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

Abstract

The invention relates to a microfluidic device (10) comprising: - a support (20) comprising a substrate (25) made of an elastically deformable material; - a study chamber (30, 30a, 30b) extending inside the support (20) having at least one upper wall (32) at least partially formed by the substrate (25), at least one lower wall (34) and at least two side walls (33); - at least one substrate deformation chamber (40, 40a, 40b) at least partially formed by the substrate (25), fluidically independent of the study chamber (30, 30a, 30b) and at least partially extending inside the support (20) along one of the side walls (33) of the study chamber (30), the device being configured so that the deformation of the deformation chamber (40, 40a, 40b) causes the mechanical straining of at least one portion of the upper wall (32) in the study chamber (30).

Description

[0001] Description

[0002] Title: Microfluidic device for studying and / or manipulating a study medium and / or at least one micro-object.

[0003] 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.

[0004] Technical field

[0005] Today, there are various methods for mechanically manipulating and characterizing micro-objects, particularly individual cells or cell aggregates. These methods are particularly used in the field of mechano-biology. These methods are used, for example, to characterize mechanical properties or to analyze the reactions of objects to mechanical stimuli.

[0006] Prior art

[0007] Known methods in this field include micropipette aspiration, atomic force microscopy, or compression using parallel plates mechanically moved between them. Many devices have been used for two-dimensional studies. Such devices can only characterize a single object at a time and are complicated to implement. In addition, optical analysis, especially in the case of two parallel plates, can be complex.

[0008] Furthermore, there are now many types of microfluidic devices that can perform different operations, including the control or manipulation of fluids in small volumes, typically on the microliter scale or less, and / or small objects, typically on the millimeter scale or less, in a fluid.

[0009] It is notably known from the article Y.-J. Liu et al., “Confinement and Low Adhesion Induce Fast Amoeboid Migration of Slow Mesenchymal Cells f Cell, vol. 160, no. 4, pp. 659-672, Feb. 2015, doi: 10.1016 / j.cell.2015.01.007 a microfluidic device comprising a chamber with 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 rise or fall of the rigid wall in the central zone. This makes it possible in particular to confine the cells in compartments that form when the rigid wall is in the low position. Such a device is only described for the study of single cells and requires good dispersion of the cells in the central zone. In addition, 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 or to multiplex several chambers.

[0010] International application WO2020084148 discloses a microfluidic device comprising a chamber for receiving material to be mechanically stimulated and a row of compression chambers separated from the receiving chamber by an elastic membrane. The compression chambers are pressurized independently of each other and are connected to each other 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 causes 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. In addition, the achievable compression range is limited.Moreover, the deformation is localized in regions close to the elastic membrane and is not transmitted to objects further in the microchannel.

[0011] There is therefore a need for a microfluidic device that is simple to manufacture, inexpensive, easy to use and offers great versatility of application allowing the application of a mechanical action on an object in order to determine its mechanical properties or response to a mechanical stimulus.

[0012] 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 formed from several cell types or biopsy samples from fibrous tissue, which exhibits anisotropy without mechanical response.

[0013] In this regard, there is a particular need, particularly in the pharmaceutical or diagnostic field, for devices capable of characterizing a heterogeneous structure, such as a biopsy. There is also a particular need for devices suitable for high-throughput and / or multiplex practice.

[0014] The invention aims to satisfy these needs.

[0015] Statement of the invention

[0016] The invention meets this need by a microphylic device comprising:

[0017] - a support comprising a substrate made of an elastically deformable material,

[0018] - 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 lower wall and at least two side walls,

[0019] - at least one support deformation chamber formed at least partially by the substrate, fluidly independent of the study chamber and extending at least partially in 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 portion of the upper wall of the study chamber in the study chamber, in particular by deformation of the upper wall.

[0020] Spatial concepts such as "upper" or "lateral" are to be understood in relation to each other. It is of course understood that the device can be oriented in all directions and that there is an orientation of the device having the above orientation characteristics.

[0021] By "made of an elastically deformable material" is meant that the substrate has a capacity for reversible deformation by application of a force. Preferably, the Young's modulus of the substrate is less than or equal to 1 GPa, better still less than or equal to 0.1 GPa.

[0022] The fact that the study chamber and the deformation chamber are arranged at least partially laterally relative to each other makes manufacturing easier 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.

[0023] In the invention, the 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 in the substrate local deformation stresses in the volume of the substrate, in particular at the upper wall of the study chamber formed by the substrate and results in the application of a local stress of the upper wall in the study chamber. The stress of the upper wall in the study chamber is therefore obtained indirectly by the deformation of the deformation chamber and depends on the local deformation stresses in the substrate generated by the deformation of the deformation chamber.

[0024] 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 in the study chamber which is simple to implement and at low cost by a simple deformation of the deformation chamber. In addition, the dimensions of the accessible study chamber allow to have a large quantity of objects of a sample simultaneously in the study chamber, which allows a study or manipulation of several micro-objects simultaneously whether they are two-dimensional micro-objects of the single cell type or three-dimensional micro-objects of the aggregate type.

[0025] The 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 suitable for such use.

[0026] The simple structure of this device allows for a great adaptability of the device for a large number of applications, in particular by simple choice of the shapes, structures and dimensions of the study and deformation chambers, 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 environment for direct or indirect stimulation of fluids, gels and / or micro-objects and by the simplicity of use of the device.

[0027] The term "pressure" here refers in particular to gauge pressure, i.e. pressure relative to ambient air pressure. Thus, a pressure of -600 mbar indicates that the pressure of the pressure chamber is 600 mbar below the ambient air pressure, for example 600 mbar below atmospheric pressure (1.01321 bar). In general, the ambient air pressure may be atmospheric air pressure. However, the device may operate in a pressurized environment, such as a high-pressure room, in which the ambient pressure is, for example, approximately twice the atmospheric pressure (2.02642 bar). The term "pressure" here may refer to both positive and negative pressures. The term "positive pressure" refers to a pressure higher than ambient pressure. Similarly, the term "negative pressure" refers to a pressure lower than ambient pressure.It is clear to the skilled person that the absolute value of the vacuum cannot exceed the ambient pressure. It is also clear to the skilled person that different pressures will be selected for different materials, i.e. the stiffer the material, the more 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 may be chosen in the range of +1000 to -1000 mbar (relative to the ambient pressure), better in the range of -100 to -800 mbar.

