Microfluidic device for studying and / or manipulating a study medium and / or at least one micro-object.

Abstract

Microfluidic device for studying and / or manipulating a study medium and / or at least one micro-object.The invention relates to a microfluidic device (10) comprising: - a support (20) having a substrate (25) made of an elastically deformable material, - a study chamber (30, 30a, 30b) extending into the support (20) having at least one upper wall (32) formed at least partially by the substrate (25), at least one lower wall (34) and at least two lateral walls (33), - at least one deformation chamber (40, 40a, 40b) of the substrate formed at least partially by the substrate (25), fluidically independent of the study chamber (30, 30a, 30b) and extending at least partially into the support (20) and at least partially along one of the lateral walls (33) of the study chamber (30), the device being configured such that the deformation of the deformation chamber (40, 40a, 40b) generates the application of a stress mechanics of at least part of the upper wall (32) in the study chamber (30). Figure for the abstract: Fig. 1.

Description

Title of the invention: Microfluidic device for studying and / or manipulating a study medium and / or at least one micro-object.

[0001] The present invention relates to a microfluidic device for studying and / or manipulating a study medium and / or a micro-object in a study chamber having at least one elastically deformable upper wall. The invention also relates to a method for studying and / or manipulating at least one micro-object using the microfluidic device. technical field

[0002] Various methods now exist for mechanically manipulating and characterizing micro-objects, particularly individual cells or cell aggregates. These methods are notably used in the field of mechanobiology. For example, they are used to characterize mechanical properties or to analyze the reactions of objects to mechanical stimuli. Previous technique

[0003] Known methods in this field include, for example, micropipette aspiration, atomic force microscopy, and compression using parallel plates moved relative to each other mechanically. Numerous devices have been used for two-dimensional studies. Such devices only allow the characterization of a single object at a time and are complicated to implement. Furthermore, optical analysis, particularly in the case of two parallel plates, can be complex.

[0004] Moreover, there are now many types of microfluidic devices that allow different operations to be carried out, including the control or manipulation of small volume fluids, typically on the scale of microlitres or less, and / or of small objects, typically on the scale of millimetres or less, in a fluid.

[0005] A microfluidic device comprising a chamber having a central compression zone of reduced height with a rigid upper wall and a peripheral zone that can be pressurized or depressurized so as to control the raising or lowering of the rigid wall in the central zone is known in particular from the article by Y.-J. Liu et al., “Confinement and Low Adhesion Induce Fast Amoeboid Migration of Slow Mesenchymal Cells,” Cell, vol. 160, no. 4, pp. 659–672, Feb. 2015, doi: 10.1016 / j.cell.2015.01.007. This makes it possible, in particular, to confine cells in compartments that form when the rigid wall is in the lowered position. Such a device is described only for the study of single cells and requires good dispersion of cells in the central zone. Furthermore, the fluidic continuity between the central zone and the peripheral zone does not allow the use of such a device with a fluid flow in the chamber nor to multiplex several chambers.

[0006] A microfluidic device comprising a receiving chamber for a material to be mechanically stimulated and a row of compression chambers separated from the receiving chamber by an elastic membrane is known from international patent WO2020084148. The compression chambers are pressurized independently of each other and are connected by a chamber separator. In this device, the receiving chamber is deformed by the direct deformation of the membrane by the compression chambers. A positive pressure in a compression chamber leads to a reduction in the volume of the compression chamber by deformation of the membrane towards the receiving chamber, and vice versa. Such a device, due to the presence of a thin elastic membrane between the receiving chamber and the compression chambers, is complicated to manufacture and fragile. Furthermore, the achievable compression range is limited.Furthermore, the deformation is localized in regions close to the elastic membrane and is not transmitted to objects further along the microchannel.

[0007] There is therefore a need for a microfluidic device that is simple to manufacture, inexpensive, easy to use and has a wide range of applications allowing the application of a mechanical action on an object in order in particular to determine its mechanical properties or its response to a mechanical stimulus.

[0008] In particular, there is a need for devices that can be adapted to the study of complex and heterogeneous biological materials, such as cancerous tissues, which are known to have distinct mechanical properties and may in particular be made up of several cell types, or biopsy samples from fibrous tissue, which exhibits anisotropy without mechanical response.

[0009] In this regard, there is a particular need, especially in the pharmaceutical or diagnostic field, for devices capable of characterizing a heterogeneous structure, such as a biopsy.

[0010] There is also a particular need for devices suitable for high-speed and / or multiplex operation.

[0011] The invention is intended to satisfy these needs. Description of the invention

[0012] The invention addresses this need by means of a microfluidic device comprising: • a support comprising a substrate made of an elastically deformable material, • a study chamber extending into the support having at least one upper wall formed at least partially, preferably totally, by the substrate, at least one bottom wall and at least two side walls, • at least one support deformation chamber formed at least partially by the substrate, fluidly independent of the study chamber and extending at least partially into the support at least partially along one of the side walls of the study chamber, the device being configured so that the deformation of the deformation chamber generates the application of a mechanical stress of at least a part of the top wall of the study chamber into the study chamber, in particular by deformation of the top wall.

[0013] Spatial concepts such as "superior" or "lateral" are to be understood in relation to each other. It is understood that the device can be oriented in all directions and that there exists an orientation of the device having the orientation characteristics described above.

[0014] By "in an elastically deformable material," it is understood that the substrate has a reversible deformation capacity upon application of a force. Preferably, the Young's modulus of the substrate is less than or equal to 1 GPa, better less than or equal to 0.1 GPa.

[0015] The fact that the study chamber and the deformation chamber are arranged at least partially laterally to each other facilitates manufacturing and reduces costs. The use of a simple mold is possible. There is no need for a thin membrane that could weaken the structure or complicate its manufacture.

[0016] In the invention, deformation of the deformation chamber is possible because it is formed at least partially by the elastically deformable substrate. Such deformation of the deformation chamber generates local strain stresses in the substrate volume, particularly at the upper wall of the study chamber formed by the substrate, and results in the application of a local stress on the upper wall of the study chamber. The stress on the upper wall of the study chamber is therefore obtained indirectly by the deformation of the deformation chamber and depends on the local strain stresses in the substrate generated by the deformation of the deformation chamber.

[0017] Such a device allows the study and / or manipulation of a fluid, gel, and / or micro-object in the study chamber by mechanical action of the upper wall of the study chamber. This is simple to implement and inexpensive, achieved by simply deforming the chamber. Furthermore, the accessible dimensions of the study chamber allow for a large number of objects from a sample to be held simultaneously, thus enabling the study or manipulation of several micro-objects at the same time, whether they are two-dimensional micro-objects such as single cells or three-dimensional micro-objects such as aggregates.

[0018] Lateral positioning also allows easy observation of the study chamber from above or below perpendicular to the upper wall and observation of the effect of stress on a fluid, gel and / or micro-object in this axis, in particular by microscopy and the size of the device is also particularly suited to such use.

[0019] The simple structure of this device allows for great adaptability of the device for a large number of applications, in particular by simple choice of shapes, structures and dimensions of the study and deformation chamber, by the precision of control of the stress generated by the upper wall, in particular by the control of the pressure applied in the deformation chamber, by the adaptability of a study medium for direct or indirect stimulation of fluids, gels and / or micro-objects and by the simplicity of use of the device.

[0020] The term "pressure" here refers specifically to gauge pressure, that is, pressure relative to ambient air pressure. Thus, a pressure of -600 mbar indicates that the pressure in the pressure chamber is 600 mbar below ambient air pressure, for example, 600 mbar below atmospheric pressure (1.01321 bar). In general, ambient air pressure can be atmospheric pressure. However, the device can operate in a pressurized environment, such as a high-pressure room, in which the ambient pressure is, for example, approximately twice atmospheric pressure (2.02642 bar). The term "pressure" here can refer to both positive and negative pressures. The term "positive pressure" refers to a pressure greater than ambient pressure. Similarly, the term "negative pressure" refers to a pressure less than ambient pressure.It is clear to those skilled in the art that the absolute value of the vacuum cannot exceed the ambient pressure. It is also clear to those skilled in the art that different pressures will be selected for different materials; that is, the stiffer the material, the higher the pressure may be required to achieve a desired material deformation (and vice versa). For example, for a material with a stiffness comparable to PDMS, the pressure can be chosen in the range of +1000 to -1000 mbar (relative to ambient pressure), or better yet, in the range of -100 to -800 mbar.

[0021] Such a device allows the application of mechanical stimulus(s) to a study medium and / or one or more micro-objects. Such a device can generate mechanical stimuli, for example, one or more sequences of mechanical stimuli. Mechanical stress

[0022] In certain embodiments, the device is configured so that the deformation of the deformation chamber generates a deformation of the upper wall of the study chamber in the direction of an increase in volume or a decrease of the volume of the study chamber, the mechanical stress exerted by the upper wall being a compressive stress in the direction of a decrease in the volume of the study chamber, or a relaxation stress in the direction of an increase in the volume of the study chamber, said deformation of the upper wall generating the application of a mechanical stress in the study chamber.

[0023] Preferably, the device is configured such that a reduction in the volume of the deformation chamber, in particular by applying negative pressure in the deformation chamber, generates a deformation of the upper wall of the study chamber in the direction of a reduction in volume, in particular a reduction in height, of the study chamber, and such that an increase in the volume of the deformation chamber, in particular by applying positive pressure in the deformation chamber, generates a deformation of the upper wall of the study chamber towards an increase in volume, in particular an increase in height of the study chamber. The upper wall can take on a curved shape in at least one cross-section, preferably in at least two orthogonal cross-sections, when the deformation chamber is deformed.The curved shape can be convex towards the study chamber, reducing the volume of the deformation chamber, and concave towards the study chamber, increasing the volume of the deformation chamber. Such deformation facilitates implementation because the volume reduction of the study chamber is achieved by applying negative pressure, which is easier to maintain in a microfluidic device. Applying positive pressure can more easily cause the support to break, particularly at the junction between several layers, such as a rigid plate and the substrate. Direct deformation by a deformation chamber, as described in the aforementioned prior art, would have the opposite effect, as in this case, an increase in the volume of the deformation chamber generates a deformation of the study chamber in the direction of a volume reduction.

