresponds to the 5 min time point, while the right image shows the aggregates formed after 20 min. Thus, the white particles are gradually concentrated with the optical exclusion of the red particles.

[0072] The photographs in Figure 22 relate to the various stages of forming "purified" aggregates in acoustic levitation by optical displacement of a mixture of cells with a flow. The experiment shows the displacement of red blood cells by applying a signal at a wavelength that allows this displacement.

[0073] The cells include red blood cells ( / 100) and MDA cancer cells ( / 100). Illumination is obtained with a 460 nm signal at 80% of maximum power, with a magnification of 10x. The acoustic frequency is 1.59 MHz with an amplitude of 10 V. The flow rate is 0.15 ml / h.

[0074] Figure 23 shows the optical exclusion effect applied to a multi-node cavity. In the left picture, we can see the agglomerates of red particles during acoustic levitation in a microfluidic chip. The agglomerates form a continuous layer in the shape of a filled circle. In the middle picture, lighting is applied, reaching the first layer first. It starts by clearing particles from the central area to form a crown. Starting from the bottom (close to the lighting), the layers turn into crowns one after the other, thus freeing up a light passage for the upper layers each time. In the photo on the right, the layer has almost entirely been transformed into a crown. The aggregates are formed with 15 μm particles of red polystyrene. Illumination is obtained with a 460 nm signal at 60% of maximum power with a magnification of 10 times. The acoustic frequency is 1.91 MHz with an amplitude of 9 V. Illumination sequence: 460 off-on, i.e. first the aggregates are formed in white light (460 off), followed by illumination with 460 nm (on) to form a crown of particles in acoustic levitation.

[0075] In Fig. 24 the layered ring structure can be seen. The mixture of particles includes red polystyrene particles with a diameter of 15 μm and white polystyrene particles with a diameter of 40 μm. Illumination is obtained with a 460 nm signal at 60% of the maximum power with a magnification of 10 times. The acoustic frequency is 1.91 MHz with an amplitude of 9 V. Illumination sequence: 460 on-off, i.e. acoustic and optical are activated simultaneously, which expels the red particles from the center. Then the illumination at 460 nm is switched off, which allows the red particles to form aggregates on the central aggregate.

Claims

1. A microfluidic chip capable of carrying out the manipulation of cells and / or the structuring and / or culturing of said cells, - a block (5) made of a biocompatible material, - passage channels (7, 8) made in said block for the passage of cells immersed in a liquid; a resonant cavity (6) made in said block (5), connected to said through-channel and including a wall for containing said cells originating from said through-channel, so that said cells are no longer subject to the flow in said through-channel; - an acoustic wave generator (12) capable of forming at least one cell aggregate (18, 25) in acoustic levitation within said resonant cavity; - at least one optical emitter (13) capable of irradiating cells in the resonant cavity through at least one wall of the resonant cavity and simultaneously with the generation of acoustic waves in such a way as to structure the at least one cell aggregate, by a technique that optically excludes a portion of the cells, so that only the cells that are not sensitive to the irradiation form aggregates in a layer.

2. 2. The chip according to claim 1, further comprising at least one second light emitter (13'), the two light emitters being arranged to emit through two opposite walls of the resonant cavity (6), respectively.

3. The two light emitters emit light at different wavelengths (λ ｏｐｔ １ , λ ｏｐｔ ２ 3. The chip of claim 2, wherein the chip emits at 100 .mu.m.

4. 4. The chip according to claim 1, wherein the acoustic generator comprises upper and lower elements on two opposing walls that sandwich at least a part of the resonant cavity, the upper element being fixed or removable and being an upper transducer or an acoustic reflector (11) and the lower element being a lower transducer (12), the upper and lower transducers being capable of emitting the acoustic waves, and the upper and / or lower elements being transparent to a light beam provided to illuminate the cells.

5. 5. The chip according to claim 4, characterized in that the resonant cavity (6) is closed at its lower end by the lower transducer (12).

6. The chip according to claim 4, characterized in that the lower transducer (12) is arranged inside the resonant cavity (6).

7. 7. The chip according to claim 6, characterized in that the resonant cavity (6) has at least one stop (14, 15) for blocking the head of the lower transducer once it has been inserted into the resonant cavity.

8. The chip according to any one of claims 4 to 7, characterized in that the lower transducer and / or the upper transducer are designed from a material (19) that allows the passage of an optical beam provided for irradiating the cells from outside the resonant cavity.

9. The chip according to any one of claims 4 to 8, characterized in that the lower transducer and / or the upper transducer have the shape of a ring (20) and allow an optical beam provided for irradiating the cells from at least outside the resonant cavity to pass inside the ring.

10. The chip according to any one of claims 1 to 4, characterized in that the resonant cavity is closed at its lower end by a fixed or removable film (16) that is transparent to the sound waves originating from the lower transducer arranged outside the resonant cavity.

11. The chip according to any one of claims 1 to 3, characterized in that the acoustic wave generator is a transducer (24) in the shape of a hollow cylinder arranged externally around the resonant cavity or forming a side wall of the resonant cavity, the upper and / or lower walls of the cavity being made of a material that is transparent to an optical beam provided to irradiate the cells from outside the cavity.