[0028] 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 allow the generation of mechanical stimuli, for example one or more sequences of mechanical stimuli.

[0029] Mechanical constraint

[0030] In some 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 in volume of the study chamber, the mechanical stress exerted by the upper wall being a compression stress in the direction of a decrease in 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.

[0031] Preferably, the device is configured so that a reduction in volume of the deformation chamber, in particular by the application of a negative pressure in the deformation chamber, generates a deformation of the upper wall of the study chamber in the direction of a reduction in the volume, in particular a reduction in the height, of the study chamber and so that an increase in volume of the deformation chamber, in particular by the application of a positive pressure in the deformation chamber, generates a deformation of the upper wall of the study chamber towards an increase in the volume, in particular an increase in the height of the study chamber. The upper wall can take a curved shape according to at least one cross-section, better according to at least two orthogonal cross-sections, when the deformation chamber is deformed.The curved shape may be convex towards the study chamber with volume reduction of the deformation chamber and concave towards the study chamber with volume increase of the deformation chamber. Such deformation facilitates implementation because the volume reduction of the study chamber is done by applying a negative pressure which is easier to maintain in a microfluidic device, the application of a positive pressure being able to more easily generate a rupture of the support in particular at the junction between several layers, in particular a rigid plate and the substrate. A direct deformation by a deformation chamber as mentioned in the aforementioned prior arts would go in the opposite direction because, 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 of the study chamber.

[0032] Preferably, the device is configured so that the side walls of the study chamber do not deform by a deformation greater than 5%, better still greater than 3%, better still do not deform substantially. This makes it possible to have a deformation only on the height more easily controllable and more effective because as explained previously a lateral deformation would be in the opposite direction of the deformation of the upper wall.

[0033] Preferably, the device is configured so that the deformation of the upper wall of the study chamber towards the inside of the study chamber, at a pressure in the deformation chamber of between -100 and -800 mbar, is greater than or equal to 10%, better still greater than or equal to 20%, even better still greater than or equal to 40%, even better still greater than or equal to 60% of the height of the study chamber.

[0034] In some embodiments, the device is configured to exert a force, preferably a compressive force, on the contents of the study chamber, including the study medium and / or one or more micro-objects. Such a force may generate a stimulus on the study medium and / or the one or more micro-objects.

[0035] Control unit

[0036] Preferably, the device comprises a unit for controlling the deformation of the deformation chamber, in particular by applying a positive or negative pressure in the deformation chamber, fluidically connected to the deformation chamber, in particular by a channel. The deformation control unit can be configured to apply a negative pressure in the deformation chamber of the substrate. The control unit can be a programmable pressure source.

[0037] The channel connecting the control unit to the deformation chamber may extend at least partially into the substrate.

[0038] The deformation chamber may 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 an inlet and / or outlet flow controller for a deformation chamber having a fluid inlet and outlet.

[0039] Preferably, the deformation chamber is filled with a gas and the pressure of the gas in the deformation chamber is controlled by the control unit, the control unit being a pressure controller of the gas in 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 the flows into and out of the deformation chamber.

[0040] Study room

[0041] The device may comprise a supply channel for the study chamber, in particular opening outwards, for supplying the study chamber with a study medium and / or an outlet channel for the study chamber for extracting the study medium from the study chamber.

[0042] The study chamber may include one or more capillary traps in the form of protruding or recessed reliefs extending over one of the internal walls, in particular the upper wall or a lower wall.

[0043] The device may comprise an additional chamber upstream and / or downstream of the study chamber, fluidically connected to the study chamber. The additional chamber may be of a greater height than the study chamber.

[0044] Without pressure in the deformation chamber, the study chamber may be of substantially polygonal section, in particular rectangular. Preferably, the study chamber has a ratio of its greatest width at the base to its height greater than or equal to 5, better still greater than or equal to 8, even better still greater than or equal to 10.

[0045] The study room can have a constant height in section.

[0046] The height of the study chamber may be constant along its length. Alternatively, it may vary. Such variation along the length may allow micro-objects to be classified according to their size or may guide and facilitate the movement of micro-objects from one end of the study chamber to the other.

[0047] Preferably, the height of the study chamber is less than or equal to 5 mm, better still less than or equal to 1 mm, even better still less than or equal to 500 μm, even better still less than or equal to 200 μm.

[0048] Preferably, the width of the study chamber is less than or equal to 5 cm, better still less than or equal to 3 cm, even better still less than or equal to 1 cm, even better still less than or equal to 3 mm.

[0049] The width of the study chamber can be constant along the entire length of the study chamber or vary. Varying the width can help guide microobjects into the study chamber or classify them according to their sizes.

[0050] Deformation chamber

[0051] Preferably, the deformation chamber extends in the support opposite a plane defined by one of the side walls of the study chamber over at least part of the height of the study chamber. The deformation chamber may extend opposite one of the side walls over a height greater than or equal to 30%, better still 50%, even better still 70%, even better still 80%, of the maximum height of the deformation chamber. The deformation chamber may extend opposite one of the side walls of the study chamber over its entire height. The fact that the greater part of the deformation chamber extends opposite one of the side walls makes it easier to manufacture and improves the deformation in the sense described above. Indeed, too great an extension above the study chamber would be detrimental to the desired deformation described above.

[0052] 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, more preferably greater than or equal to 10. Such a height ratio makes it possible to have a good deformation capacity of the upper wall of the study chamber. Preferably, the deformation chamber has an extension along the upper wall of the study chamber above the latter a width less than or equal to 50%, more preferably 40%, even more preferably 30%, even more preferably 20% of the width of the study chamber or has no such extension.

[0053] 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, better still less than or equal to 50 times the height of the study chamber, better still less than or equal to 40 times the height of the study chamber, even better still less than or equal to 20 times the height of the study chamber. Such a thickness makes it possible to limit the direct deformation of the study chamber by the deformation chamber, which would go in the opposite direction to that sought, as explained previously.