[0024] Preferably, the device is configured so that the side walls of the study chamber do not deform by more than 5%, better than 3%, or better still, do not deform substantially. This allows for deformation only along the height, which is more easily controlled and more efficient because, as explained previously, lateral deformation would be in the opposite direction to the deformation of the upper wall.

[0025] Preferably, the device is configured so that the deformation of the upper wall of the study chamber towards the interior of the study chamber, at a pressure in the deformation chamber between -100 and -800 mbar, is greater than or equal to 10%, better greater than or equal to 20%, even better greater than or equal to 40%, even better greater than or equal to 60% of the height of the study chamber.

[0026] In certain embodiments, the device is configured to exert a force, preferably a compressive force, on the contents of the study chamber, in particular on the study medium and / or one or more micro-objects. Such a force can generate a stimulus on the study medium and / or the micro-object(s). Control unit

[0027] Preferably, the device includes a deformation control unit for the deformation chamber, in particular by applying positive or negative pressure in the deformation chamber, fluidly connected to the deformation chamber, in particular by a channel. The deformation control unit can be configured to apply negative pressure in the substrate's deformation chamber. The control unit can be a programmable pressure source.

[0028] The channel connecting the control unit to the deformation chamber can extend at least partially into the substrate.

[0029] The deformation chamber can be filled with a fluid, the control unit being a controller of the quantity of fluid in the deformation chamber in the case of a deformation chamber with a single inlet or a controller of flow inlet and / or outlet for a deformation chamber having one inlet and one outlet of fluid.

[0030] Preferably, the deformation chamber is filled with a gas, and the gas pressure in the deformation chamber is controlled by the control unit, the control unit being a gas pressure controller for the deformation chamber. Alternatively, the deformation chamber is filled with a liquid, and the amount of liquid in the deformation chamber is controlled by the control unit; the pressure in the deformation chamber depends on the amount of liquid present in the deformation chamber or on the inlet and outlet flows of the deformation chamber. Study chamber

[0031] The device may include a supply channel for the study chamber, in particular opening to the outside, to supply the study chamber with a study medium and / or an outlet channel for the study chamber to extract the study medium from the study chamber.

[0032] The study chamber may include one or more capillary traps in the form of raised or recessed reliefs extending on one of the internal walls, in particular on the upper or lower wall.

[0033] The device may include an additional chamber upstream and / or downstream of the study chamber, fluidly connected to the study chamber. The additional chamber may be taller than the study chamber.

[0034] Without pressure in the deformation chamber, the study chamber can have a substantially polygonal cross-section, in particular rectangular.

[0035] Preferably, the study chamber has a ratio of its greatest width at the base to its height greater than or equal to 5, better greater than or equal to 8, even better greater than or equal to 10.

[0036] The study chamber may have a constant height in section.

[0037] The height of the study chamber can be constant along its length. Alternatively, it can vary. Such a variation in length can allow micro-objects to be classified according to their size or to guide and facilitate the movement of micro-objects from one end of the study chamber to the other.

[0038] Preferably, the height of the study chamber is less than or equal to 5 mm, better less than or equal to 1 mm, even better less than or equal to 500 pm, even better less than or equal to 200 pm.

[0039] Preferably, the width of the study chamber is less than or equal to 5 cm, better less than or equal to 3 cm, even better less than or equal to 1 cm, even better less than or equal to 3 mm.

[0040] The width of the study chamber can be constant along its entire length or vary. A variation in width can be used to guide micro-objects within the study chamber or to classify them according to their size. Deformation chamber

[0041] Preferably, the deformation chamber extends into the support opposite a plane defined by one of the side walls of the test chamber over at least a portion of the test chamber's height. The deformation chamber may extend opposite one of the side walls to a height greater than or equal to 30%, preferably 50%, even better 70%, or even better 80%, of the maximum height of the deformation chamber. The deformation chamber may extend opposite one of the side walls of the test chamber along its entire height. Having the greater part of the deformation chamber extend opposite one of the side walls facilitates manufacturing and improves the deformation in the direction described above. Indeed, too great an extension above the test chamber would impair the desired deformation described above.

[0042] Preferably, the deformation chamber extends over a height greater than or equal to that of the study chamber in the substrate, the height ratio preferably being greater than or equal to 5, better greater than or equal to 10. Such a height ratio allows for good deformation capacity of the upper wall of the study chamber.

[0043] Preferably, the deformation chamber has an extension along the upper wall of the study chamber above the latter with a width less than or equal to 50%, better 40%, even better 30%, even better 20% of the width of the study chamber or does not have such an extension.

[0044] Preferably, the smallest substrate thickness between the study chamber and the deformation chamber is greater than or equal to the height of the study chamber. Preferably, the smallest substrate thickness between the study chamber and the deformation chamber is less than or equal to 100 times the height of the study chamber, more preferably less than or equal to 50 times the height of the study chamber, more preferably less than or equal to 40 times the height of the study chamber, and even more preferably less than or equal to 20 times the height of the study chamber. Such a thickness limits the direct deformation of the study chamber by the deformation chamber, which would be contrary to the desired outcome, as explained above.

[0045] The width of the deformation chamber may be less than or equal to 5 cm, better less than or equal to 3 cm, even better less than or equal to 1 cm.

[0046] The height of the deformation chamber may be less than or equal to 1 cm, preferably less than or equal to 5 mm.

[0047] The deformation chamber can be substantially in the shape of an inverted trapezoid, the base of the deformation chamber being formed by the short side of the trapezoid.

[0048] Preferably, the deformation chamber contains air. Alternatively, it may contain a liquid, and the deformation of the deformation chamber may depend on the liquid pressure within the deformation chamber, as explained previously. Support

[0049] Preferably, the substrate is at least partially transparent, better totally transparent.

[0050] The support may include a rigid base in contact with the substrate. Preferably, the rigid base is at least partially transparent, better still totally transparent.

[0051] The base may be made of glass or a transparent plastic material. The rigid base may be flat. Alternatively, the base may be formed from a well or a multi-well plate.

[0052] According to one embodiment, the rigid base forms at least one wall of the deformation chamber. According to a non-mutually exclusive embodiment, the rigid base forms at least one wall of the study chamber. According to a non-mutually exclusive embodiment, the rigid base forms at least one wall of both the deformation chamber and the study chamber.

[0053] The deformation chamber and the study chamber can be completely delimited by the substrate and the rigid base. Preferably, they are formed within the substrate and closed at their base by the flat rigid base. Such a structure allows for easy fabrication of the fluidic device by manufacturing the substrate and the chambers within the substrate, in particular by molding, 3D printing, or any other technique, and then fixing the substrate onto the rigid base to close the chambers.

[0054] Alternatively, they are formed partially by the substrate and by recessed surfaces in the rigid base. Alternatively still, they are formed entirely in the substrate.

[0055] The substrate comprises an elastomer, in particular selected from PDMS, Flexdym™, latex, rubber, and any other elastomer and mixtures thereof. The substrate may comprise a crosslinking agent.

[0056] The substrate may be homogeneous throughout its volume. Alternatively, the substrate may have areas of different densities or voids within its mass, particularly in the upper wall. This can allow control of the deformation of the substrate and therefore of the upper wall according to a predetermined deformation. Analysis system

[0057] The microfluidic device may include, or be functionally coupled to, an analysis system configured to analyze the microfluidic device, in particular the study chamber, including the study medium or the micro-object(s) within the study chamber. The analysis system may be functionally coupled to the deformation control unit of the deformation chamber; in particular, the analysis system may provide an analysis signal to the control unit. The control unit may command the deformation of the deformation chamber(s) based on the analysis signal.

[0058] The analysis system in the study chamber may include an optical imaging system, in particular an optical or electron microscope, or a confocal imaging system, or a system for measuring electrical properties, in particular impedance, the conductivity of the study medium, or the electrical action of a cell unit during mechanical stimulation. Preferably, the analysis system is arranged on or below the study chamber and is configured to take images of the fluid, gel, and / or one or more micro-objects in the study chamber. The analysis system may be configured to detect static or dynamic information of the fluid, gel, and / or one or more micro-objects in the study chamber. Plurality of deformation chambers

[0059] In some embodiments, the device comprises at least two substrate deformation chambers formed at least partially by the substrate, independent of the study chamber and extending at least partially each along a lateral wall of the study chamber.

[0060] In some embodiments, two deformation chambers extend on either side of the study chamber.

[0061] The two deformation chambers of the support can be configured to extend over the upper wall of the study chamber by less than 90%, better to less than 80%, even better to less than 70%, even better to less than 50%, of the upper wall of the study chamber, and even better not to have any super- position with the study room.

[0062] In some embodiments, two deformation chambers extend along the study chamber.

[0063] In certain embodiments, two deformation chambers extend along the study chamber and are spaced such that their stress application areas in the substrate are disjoint at a zone on the upper wall of the study chamber. This allows for a non-uniform and controllable stress in the study chamber via the upper wall.

[0064] In certain embodiments, two deformation chambers extend along the study chamber and are spaced apart by such a distance that their stress application field in the substrate overlaps at least partially at the upper wall of the study chamber.

[0065] The deformation chambers may be of identical or different shape and volume.

[0066] In certain embodiments, two deformation chambers are controlled at the same pressure by the same deformation control unit, which controls them simultaneously. Preferably, the two deformation chambers are identical and arranged symmetrically with respect to the chamber under study. Thus, the deformation.

[0067] In certain embodiments, two deformation chambers are controlled independently of each other, in particular by being linked to two different deformation control units or by being controlled differently by a single deformation control unit. Such chambers may be located along the same study chamber. Such deformation chambers can allow control of the pressure and / or deformation of the upper wall according to a complex displacement.

[0068] The device may comprise a plurality of independent study chambers or, preferably, fluidly connected chambers in series or parallel. The stress through the upper wall in each study chamber, in particular the upper wall deformation, is generated by the same deformation chamber(s) or by separate deformation chambers, whether fluidly connected or not. In the case of separate deformation chambers, they may be identical. Alternatively, they may have different shapes so as to generate different upper wall deformations at the same pressure.