12. Chip according to any one of claims 1 to 11, characterized in that the resonant cavity (6) is a cylinder, the side walls of which are constituted by the blocks.

13. The chip of claim 12, wherein the through channel opens into the resonant cavity at an upper end of the cylindrical side wall.

14. The chip described in claim 13, characterized in that the resonant cavity has a stop positioned so that the head of the lower transducer (12) can be inserted to reduce the height of the usable volume in the resonant cavity until the height of the usable volume is equal to the height of the passing channel.

15. The chip according to any one of claims 1 to 14, characterized in that the passage channels (7, 8) are made on the upper surface of the block, and a connected or removable strip (11) covers all of the surface of the block, including the upper end of the resonant cavity, the strip being transparent to an optical beam provided to illuminate the cells in the resonant cavity from the outside, and the strip acts as a reflector to reflect the sound waves from the transducer inside the resonant cavity when a transducer is placed on the opposite side.

16. The passage channels (7, 8) are made on the upper surface of the block, and a connected or removable strip (11) covers all of the surface of the block including the upper end of the resonant cavity, the strip being transparent to an optical beam provided to irradiate the cells in the resonant cavity from the outside, and the strip acts as a reflector to reflect the sound waves from the transducer inside the resonant cavity when a transducer is placed on the opposite side; the through channels (7, 8) and the cylinder are arranged perpendicular to each other, and said block further comprises inside two microchannels (9, 10) passing from one side to the other, parallel to said cylinder (6) and connected respectively to the two free ends of said through-channels (7, 8); 14. The chip according to claim 12 or 13, characterized in that a first microchannel (9) is intended for the arrival of cells into the passage channel and a second microchannel (10) is intended for the egress of cells from the passage channel.

17. 5. The chip according to claim 4, characterized in that the acoustic reflector (11) is a strip made of glass, polymethylmethacrylate (PMMA), quartz, silicon, polydimethylsiloxane (PDMS) or cyclic olefin copolymer (COC).

18. 5. The chip according to claim 4, characterized in that the sound wave reflector (11) is designed starting from the same material as the material of the block and that the sound wave reflector (11) has an inner surface that is treated to reflect sound waves.

19. A chip according to any one of claims 1 to 18, characterized in that it comprises a plurality of microchannels (22, 23) made in the thickness of the block and leading to the resonant cavity for the entry and / or exit of cells.

20. The chip according to any one of claims 1 to 19, characterized in that the height of the resonant cavity is a function of the number of pressure nodes created and the wavelength of the acoustic waves generated by the acoustic wave generator.

21. Chip according to any one of the preceding claims, characterized in that the block (5) is made from polydimethylsiloxane (PDMS) or from cyclic olefin copolymer (COC).

22. The chip according to any one of the preceding claims, characterized in that the resonant cavity is dimensioned with a height greater than the diameter of the through channel leading to the resonant cavity.

23. Chip according to any one of the preceding claims, characterized in that the resonant cavity (6) has a diameter between 1 and 50 mm, the height of the resonant cavity is comprised between 5 and 15 mm and the height of the through channels is equal to 450 μm.

24. The chip according to any one of claims 1 to 23, further comprising at least one additional microchannel made in the block in the same plane as the through-channel.

25. A method for manipulating cells in acoustic levitation in a microfluidic chip according to any one of claims 1 to 24, said method comprising: - injecting cells into the resonant cavity (6) through the inlets of the passage channels (7, 8); - generating acoustic waves for acoustically levitating the injected cells so as to form cell aggregates (18) in at least one layer; - at least one phase of irradiating said cells while simultaneously maintaining said acoustic waves so as to manipulate said cells according to the optical exclusion principle.

26. 26. The method of claim 25, wherein the injected cells each have different properties, the steps of generating and irradiating acoustic waves comprising: - generating sound waves to acoustically levitate the injected cells, and at the same time applying a light beam with a wavelength allowing said principle of elimination of a portion of the cells, so that only the cells that are not sensitive to said wavelengths form aggregates in one layer, - maintaining the acoustic waves and stopping the light beam, and allowing the cells sensitive to the wavelength of the light beam to form subsequent aggregates around the aggregates already formed, thereby obtaining radially structured aggregates.

27. 27. A method according to claim 25 or 26 for producing a three-dimensional structure formed of a plurality of layers of aggregates, comprising the steps of: - the step of injecting comprises injecting cells into the resonant cavity via one or more inlets; - a method wherein the step of generating acoustic waves further comprises generating acoustic waves for acoustically levitating multiple aggregates of injected cells at multiple levels, the levels being acoustic pressure nodes, the number of acoustic pressure nodes being a function of the wavelength of the acoustic waves and the height of the resonant cavity.

28. A method according to any one of claims 25 to 27, further comprising the step of carrying out cell culture whilst holding the obtained aggregate or aggregates motionless in acoustic levitation for the duration of the culture.

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