[0054] The width of the deformation chamber can be less than or equal to 5 cm, better still less than or equal to 3 cm, even better less than or equal to 1 cm.

[0055] The height of the deformation chamber can be less than or equal to 1 cm, better less than or equal to 5 mm.

[0056] The deformation chamber may be substantially in the shape of an inverted trapezoid, with the base of the deformation chamber being formed by the short side of the trapezoid.

[0057] 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 in the deformation chamber as explained previously.

[0058] Support

[0059] Preferably, the substrate is at least partially transparent, more preferably completely transparent.

[0060] The support may comprise a rigid base in contact with the substrate. Preferably, the rigid base is at least partially transparent, more preferably completely transparent.

[0061] The base may be made of glass or a transparent plastic material. The rigid base may be flat. Alternatively, the base is formed from a well in a multi-well plate.

[0062] 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 the deformation chamber and the study chamber.

[0063] The deformation chamber and the study chamber may be completely delimited by the substrate and the rigid base. Preferably, they are formed in the substrate and closed at their base by the planar rigid base. Such a structure allows for easy fabrication of the fluidic device by manufacturing the substrate and the chambers in the substrate, in particular by molding, 3D printing or any other technique, then fixing the substrate on the rigid base to close the chambers.

[0064] Alternatively, they are formed partially by the substrate and by recessed surfaces in the rigid base. Alternatively, they are formed entirely in the substrate.

[0065] The substrate comprises an elastomer, in particular chosen from PD MS, Flexdym™, latex, rubber, and any other elastomer and mixtures thereof. The substrate may comprise a crosslinking agent.

[0066] The substrate may be homogeneous in its volume. Alternatively, the substrate may have areas of different density or hollows in its mass, particularly at the level of the upper wall. This may make it possible to control the deformation of the substrate and therefore of the upper wall according to a predetermined deformation.

[0067] Analysis system

[0068] The microfluidic device may comprise or be operatively coupled to an analysis system configured to analyze the microfluidic device, in particular the study chamber, in particular the study medium or the micro-object(s) in the study chamber. The analysis system may be operatively 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 control the deformation of the deformation chamber(s) based on the analysis signal.

[0069] The analysis system in the study chamber may comprise 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 the impedance, the conductivity of the study medium or an electrical action of a cellular unit during mechanical stimulation. Preferably, the analysis system is arranged on or under 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.

[0070] Plurality of deformation chambers

[0071] 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 each extending at least partially along a side wall of the study chamber.

[0072] In some embodiments, two deformation chambers extend on either side of the study chamber.

[0073] The two deformation chambers of the support can be configured to extend above the upper wall of the study chamber by less than 90%, more preferably less than 80%, more preferably less than 70%, more preferably less than 50%, of the upper wall of the study chamber and more preferably not to have an overlap with the study chamber

[0074] In some embodiments, two deformation chambers extend along the study chamber.

[0075] In some embodiments, two deformation chambers extend along the study chamber and are spaced apart by a distance such that their stress application fields in the substrate are disjoint at an area of ​​the top wall of the study chamber. This may allow for stress in the study chamber from the top wall to be non-uniform along the study chamber and to be controllable.

[0076] In some embodiments, two deformation chambers extend along the study chamber and are spaced apart by a distance such that their stress application range in the substrate at least partially intersects at the top wall of the study chamber.

[0077] The deformation chambers can be of the same or different shape and volume.

[0078] In some embodiments, two deformation chambers are controlled at the same pressure by the same deformation control unit controlling them simultaneously. Preferably, the two deformation chambers are identical and arranged symmetrically with respect to the study chamber. Thus, the deformation.

[0079] In some embodiments, two deformation chambers are deformation controlled independently of each other, such as by being linked to two different deformation control units or by being controlled differently by a single deformation control unit. Such chambers may be along a single study chamber. Such deformation chambers may be used to control the pressure and / or deformation of the upper wall according to a complex displacement.

[0080] The device may comprise a plurality of independent study chambers or preferably fluidly connected in series or in parallel with each other, the stress by the upper wall in each study chamber, in particular the deformation of the upper wall, being generated by the same deformation chamber(s) or by separate deformation chambers fluidly connected with each other or not. In the case of separate deformation chambers, they may be identical. Alternatively, they are of different shapes so as to generate different deformations of the upper wall at identical pressure.

[0081] The person skilled in the art understands from the above that the possibilities for combining and arranging deformation chambers and study chambers are multiple and the person skilled in the art will know how to adapt the structure of the microfluidic device to the application that he wishes to implement.

[0082] The microfluidic device may be symmetrical about a median plane transverse to the support.

[0083] Study environment

[0084] The study chamber may comprise a fluid study medium, in particular a liquid, or a gelled 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 the deformation of the upper wall to one or more micro-objects extending into the gel.

[0085] The gel may be a hydrogel, in particular selected from the group comprising agarose, collagen, agar gum, Matrigel™, gelatin, so-called “crosslinked” gels such as Polyethylene Glycol (PEG), or other types of hydrogels. According to certain embodiments, the study chamber comprises a study medium distinct from a gel, in particular distinct from a hydrogel.

[0086] The gel may be homogeneous within the study chamber. Alternatively, the gel is of varying density within the volume of the study chamber, including may have a density gradient from the center of the study chamber toward the side walls.

[0087] 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.

[0088] The device may be configured so that deformation of the upper wall of the study chamber generates a flow of the study medium from an inlet to an outlet in the study chamber, thereby acting in the manner of a diaphragm pump. In this case, the study chamber may substantially have a portion of a substantially frustoconical shape, the deformation chamber extending along a side wall on the large base side of the frustoconical shape, the largest deformation of the upper wall generated by the deformation of the deformation chamber being closer to the large base than to the small base. Thus, the study medium is pumped from the large base to the small base.

[0089] Micro-object

[0090] The device may comprise at least one micro-object, better still a plurality of micro-objects in the study chamber, in particular in the study environment.

[0091] Preferably, the micro-object(s) are deformable.