[0069] A person skilled in the art understands from the above that the possibilities for combining and arranging deformation chambers and study chambers are numerous and a person skilled in the art will be able to adapt the structure of the microfluidic device to the application he wishes to implement.

[0070] The microfluidic device can be symmetrical with respect to a median plane transverse to the support. Study environment

[0071] The study chamber may contain a fluid, in particular liquid, or gelled study medium. In the case of a gel, it preferably has a lower Young's modulus than the substrate. Such a gel allows the transfer of deformation from the upper wall to one or more micro-objects extending within the gel.

[0072] The gel may be a hydrogel, in particular chosen from the group including agarose, collagen, agar gum, Matrigel™, gelatin, so-called "cross-linked" gels such as Polyethylene Glycol (PEG), or other types of hydrogels.

[0073] According to some embodiments, the study chamber comprises a study medium distinct from a gel, in particular distinct from a hydrogel.

[0074] The gel may be homogeneous in the study chamber. Alternatively, the gel may have a variable density within the volume of the study chamber, in particular, it may exhibit a density gradient from the center of the study chamber towards the side walls.

[0075] The gel can completely fill the study chamber, or alternatively can also consist of islands separated from each other which are positioned randomly or in a well controlled manner in the plane of the study chamber.

[0076] The device can be configured so that the deformation of the upper wall of the study chamber generates a flow of the study medium from an inlet to an outlet within the study chamber, thus acting like a diaphragm pump. In this case, the study chamber can substantially have a portion of a substantially frustoconical shape, with the deformation chamber extending along a lateral wall on the side of the larger base of the frustoconical shape. The greatest deformation of the upper wall generated by the deformation of the deformation chamber is closer to the larger base than to the smaller base. Thus, the study medium is pumped from the larger base to the smaller base. Micro-object

[0077] The device may include at least one micro-object, or better yet a plurality of micro-objects in the study chamber, particularly in the study environment.

[0078] Preferably, the micro-object(s) are deformable.

[0079] In some embodiments, the height of at least one micro-object is less than 100 pm. In some embodiments, the height of at least one micro-object is greater than 100 pm.

[0080] In certain embodiments, the height of at least one micro-object is between 100 pm and 1000 pm; for example, between 100 pm and 900 pm, for example, between 100 pm and 800 pm, for example, between 100 pm and 700 pm, for example between 100 pm and 600 pm, for example between 100 pm and 500 pm, for example between 100 pm and 400 pm, for example between 100 pm and 300 pm, for example between 100 pm and 200 pm in height.

[0081] In some embodiments, the height of the micro-object(s) is greater than or equal to the height of the study chamber.

[0082] In some embodiments, the height of the micro-object(s) is less than the height of the study chamber. Preferably, in this case, the study medium includes a gelling agent.

[0083] The micro-object(s) may be chosen from microdroplets, gelled microunits, including those comprising a hydrogel and which may be in the form of gelled microdroplets, and / or any biological material, such as cellular units, including cells or cellular aggregates, such as spheroids or organoids, or cells or cell aggregates from a biopsy of patient tissue, or any type of culture medium.

[0084] The microdroplets or gelled microunits may contain biological material, such as a cellular unit, in particular a cell, a cellular aggregate, such as a spheroid or an organoid, or biopsy material from a patient. In the case of several gelled microunits in the study chamber, the gelled microunits may have different rigidities.

[0085] According to a particular embodiment, the micro-object(s) comprise or consist of biological material, and / or a medium suitable for the culture of biological material such as any culture medium capable of comprising one or more nutrients (for example, proteins, peptides, amino acids, and / or any carbon source).

[0086] The term “biological material” may refer to any type of cell, including embryonic or non-embryonic, as well as any type of extract, lysate, fraction of one or more cells, and in particular of human, animal or plant origin.

[0087] The term “culture medium” is likely to refer to any physiologically acceptable medium suitable for culturing one or more cell types, in particular any medium comprising at least one source of carbon and energy; in particular nutrients (for example peptides, polypeptides and / or amino acids, carbohydrates, essential metals and minerals, and buffering agents).

[0088] According to certain embodiments, said biological material comprises or consists of living, quiescent and / or cultured cells.

[0089] By way of non-exhaustive notice, said biological material may comprise or consist of one or more types of cells, selected from eukaryotic cells, pro- karyotes, or even viruses or viral particles.

[0090] According to certain particular embodiments, said biological material may comprise or consist of eukaryotic cells, such as mammalian cells, in particular human or non-human; for example, from humans, mice, rats, dogs, cats, cows, pigs, chickens, goats, horses, yeasts or others.

[0091] According to certain particular embodiments, said biological material may comprise or consist of non-eukaryotic cells, such as pro-karyotic cells; for example, bacteria or archaebacteria.

[0092] According to certain particular embodiments, said biological material may comprise or consist of viruses or viral particles, such as enveloped or non-enveloped viruses, RNA viruses, DNA viruses, or any other virus of the Baltimore classification; such as viruses belonging to any of groups I, II, III, IV, V, VI, or VII of said classification.

[0093] According to certain particular embodiments, said biological material may comprise or consist of one or more genetically modified, transfected cells, and / or cells whose expression of one or more nucleic acids is modulated.

[0094] According to certain particular embodiments, said biological material may comprise or consist of one or more somatic cells, or progenitor cells, for example one or more pluripotent or multipotent cells, such as embryonic cells, or cells derived from embryos.

[0095] According to certain particular embodiments, said cells may be in differentiated form, or in the process of differentiation, or even undifferentiated.

[0096] According to some embodiments, said cells may be cancerous cells, or precancerous cells, or even cells derived from cancerous tissues, of various origins.

[0097] According to certain embodiments, said cells (for example cancer cells) may be derived from one or more tissues or organs selected from a list consisting of: adrenal glands, bladder, blood, bone, bone marrow, brain, cartilage, uterus, cervix, endometrium, cornea, esophagus, gastrointestinal tract, nervous system, liver, lung, lymphatic tissue, muscle, heart, pancreas, pituitary gland, prostate, testicle, kidney, salivary gland, skin, thyroid, immune system, epithelial, endothelial, mesothelial.

[0098] According to certain particular embodiments, said cells may be chosen from blood cells and / or immune system cells, in particular chosen from a list consisting of: erythrocytes, platelets, T lymphocytes, B lymphocytes, leukocytes, dendritic cells, macrophages.

[0099] According to certain embodiments, said cells may be derived from patients or individuals suffering from one or more pathologies.

[0100] According to certain embodiments, said biological material comprises or consists of a three-dimensional cell culture.

[0101] According to certain embodiments, said biological material and / or culture medium may include one or more compounds of interest, for example one or more pharmaceutical actives, such as one or more antibodies, or antibody fragments.

[0102] According to some embodiments, said biological material comprises or consists of one or more spheroids.

[0103] According to some embodiments, said biological material comprises or consists of one or more organoids.

[0104] The term “spheroid” is capable of designating any aggregate, of any type of cell, such as an aggregate consisting of a single cell type or a plurality of cell types, capable of growing three-dimensionally, in suspension, and capable of developing interactions with one or more cell types and / or a three-dimensional matrix, such as a Matrigel™, or any type of suitable culture medium.

[0105] The term “organoid” is likely to designate any cellular structure obtained by expansion of one or more specific cell types of a given tissue, and capable, in whole or in part, of self-organizing and / or differentiating. Method of study or manipulation

[0106] The invention also relates to a method for studying or manipulating at least one study medium or micro-object, using the microfluidic device as described above, comprising introducing the study medium and / or micro-object into the study chamber or one of the study chambers, applying pressure, in particular negative pressure, into the deformation chamber(s) generating a stress through the upper wall of the study chamber in the study chamber, preferably a deformation of the upper wall, in particular in the direction of a reduction in the height of the study chamber, the stress through the upper wall in the study chamber applying a mechanical stimulus to the study medium or micro-object or generating a displacement of the study medium or micro-object in the study chamber.

[0107] The mechanical stimulus can be compression, depression and / or mechanical force.

[0108] In some embodiments, the method involves studying or moving one or more micro-objects contained in a study medium within the study chamber.

[0109] In some embodiments, the method involves the displacement of a study medium, particularly a fluid, within the study chamber. The deformation of the upper wall can generate a stress on the fluid study medium within the study chamber, resulting in a displacement field within the study medium contained in the chamber. This displacement field can be unidirectional, which generates a general fluid flow in one direction or be more diffuse, depending on the applications sought.

[0110] Preferably, the pressure applied in the deformation chamber is between +1000 and -1000 mbar, better between 0 and -800 mbar.

[0111] During the application of pressure in the deformation chamber, the upper wall can take on a curved shape along at least one cross-section, preferably along at least two orthogonal cross-sections. The curved shape can be convex, in particular parabolic, towards the chamber under study.

[0112] Preferably, during the application of pressure in the deformation chamber, the side walls of the study chamber do not deform by a deformation greater than 10%, better greater than 5%, better not deform substantially.

[0113] The method may involve introducing one or more micro-objects into the study chamber, particularly into a fluid medium contained within the study chamber. The introduction of the micro-object into the study chamber may be accomplished by pipetting the micro-object into the study chamber with a fluid, in particular a liquid.

[0114] The study medium may be a fluid circulating at least at the time of introduction of the micro-object(s) into the device and / or at the time of application of pressure in the deformation chamber(s). The micro-object(s) may be introduced into the study chamber along with the study medium.

[0115] The process may include the introduction of one or more chemical or physiological elements into the study medium, in particular the introduction of a hardening agent, in particular a gelling agent, after the introduction of the micro-object(s).

[0116] The process may include a step of introducing a curing agent into the test chamber and curing the curing agent. The curing of the curing agent may involve one or more of the following: polymerization, heating, denaturation, irradiation, such as with radiation and / or light, in particular UV light, gelation, physical crosslinking, such as (reversible) curing by ionic interactions and enzymatic crosslinking. It will be obvious to those skilled in the art which curing methods are suitable for a particular curing agent.

[0117] The study medium may include a gelling agent, in particular a hydrogel, introduced with the micro-object(s) into the study chamber or afterward into the study medium contained within the study chamber. The method may include gelling the study medium before deformation of the upper wall. Preferably, the study medium after gelling has a lower Young's modulus than the substrate. This allows, in particular, the immobilization of the micro-object(s). The fact that the study medium is gelled allows, in particular, the transfer of the upper wall deformation by compression of the gel to one or more micro-objects extending within the gel. In this case, the process may involve observing the movement of the micro-object(s) in the gel during or after the deformation of the upper wall.

[0118] The micro-object(s) may be selected from microdroplets, hydrogel micro-units, or any biological material as previously mentioned. The sample may be a cell sample. The cell sample may be a sample from a patient biopsy.

[0119] In the case of several gelled microunits in the study chamber, the gelled microunits may have different stiffnesses.

[0120] The device may include a study chamber having a continuously varying height, in particular decreasing continuously, or varying in discrete steps over its length or width, and the method may include introducing into the study chamber a plurality of deformable micro-objects of different heights all greater than or equal to the smallest height of the study chamber, the method including the automatic classification of the micro-objects in the study chamber according to their height, the deformation of the upper wall of the study chamber applying a different force on the micro-objects according to their position in the study chamber.

[0121] The method may be a mechanical deformation process of the micro-object(s). The deformation of the micro-object(s) in the study chamber may be direct, by compression of the upper wall on the micro-object(s) during its deformation. In this case, it is preferable that the height of the object(s) be greater than or equal to the height of the study chamber. Alternatively, the deformation of the micro-object(s) in the study chamber is indirect, in particular via the study medium, preferably a gel, in particular a hydrogel as described above.

[0122] The method may include immobilizing the object(s) before deformation of the upper wall, in particular by compression between the upper and lower walls of the study chamber before deformation, by the use of one or more capillary traps in the study chamber or by gelling of the study medium.

[0123] The method may include applying a deformation cycle to the deformation chamber(s) to generate a deformation cycle of the upper wall, particularly in the direction of a reduction in the height of the chamber under study, and thus applying a series of stimuli to the object(s). A "deformation cycle to the deformation chamber(s)" is understood to mean the application of a series of several successive deformations and / or relaxations to the compression chamber(s) according to a predetermined pressure diagram for each of the deformation chambers. In some embodiments, the pressure cycle may include an identical, repetitive sequence of pressure, particularly negative pressure, and relaxation for a predetermined time. In some embodiments, the A pressure cycle can consist of a predetermined sequence of different pressures, including negative pressures, and / or relaxations over a predetermined time. Such a deformation cycle allows the medium under study and / or the object(s) to be stimulated according to a sequence of identical or different stimuli.

[0124] The method may include the detection of the behavior of the micro-object(s) in the study medium during or following the deformation or deformation cycle of the upper wall, in particular the detection of the global displacement of the object(s), the local displacement of a part of the micro-object(s), in particular of one or more cell nuclei of one or more cell units, the global deformation of the micro-object(s), in particular the instantaneous radial deformation, and / or the local deformation of a part of the micro-object(s), in particular of one or more cell nuclei of one or more cell units during and / or after the deformation of the upper wall of the study chamber and / or after one or more deformation cycles of the upper wall of the study chamber.

[0125] The method may include gelling the medium before or after deformation of the upper wall or after one or more cycles of upper wall deformation. The method may include cyclic deformation of the object(s), gelling of the liquid in the study chamber, and detection of the movement of the micro-object(s) in the study chamber.

[0126] The method may include the detection, in particular optical, of lateral deformation, in particular instantaneous radial deformation, and / or of local deformation of the micro-object(s), in particular of the nucleus of a cell of a cell aggregate or of a single cell, in response to the deformation of the upper wall of the study chamber.

[0127] The method may include comparing the detected global or local displacement and / or detected local or global deformation of the micro-object(s) with the global or local displacement and / or global or local deformation of a real or simulated reference sample at the same position in the study chamber.

[0128] The process may include determining the rheological and / or biological properties of the micro-object(s) or a portion thereof by determining the deformation of the micro-object(s) or the portion thereof and comparing it with the deformation of a reference object whose rheological and / or biological properties are known, in particular that of the study medium containing the micro-object(s) or a portion thereof whose rheological properties are known. For example, the process may include introducing gelled microunits of known different stiffnesses, each containing a cell unit to be studied, and determining the rheological properties of the cell unit by determining the deformation of the cell unit within the gelled microunits. and comparison with the deformation of the hydrogel of the gelled micro-units at the same position whose rheological properties are known.

[0129] The method may involve deforming the study medium and / or pumping the study medium into the study chamber. Deforming the upper wall of the study chamber can generate a flow of the study medium from an inlet to an outlet within the chamber, thus acting like a diaphragm pump. This deformation and / or pumping of the study medium can be carried out with or without micro-objects in the study medium.

[0130] According to certain specific objects of said process, the invention also relates to a method for studying or manipulating, in particular, biological material; comprising the steps of: a. provision of a microfluidic device as described above including at least one biological material in the study chamber; b. application of pressure, in particular negative pressure, in the deformation chamber generating a deformation of the upper wall of the study chamber; c. detection of said biological material in the study chamber; preferably characterized in that said biological material is brought into contact with one or more ligands or compounds of interest, before, during, or after said detection step. Brief description of the drawings

[0131] [Fig-1] schematically represents an example of a microfluidic device according to the invention,

[0132] [Fig.2] schematically represents a cross-section of the micro device fluidic of the [Fig.l],

[0133] [Fig.3] schematically represents a section of the microfluidic device of the [Fig. 1] during the application of negative pressure in the deformation chamber,

[0134] [Fig.4A] is in cross-section a variant of a microfluidic device,

[0135] [Fig.4B] is in cross-section a variant of a microfluidic device,

[0136] [Fig.4C] is in cross-section a variant of a microfluidic device,

[0137] [Fig.5A] is in cross-section a variant of a microfluidic device during the application of negative pressure in the deformation chamber,

[0138] [Fig.5B] is in cross-section a variant of a microfluidic device during the application of negative pressure in the deformation chamber,

[0139] [Fig.6] is in cross-section a variant of a microfluidic device,

[0140] [Fig.7A] is a longitudinal section of the study chamber of the microfluidic device of [Fig.1], the study chamber containing a study medium and micro- objects,

[0141] [Fig.7B] is a longitudinal section of the study chamber of a variant of a microfluidic device, the study chamber containing a study medium and micro-objects,

[0142] [Fig.7C] is a longitudinal section of the study chamber of a variant of a microfluidic device,

[0143] [Fig.8] is in cross-section a variant of a microfluidic device,

[0144] [Fig.9] represents in cross-section the device of the [Fig.8], a pressure negative pressure being applied in the deformation chambers,

[0145] [Fig.1OA] represents in section the stress field applied in the substrate when a negative pressure is applied in a variant of a microfluidic device,

[0146] [Fig. 10B] represents the deformation at the center of the study chamber as a function of the negative pressure applied in the deformation chambers,

[0147] [Fig. 1 IA] represents in section the stress field applied in the substrate when a positive pressure is applied in a variant of a microfluidic device,

[0148] [Fig. 1 IB] is a graph representing the deformation of the upper wall at the center of the study chamber as a function of the positive pressure applied in the deformation chambers,

[0149] [Fig. 12] represents a variant of a microfluidic device according to the invention,

[0150] [Fig. 13] is a graph representing the change in area of ​​micro-objects viewed from above at the center of the study chamber and laterally in the study chamber as a function of the negative pressure applied in the deformation chambers,

[0151] [Fig. 14] represents a variant of a microfluidic device according to the invention,

[0152] [Fig. 15] is a microscopy image of the study chamber containing a study medium and micro-objects,

[0153] [Fig. 16] is a graph representing the change in area of ​​a micro-object viewed from above as a function of time during a sinusoidal pressure cycle,

[0154] [Fig.17A] represents the radial deformation of cells in a spheroid of a first sample when pressure is applied in the deformation chambers,

[0155] [Fig.17B] represents the radial deformation of cells in a spheroid of a second sample when pressure is applied in the deformation chambers,

[0156] [Fig. 18] represents a variant of a microfluidic device according to the invention,

[0157] [Fig. 19] represents a variant of a microfluidic device according to the invention,

[0158] [Fig.20] represents in section a portion of a study chamber containing a medium study of hydrogel and micro-objects in the study medium,

[0159] [Fig.21] represents in section a portion of a study chamber containing a medium study containing hydrogel units of varying rigidities containing micro-objects,

[0160] [Fig.22] represents a variant of a microfluidic device according to the invention,

[0161] [Fig.23] represents a section along XXIII-XXIII of the device of [Fig.22], the study chamber containing a hydrogel study medium and micro-objects within the study medium, and

[0162] [Fig.24] represents a variant of a microfluidic device according to the invention. Detailed description

[0163] Figures 1 and 2 illustrate a microfluidic device 10 according to the invention comprising a support 20 including, within it, a study chamber 30 and a deformation chamber 40 which are fluidically independent of each other.

[0164] The support 20 comprises a substrate 25 made of an elastically deformable material, for example PDMS. PDMS is advantageous because of its non-toxic, elastic, and transparent characteristics, and its low cost. However, the invention is not limited to PDMS, and other elastically deformable materials may be used. The channels or chambers present on the chip may be obtained by a process comprising soft lithography and / or bonding. Preferably, the substrate 25 has a Young's modulus less than or equal to 1 GPa, preferably less than or equal to 0.1 GPa. In the illustrated example, the support 20 also comprises a rigid base 28, in particular a glass plate, carrying the substrate 25. The study chamber 30 and the deformation chamber 40 extend into cavities in the substrate 25, which are closed at their bases by the rigid base 28.

[0165] However, it could be otherwise, as illustrated for example in Figures 4A to 4C. For example, the rigid base could have one or more grooves partially forming one or more of the study and deformation chambers, as illustrated in Figures 4A and 4B. Alternatively, the substrate 25 could have cavities completely enclosed within the substrate 25, the rigid base limiting the deformation of the substrate by the base of the study chambers 30 and deformation chambers 40 and not delimiting the study or deformation chambers, as illustrated in [Fig. 4C]. Alternatively still, the rigid base is other than a glass plate. The rigid base can be any rigid support capable of bearing the substrate 25, in particular a multi-well plate or a plate made of another rigid material.