[0092] In some embodiments, the height of the at least one micro-object is less than 100 pm. In some embodiments, the height of the at least one micro-object is greater than 100 pm.

[0093] In some embodiments, the height of the 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.

[0094] In some embodiments, the height of the micro-object(s) is greater than or equal to the height of the study chamber. 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 comprises a gelling agent.

[0095] The micro-object(s) may be chosen 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 cells or cell aggregates derived from a biopsy of a patient's tissue, or any type of culture medium.

[0096] The microdroplets or gelled microunits may contain within them a biological material, such as a cellular unit, including a cell, a cellular aggregate, such as a spheroid or organoid, or biopsy specimens from a patient. In the case of multiple gelled microunits in the study chamber, the gelled microunits may be of different rigidities.

[0097] According to a particular embodiment, the micro-object(s) comprise or consist of a biological material, and / or a medium suitable for the culture of a 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).

[0098] The term "biological material" may designate any cell type, 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.

[0099] The term "culture medium" is likely to designate any physiologically acceptable medium, suitable for the cultivation of 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).

[0100] According to certain embodiments, said biological material comprises or consists of living, quiescent and / or cultured cells.

[0101] In a non-exhaustive manner, said biological material may comprise or consist of one or more types of cells, chosen from eukaryotic or prokaryotic cells, or even viruses or viral particles. 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 cells; for example, derived from humans, mice, rats, dogs, cats, cows, pigs, chickens, goats, horses, yeasts or others.

[0102] According to certain particular embodiments, said biological material may comprise or consist of non-eukaryotic cells, such as prokaryotic cells; for example bacteria or archaebacteria.

[0103] 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.

[0104] According to certain particular embodiments, said biological material may comprise or consist of one or more genetically modified, transfected cells, and / or the expression of one or more nucleic acids of which is modulated.

[0105] According to certain particular embodiments, said biological material may comprise or consist of one or more somatic cells, or even progenitor cells, for example one or more pluripotent or multipotent cells, such as embryonic cells, or even cells derived from embryos.

[0106] According to certain particular embodiments, said cells may be in differentiated form, or in the process of differentiating, or even undifferentiated.

[0107] According to certain embodiments, said cells may be cancerous or precancerous cells, or even derived from cancerous tissues, of various origins.

[0108] According to certain embodiments, said cells (for example cancer cells) can 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, liver, lung, lymphatic tissue, muscle, heart, pancreas, pituitary gland, prostate, testicle, kidney, salivary gland, skin, thyroid, immune system, epithelial, endothelial, mesothelial.

[0109] According to certain particular embodiments, said cells can be chosen from blood cells and / or from the immune system, in particular chosen from a list consisting of: erythrocytes, platelets, T lymphocytes, B lymphocytes, leukocytes, dendritic cells, macrophages.

[0110] According to certain embodiments, said cells may be derived from patients or individuals suffering from one or more pathologies.

[0111] According to certain embodiments, said biological material comprises or consists of a three-dimensional culture of cells.

[0112] According to certain embodiments, said biological material and / or culture medium is likely to comprise one or more compounds of interest, for example one or more pharmaceutical active ingredients, such as one or more antibodies, or antibody fragments.

[0113] According to certain embodiments, said biological material comprises or consists of one or more spheroids.

[0114] According to certain embodiments, said biological material comprises or consists of one or more organoids.

[0115] The term "spheroid" is likely to designate 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 in a three-dimensional manner, 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.

[0116] The term "organoid" is likely to designate any cellular structure obtained by expansion of one or more cell types specific to a given tissue, and capable, in whole or in part, of self-organizing and / or differentiating.

[0117] Process of study or manipulation

[0118] The invention also relates to a method for studying or manipulating at least one study medium or a micro-object, using the microfluidic device as described above, comprising the introduction of the study medium and / or the micro-object into the or one of the study chambers, the application of a pressure, in particular negative, in the deformation chamber(s) generating a stress by 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 by the upper wall in the study chamber applying a mechanical stimulus to the study medium or the micro-object or generating a displacement of the study medium or the micro-object in the study chamber.

[0119] The mechanical stimulus can be compression, depression and / or mechanical force.

[0120] In some embodiments, the method comprises studying or moving one or more micro-objects contained in a study medium in the study chamber.

[0121] In some embodiments, the method comprises moving a study medium, particularly a fluid, in the study chamber. The deformation of the upper wall may generate a stress on the fluid study medium in the study chamber which causes a displacement field in the study medium contained in the study chamber. This displacement field may be unidirectional, which generates a general fluid flow in one direction, or be more diffuse, depending on the desired applications.

[0122] Preferably, the pressure applied in the deformation chamber is between +1000 and -1000 mbar, better between 0 and -800 mbar.

[0123] During the application of pressure in the deformation chamber, the upper wall may take a curved shape along at least one cross-section, more preferably along at least two orthogonal cross-sections. The curved shape may be convex, in particular parabolic, towards the study chamber.

[0124] 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%, more preferably greater than 5%, more preferably do not deform substantially.

[0125] The method may comprise introducing one or more micro-objects into the study chamber, in particular into a fluid medium contained in the study chamber. The introduction of the micro-object into the study chamber may be carried out by pipetting the micro-object into the study chamber with a fluid, in particular a liquid.

[0126] 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 with the study medium. The method may comprise 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).

[0127] The method may comprise a step of introducing a curing agent into the study chamber, and curing the curing agent. The curing of the curing agent may comprise 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 crosslinks. It will be apparent to those skilled in the art which curing methods are suitable for a particular curing agent.

[0128] The study medium may comprise 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 method may comprise 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 makes it possible in particular to immobilize the micro-object(s). The fact that the study medium is gelled makes it possible in particular to transfer the deformation of the upper wall by compression of the gel to one or more micro-objects extending in the gel. The method may comprise in this case the observation during or after deformation of the upper wall of the displacement of the micro-object(s) in the gel.

[0129] The micro-object(s) may be selected from microdroplets, gelled hydrogel microunits, or any biological material as previously mentioned. The sample may be a cell sample. The cell sample may be a patient biopsy sample.