[0166] In all cases, the study chamber 40 comprises at least one upper wall 32 formed by the substrate 25, two side walls 33 and a lower wall 34, as illustrated in the figures.

[0167] The study chambers 30 and deformation chambers 40 are arranged in the support so as to extend laterally relative to each other, at least partially. In the examples illustrated in Figures 1 to 4, the deformation chamber 40 has a rectangular cross-section and extends laterally over its entire height H relative to the study chamber 30. However, the shape of the deformation chamber 40 is not limited to a rectangular shape in cross-section. As illustrated in Figures 5A and 5B, it can take on another shape in cross-section, in particular a substantially polygonal shape, in particular a substantially convex quadrilateral, for example substantially trapezoidal, with the shorter side forming the upper end, as illustrated in [Fig. 5A], or with the shorter side forming the base, as illustrated in [Fig. 5B].One or more of the edges may be rounded, particularly where they form an acute angle in the substrate, as illustrated in [Fig. 5B]. In addition, the deformation chamber 40 may extend partially above the study chamber 30, particularly the upper wall 32, as illustrated in [Fig. 5B].

[0168] The study chamber 30 may have in cross-section a substantially polygonal shape, in particular a convex quadrilateral, in particular a rectangular one.

[0169] The greatest width w at the base of the study chamber 30 may be between 200 µm and 5 cm, preferably between 300 µm and 1 cm, for example, approximately 4 mm, and the greatest height h of the cross-section of the study chamber 30 may be less than its greatest width w at the base and is preferably less than or equal to 10 mm, preferably less than or equal to 500 µm. The greatest height h may be defined, in particular, according to the object(s) to be studied in the study chamber 30 between the upper wall 32 and the lower wall 34. Preferably, the ratio of the greatest width w to the greatest height h of the study chamber is greater than or equal to 5, preferably 10. The dimensions of the cross-section of the study chamber 30 are chosen, in particular, according to the object(s) to be studied in the study chamber and the study procedure envisaged, as will become clear from the examples below.

[0170] The greatest width W at the base of the deformation chamber 40 can be between 200 µm and 5 cm, preferably between 300 µm and 1 cm, for example approximately 5 mm, and the greatest height H of the cross-section of the study chamber 30 is preferably less than its greatest width W at the base and is preferably less than or equal to 10 mm, preferably less than or equal to 7 mm, for example approximately 3 mm. The greatest height H is defined in particular according to the object(s) to be studied in the study chamber 30. Preferably, the ratio of the greatest width w to the greatest height h of the study chamber is greater than or equal to 5, preferably 10.

[0171] The height H of the deformation chamber 40 is greater than or equal to the height h of study room 30. The height ratio is preferably greater than or equal to 5, better greater than or equal to 10.

[0172] The deformation chamber 40 and the study chamber 30 are separated from each other by a thickness e of substrate 25. The smallest thickness e of substrate between the study chamber and the deformation chamber is greater than or equal to the height h of the study chamber 30. Preferably, the smallest thickness e of substrate between the study chamber and the deformation chamber is less than or equal to 40 times the height of the study chamber, even better less than or equal to 20 times the height h of the study chamber 30. It is for example approximately equal to 2 mm.

[0173] It is understood that the dimensions and shapes are to be adapted according to the object(s) to be studied in the study room and the study method envisaged, as will become clear from the examples below.

[0174] In the example of [Fig. 1], the study chamber 30 is connected at its two longitudinal ends to a fluid inlet and outlet and by fluidic circulation channels 34. Such channels 34 may extend into the substrate 25 and / or to the interface between the substrate 25 and the rigid base 28. They may fluidly connect the study chamber 30 to the outside and / or to other fluidic devices in series with the study chamber 30. The other devices may be external to the support 20 of the present microfluidic device or be included in the support 20, in particular upstream or downstream of the study chamber 30. The invention is not limited to such connections. It is perfectly conceivable to have a study chamber 30 with only a fluid inlet or with none at all, particularly in the case of a study in a stationary study environment of one or more micro-objects enclosed in the study chamber.

[0175] The deformation chamber 40 is preferably fluidically connected to a deformation control unit 45, in particular to a pressure control unit in the deformation chamber, by means of a channel 42 extending into the substrate 25 and / or to the interface between the substrate 25 and the rigid base 28, as illustrated in Figures 1 and 15. Such a control unit 45 makes it possible to apply positive or negative pressure in the deformation chamber 40, thereby deforming it, as illustrated in particular in [Fig. 3]. Such deformation of the deformation chamber generates stresses in the substrate 25, which deform the upper wall 32 of the study chamber 30 by stress transmission within the elastically deformable substrate 25.As illustrated in Figures 3, 5A, 5B, 9, and 10A, in the case of negative pressure in the deformation chamber 40, the volume of the study chamber 30 decreases due to the lowering of the center of the upper wall 32. In this case, the upper wall 32 takes on a curved shape towards the interior of the study chamber 30 in at least two orthogonal directions. Conversely, as illustrated... In [Fig. 1 IA], under positive pressure, the volume of the study chamber 30 increases due to the elevation of the center of the upper wall 32. In this case, the upper wall 32 takes on a curved shape outwards from the study chamber 30. The maximum deformation d of the upper wall 32, particularly at its center, depends in particular on the absolute pressure in the deformation chamber, as illustrated in the graphs of Figures 10B and 11B, and on the parameters of the device, including size, shape, and flexibility, and is easily determined by comparative tests within the capabilities of those skilled in the art. For example, the maximum deformation d3 of the upper wall 32 in the case of an inverted trapezoidal deformation chamber, as shown in [Fig. 5B], is greater than in the case of a rectangular deformation chamber, as shown in [Fig. 1 IA].2] for example, itself larger than that in the case dl of a non-inverted trapezoidal deformation chamber, according to [Fig.5A] for example. .

[0176] Preferably, the pressure in the deformation chamber is between -100 and 1000 mbar, better between -100 and -500 mbar.

[0177] During the application of pressure in the deformation chamber 40, for a pressure in the deformation chamber 40 below -500 mbar, the lateral walls 33 of the study chamber 30 do not deform by more than 5%, preferably more than 3%. They may buckle slightly due to the deformation of the upper wall 32. The maximum width of the study chamber 30 remains substantially constant during the application of pressure in the deformation chamber 40, in particular does not change by more than 2%, preferably not by more than 1%. This is due in particular to the distance between the deformation chamber and the study chamber, which is such that the direct deformation via the substrate 20 is negligible, and to the large dimension of the width relative to the height of the study chamber.

[0178] For example, for a pressure in the strain chamber 40 between -100 and -500, the deformation of the upper wall 32 of the study chamber 30 towards the interior of the study chamber 30 is greater than or equal to 10%, better greater than or equal to 40% of the height of the study chamber, as can be seen in [Fig.1OB] representing the height of deformation of the upper wall in micrometers with respect to the pressure applied in the strain chamber.

[0179] The study chamber may have a constant height h in cross-section, as illustrated in figures 1 to 4.

[0180] The study chamber can have a constant height h over the entire length of the study chamber 30, as illustrated in [Fig.1].

[0181] Alternatively, as illustrated in [Fig. 7B], the height h of the study chamber 30 can vary monotonically along the length of the study chamber 30. This can allow for the classification or guidance of objects to be studied within the study chamber. 30.

[0182] Alternatively, one of the walls of the study chamber, in particular the upper wall 30 or the lower wall 33, may have surface reliefs 38 in the form of concave or projecting features along its length, as illustrated in [Fig. 7C], or its width, as illustrated in [Fig. 6]. Such reliefs may be capillary traps with trapping forces that vary according to the height of the study chamber, controllable by the pressure in the deformation chamber 40. Such reliefs may trap, at a minimum negative pressure applied in the deformation chamber, or release, at a minimum positive pressure applied in the deformation chamber, the micro-object(s). Alternatively, the reliefs 38 may be guides for the micro-object(s) in the study chamber 30, particularly when they are in the form of ribs or grooves extending along the length of the study chamber 30.In this case, the guidance can be continuous, with varying degrees of strength depending on the height of the study chamber 30, or it can occur only when the height of the study chamber is below a threshold height. The guidance can be direct on the micro-object(s), or indirect, notably via a liquid study medium or a hydrogel.

[0183] The study chamber 30 can be substantially cylindrical in shape. In the example in [Fig. 1], the study chamber 30 has a substantially rectangular shape. However, it could be otherwise, and the study chamber 30 could have other shapes, in particular cylindrical with a circular base or with another base, as can be seen in particular in Figures 14, 15, and 18, or other non-cylindrical shapes.

[0184] Furthermore, the invention is not limited to a single deformation chamber 40 for deforming the upper wall 32 of a test chamber 30, as illustrated in particular in Figures 8 to 10. The device may comprise a plurality of deformation chambers 40a and 40b, in particular two substantially identical deformation chambers extending on either side of the test chamber 30. Preferably, the two deformation chambers 40a and 40b are connected to the same pressure control element 45, and the pressure is identical in both chambers 40a and 40b, as illustrated in [Fig. 14]. The presence of two deformation chambers 40a and 40b allows, in particular, for a greater deformation of the upper wall 32, as can be seen in [Fig. 9]. Alternatively, the two deformation chambers 40a and 40b are not identical and / or are not at the same pressure during the application of the pressure.

[0185] Alternatively or in addition, not illustrated, the device may include deformation chambers for deforming the upper wall according to a more complex deformation profile than a simple curve, in particular deformation chambers distributed along the length, whether spaced apart or not, and having a The pressure applied is the same or not.