[0130] In the case of multiple gelled microunits in the study chamber, the gelled microunits may be of different rigidities.

[0131] The device may comprise a study chamber having a height that varies continuously, in particular decreases continuously, or varies in discrete steps along its length or width, and the method may comprise the introduction into the study chamber of a plurality of deformable micro-objects of different heights, all greater than or equal to the smallest height of the study chamber, the method comprising 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 to the micro-objects according to their position in the study chamber.

[0132] The method may be a method of mechanical deformation 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) is 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.

[0133] 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 the study medium.

[0134] The method may comprise the application of a deformation cycle of the or each deformation chamber to generate a deformation cycle of the upper wall, in particular in the direction of a reduction in the height of the study chamber, and thus apply a series of stimuli to the object(s). By "deformation cycle of the or each deformation chamber", is meant the application of a series of several successive deformations and / or relaxations of the or each compression chamber according to a predetermined pressure diagram for each of the deformation chambers. According to certain embodiments, the pressure cycle may comprise an identical repetitive series of a pressure, in particular negative, and a relaxation for a predetermined time. According to certain embodiments, the pressure cycle may comprise a predetermined series of different pressures, in particular negative, and / or relaxations for a predetermined time.Such deformation cycles make it possible to stimulate the study environment and / or the object(s) according to a series of identical or different stimuli.

[0135] The method may comprise detecting 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 detecting the overall displacement of the object(s), the local displacement of a portion of the micro-object(s), in particular of one or more cell nuclei of one or more cell units, the overall deformation of the micro-object(s), in particular the instantaneous radial deformation, and / or the local deformation of a portion 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.

[0136] The method may include gelation of the medium before or after deformation of the upper wall or after one or more cycles of deformation of the upper wall. The method may include cyclic deformation of the object(s), gelation of the liquid in the study chamber and detection of the movement of the micro-object(s) in the study chamber.

[0137] The method may comprise the detection, in particular optical, of the lateral deformation, in particular the instantaneous radial deformation, and / or of the 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.

[0138] The method may comprise comparing the detected global or local displacement and / or the detected local or global deformation of the micro-object(s) with the global or local displacement and / or the global or local deformation of a real or simulated reference sample at the same position in the study chamber.

[0139] The method may comprise determining the rheological and / or biological properties of the micro-object(s) or a portion of the micro-object(s) by determining the deformation of the micro-object(s) or a portion of the micro-object(s) 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-objects or a portion of the micro-object(s) whose rheological properties are known. For example, the method may comprise introducing gelled micro-units of different known rigidities, each containing a cellular unit to be studied, and determining the rheological properties of the cellular unit by determining the deformation of the cellular unit in the gelled micro-units and comparing it with the deformation of the hydrogel of the gelled micro-units at the same position whose rheological properties are known.

[0140] The method may include deforming the study medium and / or pumping the study medium into the study chamber. Deforming the top wall of the study chamber may generate a flow of the study medium from an inlet to an outlet in the study chamber, thereby acting in the manner of a diaphragm pump. This deformation and / or pumping of the study medium may be done with or without a micro-object in the study medium.

[0141] According to certain particular objects of said method, the invention also relates to a method for studying or manipulating, in particular, biological material; comprising the steps of: a) providing a microfluidic device as described above comprising at least one biological material in the study chamber; b) applying a pressure, in particular negative, in the deformation chamber generating a deformation of the upper wall of the study chamber; c) detecting 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.

[0142] Brief description of the drawings

[0143] [Fig 1] schematically represents an example of a microfluidic device according to the invention,

[0144] [Fig 2] schematically represents a cross-section of the microfluidic device of Figure 1,

[0145] [Fig 3] schematically represents a section of the microfluidic device of Figure 1 during the application of negative pressure in the deformation chamber,

[0146] [Fig 4A] is in cross section a variant of microfluidic device,

[0147] [Fig 4B] is in cross section a variant of microfluidic device,

[0148] [Fig 4C] is in cross section a variant of microfluidic device,

[0149] [Fig 5A] is in cross section a variant of microfluidic device during application of negative pressure in the deformation chamber,

[0150] [Fig 5B] is in cross section a variant of microfluidic device during application of negative pressure in the deformation chamber,

[0151] [Fig 6] is in cross section a variant of microfluidic device,

[0152] [Fig 7A] is a longitudinal section of the study chamber of the microfluidic device of Figure 1, the study chamber containing a study medium and microobjects, [Fig 7B] is a longitudinal section of the study chamber of a variant microfluidic device, the study chamber containing a study medium and microobjects,

[0153] [Fig 7C] is a longitudinal section of the study chamber of a variant of microfluidic device,

[0154] [Fig 8] is in cross section a variant of microfluidic device,

[0155] [Fig 9] shows in cross section the device of figure 8, a negative pressure being applied in the deformation chambers,

[0156] [Fig 10A] represents in section the stress field applying in the substrate when a negative pressure is applied in a variant of microfluidic device,

[0157] [Fig 10B] represents the deformation at the center of the study chamber as a function of the negative pressure applied in the deformation chambers,

[0158] [Fig 11 A] represents in section the stress field applying in the substrate when a positive pressure is applied in a variant of microfluidic device,

[0159] [Fig 1 IB] is a graph representing the deformation of the upper wall in the center of the study chamber as a function of the positive pressure applied in the deformation chambers,

[0160] [Fig 12] represents a variant of microfluidic device according to the invention,

[0161] [Fig 13] is a graph representing the change in area of ​​micro-objects in top view 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,

[0162] [Fig 14] represents a variant of microfluidic device according to the invention,

[0163] [Fig 15] is a microscopy image of the study chamber containing a study medium and micro-objects,

[0164] [Fig 16] is a graph representing the change in area of ​​a micro-object in top view as a function of time during a sinusoidal pressure cycle,

[0165] [Fig 17A] represents the radial deformation of cells in a spheroid of a first sample when pressure is applied in the deformation chambers,

[0166] [Fig 17B] represents the radial deformation of cells in a spheroid of a second sample when pressure is applied in the deformation chambers, [Fig 18] represents a variant of a microfluidic device according to the invention, [Fig 19] represents a variant of a microfluidic device according to the invention, [Fig 20] represents in section a portion of a study chamber containing a hydrogel study medium and micro-objects in the study medium,

[0167] [Fig 21] represents in section a portion of a study chamber containing a study medium containing hydrogel units of different rigidities containing microobjects,

[0168] [Fig 22] represents a variant of microfluidic device according to the invention,

[0169] [Fig 23] represents a section along XXIII-XXIII of the device of Figure 22, the study chamber containing a hydrogel study medium and micro-objects in the study medium, and

[0170] [Fig 24] represents a variant of microfluidic device according to the invention.

[0171] Detailed description

[0172] 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 fluidly independent of one another.

[0173] The support 20 comprises a substrate 25 made of an elastically deformable material, for example PDMS. PDMS is advantageous due to its non-toxic, elastic and transparent characteristics, and its low cost. However, the invention is not limited to PDMS and other elastically deformable materials can be used. The channels or chambers present on the chip can be obtained by a method comprising soft lithography and / or bonding. Preferably, the substrate 25 has a Young's modulus less than or equal to 1 GPa, better still less than or equal to 0.1 GPa. In the example illustrated, 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 of the substrate 25 closed at their bases by the rigid base 28.

[0174] However, it could be otherwise as illustrated for example in Figures 4A to 4C. For example, the rigid base could comprise one or more grooves partially forming one or more study and deformation chambers, as illustrated in Figures 4A and 4B. Alternatively, the substrate 25 could comprise cavities completely included in the substrate 25, the rigid base limiting the deformation of the substrate by the base of the study 30 and deformation 40 chambers and not delimiting the study or deformation chambers, as illustrated in Figure 4C. As a further variant, the rigid base is other than a glass plate. The rigid base can be any rigid support capable of supporting the substrate 25, in particular a multi-well plate or a plate made of another rigid material.

[0175] 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.

[0176] The study chamber 30 and deformation chamber 40 are arranged in the support so as to extend laterally relative to each other at least partially. In the examples illustrated in FIGS. 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 cross-sectional shape. As illustrated in FIGS. 5A and 5B, it may take another cross-sectional shape, in particular a substantially polygonal shape, in particular a substantially convex quadrilateral shape, for example substantially trapezoidal, with a short side forming the upper end, as illustrated in FIG. 5A, or with a short side forming the base, as illustrated in FIG. 5B.One or more of the edges may be rounded, particularly when they form an acute angle in the substrate, as illustrated in Figure 5B. In addition, the deformation chamber 40 may extend partially above the study chamber 30, particularly the upper wall 32, as illustrated in Figure 5B.

[0177] The study chamber 30 may have a substantially polygonal shape in cross section, in particular a convex quadrilateral, in particular a rectangular one.

[0178] The greatest width w at the base of the study chamber 30 may be between 200 μm and 5 cm, better between 300 μm and 1 cm, for example substantially equal to 4 mm and the greatest height h of the section of the study chamber 30 may be smaller than its greatest width w at the base and is preferably less than or equal to 10 mm, better less than or equal to 500 μm. The greatest height h may be defined in particular as a function of the object(s) to be studied in the study chamber 30 between the upper 32 and lower 34 walls. Preferably, the ratio of the greatest width w to the greatest height h of the study chamber is greater than or equal to 5, better than 10. The dimensions of the section of the study chamber 30 are in particular chosen as a function of the object(s) to be studied in the study chamber and the study method envisaged as will appear clearly on reading the examples below.

[0179] The greatest width W at the base of the deformation chamber 40 may be between 200 μm and 5 cm, better between 300 μm and 1 cm, for example substantially equal to 5 mm and the greatest height H of the section of the study chamber 30 is preferably smaller than its greatest width W at the base and is preferably less than or equal to 10 mm, better less than or equal to 7 mm, for example substantially equal to 3 mm. The greatest height H is defined in particular as a function of 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, better than 10.

[0180] The height H of the deformation chamber 40 is greater than or equal to the height h of the study chamber 30. The height ratio is preferably greater than or equal to 5, better still greater than or equal to 10.

[0181] The deformation chamber 40 and the study chamber 30 are spaced apart 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 substantially equal to 2 mm.

[0182] It is understood that the dimensions and shapes must be adapted according to the object(s) to be studied in the study room and the study process envisaged, as will become clear from reading the examples below.

[0183] In the example of Figure 1, the study chamber 30 is connected at its two longitudinal ends to a fluid inlet and outlet and by fluid 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 fluidically 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 comprising only a fluid inlet or not comprising one, in particular in the case of a study in a stationary study environment of one or more micro-objects enclosed in the study chamber.

[0184] The deformation chamber 40 is preferably fluidically connected to a deformation control unit 45, in particular to a unit for controlling the pressure in the deformation chamber, by a channel 42 extending in the substrate 25 and / or at the interface between the substrate 25 and the rigid base 28, as illustrated in FIGS. 1 and 15. Such a control unit 45 makes it possible to apply a positive or negative pressure in the deformation chamber 40, which deforms it, as illustrated in FIG. 3 in particular. Such a deformation of the deformation chamber generates stresses in the substrate 25, which deforms the upper wall 32 of the study chamber 30 by transmission of stress within the elastically deformable substrate 25.As illustrated in Figures 3, 5A, 5B and 9 and 10A, in the case of a negative pressure in the deformation chamber 40, the volume of the study chamber 30 decreases by lowering the center of the upper wall 32. The upper wall 32 takes, in this case, a curved shape towards the inside of the study chamber 30 in at least two orthogonal directions. Conversely, as illustrated in Figure 11A, in the case of a positive pressure, the volume of the study chamber 30 increases by raising the center of the upper wall 32. The upper wall 32 takes, in this case, a curved shape towards the outside of the study chamber 30.The greatest deformation d of the upper wall 32, in particular at its center, depends in particular on the absolute pressure in the deformation chamber, as illustrated in the graphs of FIGS. 10B and 11B, and on the parameters of the device, in particular size, shape and flexibility and are easily determinable by comparative tests within the reach of those skilled in the art. For example, the maximum deformation d3 of the upper wall 32 in the case of a deformation chamber in the shape of an inverted trapezoid, according to FIG. 5B for example, is greater than in the case d.2 of a deformation chamber of rectangular shape, according to FIG. 2 for example, itself greater than that in the case d1 of a deformation chamber of non-inverted trapezoid shape, according to FIG. 5A for example.