[0186] In certain embodiments, particularly those illustrated in Figures 12 and 14, the device may comprise a plurality of study chambers 30a and 30b fluidically connected to each other in series, the outlet of a study chamber 30a being fluidly connected to the inlet of the study chamber 30b. Each study chamber may be laterally bordered by one or more deformation chambers controlled independently or simultaneously by one or more pressure control units 45. Study environment and micro-objects

[0187] During the study or manipulation process, a study medium 50 containing one or more micro-objects 55 may be introduced into the study chamber 30 before or during the application of pressure in the deformation chamber(s) 40. This study medium 50 may be introduced by any means commonly used in the field of microfluidics, in particular by pipetting from a reservoir containing the study medium. This study medium and / or the micro-object(s) may be stationary in the study chamber or moving from a fluid inlet to a fluid outlet during the application of pressure.

[0188] The study medium may be liquid. It may contain a hardening agent, in particular a gelling agent, for example a hydrogel. It may be hardened before, during, or after the application of pressure in the deformation chamber(s) 40. In the case of a hardened study medium, in particular a gelled one, it preferably has a lower Young's modulus than the substrate. Such a hardened study medium allows the transfer of deformation from the upper wall to one or more micro-objects extending within the study medium. The latter may be homogeneous within the study chamber 30. Alternatively, it may have a variable density within the volume of the study chamber 30, in particular exhibiting a density gradient from the center of the study chamber to the side walls.

[0189] The micro-object(s) are preferentially deformable.

[0190] The micro-object(s) may be selected from microdroplets, gelled microunits, including those containing a hydrogel and which may be in the form of gelled microdroplets, and / or any biological material, such as cellular units, including cells or cellular aggregates, such as spheroids or organoids. In the case of several gelled microunits in the study chamber, the gelled microunits may have different rigidities. The microdroplets or gelled microunits may contain biological material, including a cellular unit, such as a cell or a cellular aggregate, the cellular aggregate being, in particular, a spheroid or an organoid or derived from a patient biopsy.

[0191] According to a particular embodiment, the micro-object(s) comprise or consist of biological material, and / or a medium suitable for the culture of biological material such as any culture medium capable of comprising one or more nutrients (for example, proteins, peptides, amino acids, and / or any carbon source).

[0192] The term “biological material” may refer to any type of cell, including embryonic or non-embryonic, as well as any type of extract, lysate, fraction of one or more cells, and in particular of human, animal or plant origin.

[0193] The term “culture medium” may refer to any physiologically acceptable medium suitable for culturing one or more cell types, in particular any medium comprising at least one source of carbon and energy; in particular nutrients, for example peptides, polypeptides and / or amino acids, carbohydrates, essential metals and minerals, and buffering agents.

[0194] According to some embodiments, said biological material comprises or consists of living, quiescent and / or cultured cells.

[0195] According to some embodiments, the biological material comprises or consists of nucleic acids or fractions of nucleic acids, in particular DNA and / or RNA.

[0196] In a non-exhaustive manner, the said biological material may include or consist of one or more types of cells, chosen from eukaryotic cells, prokaryotic cells, or even viruses or viral particles.

[0197] According to certain particular embodiments, said biological material may comprise or consist of eukaryotic cells, such as mammalian cells, in particular human or non-human; for example, from humans, mice, rats, dogs, cats, cows, pigs, chickens, goats, horses, yeasts or others.

[0198] According to certain particular embodiments, said biological material may comprise or consist of non-eukaryotic cells, such as pro-karyotic cells; for example, bacteria or archaebacteria.

[0199] According to certain particular embodiments, said biological material may comprise or consist of viruses or viral particles, such as enveloped or non-enveloped viruses, RNA viruses, DNA viruses, or any other virus of the Baltimore classification; such as viruses belonging to any of groups I, II, III, IV, V, VI, or VII of said classification.

[0200] According to certain particular embodiments, said biological material may comprise or consist of one or more genetically modified, transfected cells, and / or cells whose expression of one or more nucleic acids is modulated.

[0201] According to certain particular embodiments, said biological material may comprise or consist of one or more somatic cells, or progenitor cells, for example one or more pluripotent or multipotent cells, such as embryonic cells, or cells derived from embryos.

[0202] According to certain particular embodiments, said cells may be in differentiated form, or in the process of differentiation, or even undifferentiated.

[0203] According to some embodiments, said cells may be cancerous cells, or precancerous cells, or even cells derived from cancerous tissues, of various origins.

[0204] According to certain embodiments, said cells (for example cancer cells) may be derived from one or more tissues or organs selected from a list consisting of: adrenal glands, bladder, blood, bone, bone marrow, brain, cartilage, uterus, cervix, endometrium, cornea, esophagus, gastrointestinal tract, nervous system, liver, lung, lymphatic tissue, muscle, heart, pancreas, pituitary gland, prostate, testicle, kidney, salivary gland, skin, thyroid, immune system, epithelial, endothelial, mesothelial.

[0205] According to certain particular embodiments, said cells may be selected from blood cells and / or immune system cells, in particular selected from a list consisting of: erythrocytes, platelets, T lymphocytes, B lymphocytes, leukocytes, dendritic cells, macrophages.

[0206] According to certain embodiments, said cells may be derived from patients or individuals suffering from one or more pathologies. These pathologies may include pathologies that alter the mechanical properties of tissues, such as fibrosis, cancer, or any other condition that leads to tissue scarring.

[0207] According to some embodiments, said biological material comprises or consists of a three-dimensional cell culture.

[0208] According to certain embodiments, said biological material and / or culture medium may include one or more compounds of interest, for example one or more pharmaceutical actives, such as one or more antibodies, or antibody fragments.

[0209] According to certain embodiments, said biological material comprises or consists of one or more spheroids.

[0210] According to certain embodiments, said biological material comprises or consists of one or more organoids.

[0211] The term “spheroid” may refer to any aggregate, of any type of cell, such as an aggregate consisting of a single cell type or a plurality of cell types, capable of growing three-dimensionally, in suspension, and capable of developing interactions with one or more cell types and / or a three-dimensional matrix (such as a Matrigel™) or any type of suitable culture medium.

[0212] The term “organoid” is likely to designate any cellular structure obtained by expansion of one or more specific cell types of a given tissue, and capable, in whole or in part, of self-organizing and / or differentiating.

[0213] The height of the micro-object(s) may be greater than or equal to the height of the study chamber, as illustrated in Figures 7A and 7B. Alternatively, the height of the micro-object(s) is less than the height of the study chamber 30. In this case, the study medium preferably comprises a gelling agent, in particular a hydrogel, so as to be able to gel the study medium before, during or after the application of pressure in the deformation chamber(s). Analysis system

[0214] As illustrated in [Fig. 14], the microfluidic device may include or be functionally coupled to an analysis system 60 configured to analyze the study chamber 30, in particular the study medium 50 or the micro-object(s) 55 in the study chamber 30. The analysis system may or may not be functionally coupled to the deformation control unit of the deformation chamber; in particular, the analysis system may provide an analysis signal to the control unit. The control unit may command the deformation of the deformation chamber(s) according to the analysis signal.

[0215] The analysis system 60 in the study chamber 30 may be an optical imaging system, in particular an optical or electron microscope, or a confocal imaging system as illustrated in [Fig. 14], or any other analysis system, in particular a system for measuring electrical properties, in particular impedance, conductivity of the study medium, or the electrical action of a cell unit during mechanical stimulation. Preferably, the analysis system 60 is arranged on or below the study chamber and is configured to take images of the fluid, gel, and / or one or more micro-objects in the study chamber. The analysis system 60 may be configured to detect static or dynamic information of the fluid, gel, and / or one or more micro-objects in the study chamber. Process

[0216] In certain embodiments, the deformation of the upper wall 32 of the study chamber applies a force to the micro-objects 55 in the study chamber 30, generating their deformation. It is then possible to determine, in particular by microscopy and image analysis methods, the deformation of the micro-object(s) 55. The deformation determined may be the overall deformation of the micro-object(s) or the local deformation of only a part of the micro-object(s), in particular one or a few cells of a cell aggregate or the nucleus of a cell. Such a measurement of the deformation can make it possible to determine the mechanical properties of the or micro-objects or parts of micro-objects, including their rigidity.

[0217] This deformation can be direct when the micro-object(s) are smaller than the height h of the study chamber 30 or indirectly through the study medium, the deformation of the upper wall 32 generating a volume deformation of the study medium, in particular gelled, in the study chamber 30 and thereby the deformation of the micro-object(s).

[0218] The method may include comparing the deformation of the micro-object(s) or part of the micro-object(s) with the deformation of a real or calculated reference sample in the same study chamber at the same position or an equivalent position in order to deduce a characteristic of the micro-object(s), in particular a deformability characteristic and / or a biological characteristic.

[0219] In certain embodiments, deformation of the upper wall of the study chamber causes displacement of the micro-object(s) within the study chamber 30 or of a part of the micro-object(s) within the micro-object(s), in particular of one or a few cells within a cell aggregate or of the cell nucleus. This displacement may occur during deformation due to the action of the upper wall 32 on the study medium, in particular a gel containing the micro-object(s), or on the micro-object(s).

[0220] The method may include comparing the displacement of the micro-object(s) or part of the micro-object(s) with the displacement of a reference sample in the same study chamber in order to deduce a characteristic of the micro-object(s), in particular a biological characteristic.

[0221] In some embodiments, the process may include the study of the deformation of the micro-object(s) and their displacement as described above.

[0222] In certain embodiments, the method comprises applying a pressure cycle in the deformation chamber(s) 40 corresponding to the periodic repetition, over a predetermined time, of a succession of pressure / relaxation in the deformation chamber(s) 40. The different pressures of the cycle may or may not be identical and may or may not be separated by the same time interval. Such a pressure cycle makes it possible to apply a cyclic deformation of the upper wall 32 for a determined time, which generates periodic stimulation on the micro-object(s) 55. It is then possible to study, in particular by imaging and image analysis, the reactions of the micro-object(s), especially biological material, to such cyclic stimulation.The process may involve studying the deformation of the micro-object(s) over time in correlation with the applied pressure cycle, or studying the displacement of the micro-object(s) over the application time of the pressure cycle, in correlation with the applied pressure cycle. It is also possible to study the displacement of the micro-object(s) over time in correlation with the... applied pressure cycle or the study of the displacement of the micro-object(s) during the application time of the pressure cycle, in correlation with the applied pressure cycle or after the application of the pressure cycle in reaction to the stimulus applied by the deformation cycle, particularly in the case of micro-object(s) containing biological material.