[0185] Preferably, the pressure in the deformation chamber is between -100 and 1000 mbar, better between -100 and -500 mbar.

[0186] During the application of the pressure in the deformation chamber 40, for a pressure in the deformation chamber 40 less than -500 mbar, the side walls 33 of the study chamber 30 do not deform by a deformation greater than 5%, better still greater than 3%. They may buckle slightly due to the deformation of the upper wall 32. The greatest width of the study chamber 30 is substantially constant during the application of the pressure in the deformation chamber 40, in particular does not change by more than 2%, better still does not change by more than 1%. This is notably due 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.

[0187] For example, for a pressure in the deformation chamber 40 between -100 and -500, the deformation of the upper wall 32 of the study chamber 30 towards the inside of the study chamber 30 is greater than or equal to 10%, better still greater than or equal to 40% of the height of the study chamber, as can be seen in FIG. 10B representing the height of deformation of the upper wall in micrometers relative to the pressure applied in the deformation chamber.

[0188] The study chamber may have a constant height h in cross-section, as illustrated in Figures 1 to 4.

[0189] The study chamber may have a constant height h along the entire length of the study chamber 30, as illustrated in FIG. 1.

[0190] Alternatively, as illustrated in Figure 7B, the height h of the study chamber 30 may vary along the length of the study chamber 30 monotonically. This may allow the objects to be studied to be classified or guided in the study chamber 30.

[0191] Alternatively, one of the walls of the study chamber, in particular the upper wall 30 or the lower wall 33, may comprise reliefs of surfaces 38 recessed or projecting along the length, as illustrated in FIG. 7C, or the width, as illustrated in FIG. 6. Such reliefs may be capillary traps with variable trapping forces depending on 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, in particular 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 permanent, being more or less strong depending on the height of the study chamber 30, or take place only when the height of the study chamber is below a threshold height. The guidance can be done directly on the microobject(s), or indirectly, in particular via a liquid study medium or via a hydrogel.

[0192] The study chamber 30 may be of substantially cylindrical shape. In the example of FIG. 1, the study chamber 30 has a substantially straight block 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 FIGS. 14, 15, and 18 or other non-cylindrical shape.

[0193] Furthermore, the invention is not limited to a single deformation chamber 40 for deforming the upper wall 32 of a study chamber 30. As illustrated in FIGS. 8 to 10 in particular. 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 study chamber 30. Preferably, the two deformation chambers 40a and 40b are connected to the same pressure control member 45 and the pressure is identical in the two chambers 40a and 40b, as illustrated in FIG. 14. The presence of two deformation chambers 40a and 40b makes it possible in particular to have 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.

[0194] Alternatively or additionally, not shown, the device may include deformation chambers making it possible to deform the upper wall according to a more complex deformation profile than a simple curve, in particular deformation chambers distributed along the length, being spaced or not and having an identical or different applied pressure.

[0195] In certain embodiments, in particular illustrated in Figures 12 and 14, the device may comprise a plurality of study chambers 30a and 30b fluidly 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 bordered laterally by one or more deformation chambers controlled independently or simultaneously by one or more pressure control units 45.

[0196] Study medium and micro-objects 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 usually 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 in motion from a fluid inlet to a fluid outlet during the application of pressure.

[0197] The study medium may be liquid. It may comprise 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 gelled, it preferably has a lower Young's modulus than the substrate. Such a hardened study medium allows the transfer of the deformation of the upper wall to one or more micro-objects extending in the study medium. The latter may be homogeneous in the study chamber 30. Alternatively, it is of variable density in the volume of the study chamber 30, in particular may have a density gradient from the center of the study chamber towards the side walls.

[0198] The micro-object(s) are preferentially deformable.

[0199] The micro-object(s) may be chosen from microdroplets, gelled micro-units, in particular comprising a hydrogel and which may be in the form of gelled micro-droplets, and / or any biological material, such as cellular units, in particular cells or cellular aggregates, such as spheroids or organoids. In the case of several gelled micro-units in the study chamber, the gelled micro-units may be of different rigidities. The microdroplets or gelled micro-units may contain within them a biological material, in particular 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 biopsy of a patient.

[0200] According to a particular embodiment, the micro-object(s) comprise or consist of a biological material, and / or a medium suitable for the culture of a 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). The term “biological material” is capable of designating any cell type, 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.

[0201] The term "culture medium" is likely to designate any physiologically acceptable medium, suitable for the cultivation of 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.

[0202] According to certain embodiments, said biological material comprises or consists of living, quiescent and / or cultured cells.

[0203] In some embodiments, the biological material comprises or consists of nucleic acids or nucleic acid fractions, including DNA and / or RNA.

[0204] In a non-exhaustive manner, said biological material may comprise or consist of one or more types of cells, chosen from eukaryotic or prokaryotic cells, or even viruses or viral particles.

[0205] 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, derived from humans, mice, rats, dogs, cats, cows, pigs, chickens, goats, horses, yeasts or others.

[0206] According to certain particular embodiments, said biological material may comprise or consist of non-eukaryotic cells, such as prokaryotic cells; for example bacteria or archaebacteria.

[0207] 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.

[0208] According to certain particular embodiments, said biological material may comprise or consist of one or more genetically modified, transfected cells, and / or cells in which the expression of one or more nucleic acids is modulated. 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.

[0209] According to certain particular embodiments, said cells may be in differentiated form, or in the process of differentiating, or even undifferentiated.

[0210] According to certain embodiments, said cells may be cancerous or precancerous cells, or even derived from cancerous tissues, of various origins.

[0211] According to certain embodiments, said cells (for example cancer cells) can 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, liver, lung, lymphatic tissue, muscle, heart, pancreas, pituitary gland, prostate, testicle, kidney, salivary gland, skin, thyroid, immune system, epithelial, endothelial, mesothelial.