[0223] We will now detail several examples of the use of the microfluidic device described above. Cell culture and spheroid formation

[0224] H4-II-EC3 and NIH-3T3 and MDA-MB-231 GFP type cancer cells are cultured in DMEM medium, supplemented with 10% FBS, and 1% of Penicillin and / or Streptomycin type antibiotic.

[0225] Spheroids are produced from 96-well non-adherent U-shaped plates (Corning catalogue 7007).

[0226] To obtain co-cultures of spheroids, the two cell types are mixed in different ratios depending on the desired spheroid size and the intended application. For example, a mixture of one hundred H4-II-EC3 cells and four hundred NIH-3T3 cells results in larger spheroids, and one hundred cells of each type yields smaller spheroids. The cells are co-cultured for 72 hours to form the spheroids. Microscopy

[0227] Images are captured with a motorized Nikon Ti2 spinning disk epifluorescence microscope equipped with a 20x lens. Illumination is provided by a Lumencor LED light source for epifluorescence, or by an Oxius laser array for confocal imaging, and images are captured by a Hamamatsu C13440-20CU SCMOS camera. Raw data are collected using Nikon Elements imaging software (version 5.11.01, Build 1367). Fluorescent marking

[0228] Cells can be labeled either by immunofluorescence or by standard detection kits. All reagents are introduced into the device using a pipette, without applying pressure. Actin labeling is performed with Alexa Fluor™ 488 Phalloidin (Thermofisher - Catalog number: A12379) at a dilution of 1:200. Nuclei are labeled with NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342, Invitrogen Catalog number: R37605) according to the commercial protocol. The membrane is labeled with CellBrite® Steady 650, according to the commercial protocol (Biotium catalog number: 30108). Measurement of spheroid size

[0229] The sizes of the spheroids are analyzed from the microscopy images using from a macro of the ImageJ software (FIKI). For each pressure value, the perimeter, total area, and major and minor axes of the spheroids can be measured and recorded.

[0230] Example 1: Analysis of the overall compression of spheroids as a function of position in the study chamber within the framework of a rectangular prism-shaped study chamber

[0231] A device according to [Fig. 12] is manufactured. A monolithic polydimethylsiloxane (PDMS) substrate 25 is made from a mold produced by 3D printing, the mold having reliefs corresponding to the inverted outline of the study chambers 30 and deformation chambers 40, as well as the conduits 34 and 42. The substrate 25 thus manufactured is placed on a microscope slide 28, the side of the substrate bearing the chamber impressions being in contact with the microscope slide. The device comprises two identical study chambers 30a and 30b, elongated in shape and with substantially rectangular transverse and longitudinal sections, connected to each other in series. The two study chambers 30a and 30b are each bordered on either side of their longitudinal axis by two deformation chambers 40a and 40b connected to the same pressure source by a conduit 42 leading into both deformation chambers.The two pairs of deformation chambers, each corresponding to a study chamber, are not connected to each other. In this case, they are subjected to the same pressure. The study chambers have a height approximately equal to 100 pm.

[0232] A sample of spheroids formed as previously described, with a diameter approximately equal to 130 ± 20 pm, is introduced into the study chambers 30a and 30b using a pipette fixed in the study chambers. Measurements of the visible area, by microscopy and image analysis according to the method described previously, of spheroids in the study chamber in the central position of the study chamber and in the lateral position of the study chamber are made by microscopy before and after application of different negative pressures in the deformation chambers. The results before and after application of pressure in the deformation chambers are compared to determine the percentage change in the area of ​​the spheroids representing the deformation of the spheroids by the deformation of the upper wall. [Fig.Figure 13 illustrates the average change in area as a function of pressure in the deformation chambers of spheroids in a lateral position in the study chambers (bottom curve) and in a central position in the study chambers (top curve). It can be seen that the deformation of the spheroids is a function of both the pressure applied in the deformation chambers and the position of the spheroids in the study chamber, the deformation of the upper wall 32 being more pronounced in the center of the study chamber than in a lateral position.

[0233] Example 2: Analysis of the overall behavior of spheroids during application of a pressure cycle within a cylindrical study chamber with a circular base.

[0234] A device according to [Fig. 14] is manufactured. A monolithic polydimethylsiloxane (PDMS) substrate 25 is made from a mold produced by 3D printing, the mold having reliefs corresponding to the inverted trace of the study chambers 30 and deformation chambers 40, as well as the conduits 34 and 42. The substrate 25 thus manufactured is placed on a microscope slide 28, the side of the substrate bearing the chamber impressions being in contact with the microscope slide. The device comprises two identical study chambers 30a and 30b, circular in shape when viewed from above, connected to each other in series by a conduit. The connecting conduits, inlet and outlet of the device, are taller than the height of the study chambers. The two study chambers 30a and 30b are each bordered on both sides by two deformation chambers 40a and 40b connected to the same pressure source by a conduit 42 giving into the two deformation chambers.The two pairs of deformation chambers, each corresponding to a study chamber, are not connected to each other. In this case, they are subjected to different pressures so as to simultaneously study the deformation of spheroids at two different pressures. The study chambers have a height approximately equal to 100 pm.

[0235] A sample of spheroids formed as described above, with a diameter approximately equal to 130 ± 20 pm, are introduced into the study chambers 30a and 30b using a pipette fixed in the study chambers. Measurements of the visible area, by microscopy and image analysis according to the method described above, of spheroids in the study chamber in a substantially central position of the study chamber are made by microscopy, as shown in [Fig. 15], before the application of a sinusoidal pressure cycle at a negative pressure of -300 mbar and with a frequency of 0.5 Hz. [Fig. 16] illustrates the mean change in the area of ​​the spheroids over time during the application of the pressure cycle. It appears from [Fig. 16] that the deformation of the spheroids follows the pressure cycle over time and therefore that the upper wall deforms periodically according to the pressure cycle.

[0236] Example 3: Analysis of the deformation of individual cells in spheroids when applied within a circular-based cylindrical study chamber.

[0237] In the device of Example 2, spheroid samples formed as previously described, with a diameter approximately equal to 130 ± 20 pm, are introduced into the study chambers 30a and 30b using a pipette fixed in the study chambers. It has been shown that the local deformation of individual cells and their nuclei in the spheroids, labeled by fluorescence as The previously described phenomena can be observed. It is then possible to study nuclear deformation in cells, cell deformation, and / or cell behavior / rearrangements in spheroids during spheroid deformation.

[0238] Example 4: Analysis of the deformation of individual cells in spheroids when applied within a circular-based cylindrical study chamber.

[0239] In the device of Example 2, two spheroid samples of different cell combinations and of approximately equal diameters of 130 ± 20 µm are introduced into study chambers 30a and 30b using a pipette fixed in the chambers. The first sample consists of spheroids formed solely from H4-ILEC3 cells, and the second sample consists of spheroids formed from a co-culture of H4-ILEC3 and NIH-3T3 cells. Before deformation of the upper wall, the NIH-3T3 cells are distributed in the center of the spheroids, while the H4-ILEC3 cells form a shell around them. The deformation field of the spheroids, in the plane of the bright-field image, is measured using particle image velocimetry (PIV) software, and the outward radial deformation is calculated. The graphs in figures 17A to 17C are obtained.Figure 17A shows the radial deformation of a spheroid from the first sample. Figure 17B shows the radial deformation of a spheroid from the second sample. For the first sample, containing only H4-ILEC3 cells, the deformation occurs from the center to the edge, increasing gradually. For the second sample, containing only the co-culture of H4-ILEC3 and NIH-3T3 cells, the spheroid has a static core with little deformation and a lateral region with more deformation.

[0240] The hypothesis that such a difference in behavior was specifically linked to the self-organization of these different cells into a nucleus-envelope structure, and therefore that either H4-ILEC3 cancer cells or NIH-3T3 cells could exhibit different mechanical characteristics, one being more rigid than the other, was tested.

[0241] NIH-3T3 cells were labeled with GFP before being mixed with H4-ILEC3 type cells. Observing the cells in the spheroids, it was noted that the NIH-3T3 cells moved towards the interior of the spheroid forming a nucleus, while the cancer cells were arranged at the periphery.

[0242] These results therefore suggest that NIH-3T3 cells assemble in a nucleus forming a more rigid structure compared to the lateral part made up of H4-II-EC3 cells.

[0243] In particular, quantitative data indicate that the nucleus of NIH-3T3 cells is approximately 30 to 50 times more rigid than the outer layer formed by H4-II-EC3 cells.

[0244] It is therefore possible to determine the mechanical properties of cellular arrangements in different three dimensions and of heterogeneous structure, and to determine, by comparison with the mechanical properties of known cellular arrangements, the biological properties of a cell sample to be studied. This example thus demonstrates that the device is particularly well-suited to the characterization of heterogeneous structures, as well as to the identification of a demarcation between healthy and cancerous cells.

[0245] Example 5: Microfluidic devices for a multi-well plate.

[0246] Devices according to Figures 18 and 19 are manufactured based on a multi-well plate. Since the rectangular structure is not suitable for the microfluidic device, more suitable structures have been developed. A monolithic polydimethylsiloxane (PDMS) substrate 25 is manufactured from a mold produced by 3D printing. The mold has ribs corresponding to the inverted trace of the study chambers 30 and deformation chambers 40, as well as the conduits 34 and 42. The substrate 25 thus manufactured is sized to fit the wells of a multi-well plate, with the side of the substrate bearing the chamber impressions in contact with the bottom of the well.

[0247] The device according to [Fig. 18] comprises a single, substantially circular study chamber viewed from above, connected to two sample inlet and outlet channels 34, the channels forming an acute angle with each other, and a crescent-shaped deformation chamber viewed from below, partially surrounding the study chamber. The pressure in the deformation chamber is controlled by means of a control channel 42. At -600 mbar in the deformation chamber 40, the upper wall of the study chamber deforms at its center inward toward the study chamber by approximately 43 µm.

[0248] The device according to [Fig. 19] comprises a single, substantially circular study chamber viewed from above, connected to two sample inlet and outlet channels 34, the channels being in opposition, and two identical deformation chambers on either side of the study chamber, kidney-shaped viewed from above. The pressure in the deformation chambers is controlled by means of a common control channel 42. They are therefore subjected to the same pressure. At -600 mbar in deformation chambers 40a and 40b, the upper wall 32 of the study chamber deforms at its center towards the interior of the study chamber by approximately 49 µm.

[0249] Example 6: Cyclic compression of a cell aggregate in a gelled medium.

[0250] In the device of Example 1, a sample of spheroids of metastatic breast cancer cells of type MDA-MB-231 in a liquid medium is introduced into the study chamber 30, having a diameter greater than the height of the chamber. study. Spheroids are stimulated in the liquid medium by a substantially sinusoidal pressure cycle at -350 mbar with a frequency of 0.5 Hz for 12 h in deformation chambers 40. After stimulation, the liquid medium is replaced with a basement membrane matrix, called Matrigel™, and cell migration in the matrix in response to cyclic stimulation is studied over time for 16. MDA-MB-231 cancer cells migrate outwards in the matrix, which allows the metastatic capacity of cancer cells to be studied in response to an external stimulus.

[0251] Example 7: Stimulation of spheroids in a hydrogel.

[0252] In the device of Example 1, a sample of spheroids in an agarose gel medium is studied. The spheroids are smaller than the height of the study chamber. This configuration allows for the stimulation and application of mechanical stress to the spheroids instead of imposing deformation. The upper wall deforms the hydrogel containing the spheroids, which laterally displaces the spheroids and the hydrogel within the study chamber, as illustrated in [Fig. 20]. This allows for the application of shear and / or tensile stress to the spheroids.

[0253] Such spheroid deformation dynamics within the hydrogel could be used to analytically determine the rheological properties of tissues or cell aggregates embedded within a chamber. Indeed, the amount of spheroid deformation can be extracted by comparing it to the deformation of the surrounding hydrogel, which has known rheological properties: a very soft microtissue will deform significantly, while a very rigid microtissue will remain virtually undeformed relative to the surrounding gel. It is therefore possible to test tissues with different stiffnesses by adjusting the stiffness of the surrounding hydrogel. In extreme cases, the hydrogel can be replaced by a purely viscous liquid hydrogel, such as glycerol, paraffin oil, or other liquids.

[0254] Example 8: Stimulation of spheroids in hydrogel units.

[0255] Using the methodology described above for spheroids embedded in hydrogels, it is also possible to have hydrogel units 65a to 65c in a fluid medium within the study chamber, each containing spheroids. This allows for multiplexing the ability to apply different ranges of stress in a study. As mentioned in Example 7, hydrogels of known stiffness can be used to analyze the stiffness of spheroids with respect to the rheological properties of the hydrogel. With hydrogel units, it is possible to have hydrogel units with different rheological properties in the same study chamber. These different hydrogel units with different stiffnesses, when compressed, can produce different fluxes within them, thus generating different forces. and mechanical stresses in the spheroids integrated inside as illustrated in [Fig.21].

[0256] Example 9: Microfluidic devices generating a displacement of the study medium and spheroids.

[0257] A device according to [Fig. 22] is manufactured using the same method as in Example 1. The device comprises a funnel-shaped study chamber 30 viewed from above and a deformation chamber 40 extending along the longer side of the funnel shape. The deformation chamber 40 has a cross-section substantially the same shape as the deformation chamber of [Fig. 5B]. Deformation of the deformation chamber by applying pressure within the deformation chamber generates a deformation of the upper wall 32 of the study chamber only in the enlarged part of the funnel, as illustrated in [Fig. 23]. A hydrogel containing spheroids is introduced into the study chamber.

[0258] In this design, the study chamber is pressed only from one end (adjacent to the deformation chamber), resulting in a forward thrust of the hydrogel and the spheroids embedded within the hydrogel, as illustrated in [Fig. 23]. Such deformation produces a spatially heterogeneous deformation in the hydrogel. Different device configurations can be designed to obtain different hydrogel deformations. For example, introducing 65 ridges on the edges of the study chamber, as illustrated in [Fig. 24], generates more bending-type deformations in the hydrogels between ridges in the study chamber.

[0259] The invention is not limited to the examples and embodiments described above. The features of the various embodiments described above can be combined in additional variants.

Claims

Demands

1. Microfluidic device (10) comprising: • a support (20) having a substrate (25) made of an elastically deformable material, • a study chamber (30, 30a, 30b) extending into the support (20) having at least one upper wall (32) formed at least partially, preferably totally, by the substrate (25), at least one lower wall (34) and at least two side walls (33), • at least one deformation chamber (40, 40a, 40b) of the substrate formed at least partially by the substrate (25), fluidically independent of the study chamber (30, 30a, 30b) and extending at least partially into the support (20) at least partially along one of the side walls (33) of the study chamber (30), the device being configured such that the deformation of the deformation chamber (40, 40a,40b) generates the application of a mechanical stress on at least a part of the upper wall (32) of the study chamber within the study chamber (30), in particular by deformation of the upper wall (32).

2. Device according to claim 1, wherein the Young's modulus of the substrate (25) is less than or equal to 1 GPa, preferably less than or equal to 0.1 GPa.

3. A device according to any one of the preceding claims, configured such that a reduction in the volume of the deformation chamber (40, 40a, 40b), in particular by negative pressure in the deformation chamber (40, 40a, 40b), generates a deformation of the upper wall (32) of the study chamber in the direction of a reduction in volume, in particular a reduction in height h, of the study chamber (30), and such that an increase in the volume of the deformation chamber (40, 40a, 40b), in particular by positive pressure in the deformation chamber (40, 40a, 40b), generates a deformation of the upper wall (32) of the study chamber towards an increase in volume, in particular an increase in height h, of the study chamber, said deformation of the upper wall (32) generating the application of a mechanical constraint in the study chamber (30, 30a, 30b).

4. Device according to any one of the preceding claims, wherein the deformation chamber (40, 40a, 40b) extends over a height H greater than or equal to that h of the study chamber (30, 30a, 30b) in the substrate (25), the height ratio H / h preferably being greater than or equal to 5, better greater than or equal to 10.

5. Device according to any one of the preceding claims, wherein the smallest thickness e of substrate between the study chamber (30, 30a, 30b) and the deformation chamber (40, 40a, 40b) is greater than or equal to the height h of the study chamber (30, 30a, 30b) and / or less than or equal to 100 times the height h of the study chamber (30, 30a, 30b), better less than or equal to 50 times the height h of the study chamber (30, 30a, 30b), better less than or equal to 40 times the height h of the study chamber (30, 30a, 30b), even better less than or equal to 20 times the height h of the study chamber (30, 30a, 30b).

6. Device according to any one of the preceding claims, wherein the deformation chamber (40, 40a, 40b) extends in the support (20) opposite a plane defined by one of the side walls (33) of the study chamber (30, 30a, 30b) over a height greater than or equal to 50% of its maximum height H, better, over a height greater than or equal to 70% of its maximum height H, even better over a height greater than or equal to 80% of its maximum height H.

7. Device according to any one of the preceding claims, comprising at least two deformation chambers (40a, 40b) of the substrate formed at least partially by the substrate (25), independent of the study chamber (30, 30a, 30b) and extending at least partially along each of a lateral wall (33) of the study chamber, in particular extending on either side of the study chamber (30, 30a, 30b).

8. Device according to any one of the preceding claims, comprising a plurality of study chambers (30a, 30b) fluidly connected in series with each other, the stress through the upper wall (32) in each study chamber, in particular the deformation of the upper wall, being generated by the same deformation chamber(s) (40, 40a, 40b) or by separate deformation chambers fluidly connected or not with each other.

9. Device according to any one of the preceding claims, comprising a control unit (45) of the deformation of the deformation chamber (40, 40a, 40b), in particular of the pressure in the deformation chamber, fluidly connected to the deformation chamber (40, 40a, 40b), in particular by a channel (42).

10. Device according to any one of the preceding claims, comprising or being functionally coupled to an analysis system (60) configured to analyze the microfluidic device, in particular the study chamber, including the study medium or the micro-object(s) in the study chamber (30, 30a, 30b).

11. Device according to any one of the preceding claims, wherein the study chamber (30, 30a, 30b) comprises a study medium (50) and / or at least one micro-object (55), or better, a plurality of micro-objects in the study chamber, in particular in the study medium (50).

12. Device according to claim 11, wherein the micro-object(s) (55) can be selected from microdroplets, gelled microunits, in particular comprising a hydrogel and which may be in the form of gelled microdroplets, and / or any biological material, such as cellular units, in particular cells or cellular aggregates, such as spheroids or organoids, or any type of culture medium.

13. A method for studying or manipulating at least one study medium (50) or a micro-object (55), using the microfluidic device (10) according to any one of the preceding claims, comprising introducing the study medium (50) and / or the micro-object (55) into the study chamber(s) (30, 30a, 30b), applying pressure, in particular negative pressure, into the deformation chamber(s) (40, 40a, 40b) generating stress through the upper wall (32) of the study chamber into the study chamber (30, 30a, 30b), preferably deforming the upper wall, in particular reducing the height of the study chamber, the stress through the upper wall of the study chamber applying a mechanical stimulus to the study medium (50) or the micro-object (55) or generating a displacement of the study medium (50) or of the micro-object (55) in the study chamber (30, 30a, 30b).

14. A method according to claim 13, wherein the study medium (50) comprises a gelling agent, in particular a hydrogel, introduced with the micro-object(s) into the study chamber or after into the study medium contained in the study chamber, the study medium (50) exhibiting after gelation a Young's modulus lower than the substrate (25).

15. A method according to any one of claims 13 and 14, comprising deformation of the study medium and / or pumping of the study medium into the study chamber (30, 30a, 30b), deformation of the upper wall (32) of the study chamber (30, 30a, 30b) generating in particular a flow of the study medium (50) from an inlet to an outlet in the study chamber (30, 30a, 30b), thus acting in the manner of a diaphragm pump.

Images