[0212] According to certain particular embodiments, said cells can be chosen from blood cells and / or from the immune system, in particular chosen from a list consisting of: erythrocytes, platelets, T lymphocytes, B lymphocytes, leukocytes, dendritic cells, macrophages.

[0213] In some 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.

[0214] According to certain embodiments, said biological material comprises or consists of a three-dimensional culture of cells.

[0215] According to certain embodiments, said biological material and / or culture medium is likely to comprise one or more compounds of interest, for example one or more pharmaceutical active ingredients, such as one or more antibodies, or antibody fragments.

[0216] According to certain embodiments, said biological material comprises or consists of one or more spheroids.

[0217] According to certain embodiments, said biological material comprises or consists of one or more organoids. The term "spheroid" is likely to designate any aggregate, of any type of cells, 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.

[0218] The term "organoid" is likely to designate any cellular structure obtained by expansion of one or more cell types specific to a given tissue, and capable, in whole or in part, of self-organizing and / or differentiating.

[0219] The height of the micro-object(s) may be greater than or equal to the height of the study chamber, as illustrated in FIGS. 7 A 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).

[0220] Analysis system

[0221] As illustrated in Figure 14, the microfluidic device may comprise or be operatively 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 be operatively coupled to the deformation control unit of the deformation chamber or not, in particular the analysis system may provide an analysis signal to the control unit. The control unit may control the deformation of the deformation chamber(s) based on the analysis signal.

[0222] 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 the impedance, the conductivity of the study medium or an electrical action of a cellular unit during mechanical stimulation. Preferably, the analysis system 60 is arranged on or under 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. Method

[0223] In some 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 determined deformation may be the overall deformation of the micro-object(s) or the local deformation of only a portion of the micro-object(s), in particular of one or a few cells of a cell aggregate or of the nucleus of a cell. Such a measurement of the deformation may make it possible to determine the mechanical properties of the micro-object(s) or of the portion of the micro-object(s), in particular their rigidity.

[0224] This determined deformation can be direct when the micro-object(s) are smaller than the height h of the study chamber 30 or indirectly via 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).

[0225] The method may comprise 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 to deduce therefrom a characteristic of the micro-object(s), in particular a deformability characteristic and / or a biological characteristic.

[0226] In some embodiments, the deformation of the upper wall of the study chamber causes a displacement of the micro-object(s) in the study chamber 30 or of a portion of the micro-object(s) in the micro-object(s), in particular of one or a few cells in a cell aggregate or of the cell nucleus. This displacement may take place during the deformation due to the action of the upper wall 32 on the study medium, in particular gelled medium containing the micro-object(s) or on the micro-object(s).

[0227] The method may comprise comparing the movement of the micro-object(s) or part of the micro-object(s) with the movement of a reference sample in the same study chamber to deduce a characteristic of the micro-object(s), in particular a biological characteristic.

[0228] In some embodiments, the method may include the study of the deformation of the micro-object(s) and their displacement as described previously. In some embodiments, the method includes the application of a pressure cycle in the deformation chamber(s) 40 corresponding to the periodic repetition for a predetermined time of a pressure / relaxation succession in the deformation chamber(s) 40. The different pressures of the cycle may be identical or not and spaced apart by an identical or not time. Such a pressure cycle makes it possible to apply a cyclic deformation of the upper wall 32 for a determined time, which generates a 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), in particular of the biological material to such cyclic stimulation.The method may comprise 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 time of application 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 studying the displacement of the micro-object(s) over the time of application 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, in particular in the case of micro-object(s) comprising biological material.

[0229] We will now detail several examples of use of the microfluidic device described above.

[0230] Example

[0231] Cell culture and spheroid formation

[0232] H4-II-EC3 and NIH-3T3 and MDA-MB-231 GFP cancer cells are cultured in DMEM medium, supplemented with 10% FBS, and 1% penicillin and / or streptomycin antibiotics.

[0233] Spheroids are produced from non-adherent 96-well U-shaped plates (Corning catalog 7007).

[0234] To obtain spheroid cocultures, the two cell types are mixed in different ratios depending on the desired spheroid size and the intended application. For example, a mixture of 100 H4-II-EC3 cells and 400 NIH-3T3 cells results in larger spheroids, and 100 cells of each type results in smaller spheroids. The cells are cocultured for 72 hours to form the spheroids.

[0235] Images were captured with a Nikon spinning disk Ti2 motorized epifluorescence microscope equipped with a 20x objective lens. Illumination was generated from a Lumencor LED light source for epifluorescence, or an Oxius laser set for confocal imaging, and images were captured by a Hamamatsu C13440-20CU SCMOS camera. Raw data were collected with Nikon Elements imaging software (Version 5.11.01, Build 1367). fluorescent

[0236] Cells can be labeled either by immunofluorescence or by standard detection kits. All reagents are introduced with a pipette into the device, 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 reagent, according to the commercial protocol (Biotium catalog number: 30108).

[0237] Measuring the size of spheroids

[0238] The sizes of the spheroids are analyzed from the microscopy images using a macro in ImageJ software (FIKI). For each pressure value, the perimeter, total Do, and major and minor axes of the spheroids can be measured and recorded.

[0239] Example 1: Analysis of the overall compression of spheroids as a function of the position in the study chamber in the context of a cobblestone-shaped study chamber

[0240] A device according to Figure 12 is manufactured. A monolithic polydimethylsiloxane (PDMS) substrate 25 is manufactured from a mold manufactured by 3D printing, the mold having reliefs corresponding to the inverted trace of the study chambers 30 and deformation chambers 40, as well as conduits 34 and 42. The substrate 25 thus manufactured is placed on a microscope plate 28, the side of the substrate comprising the imprints of the chambers being in contact with the microscope plate. The device comprises two identical study chambers 30a and 30b, of elongated shape and substantially rectangular transverse and longitudinal sections, connected together 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 opening 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 the same pressure. The study chambers have a height approximately equal to 100 pm.

[0241] A sample of spheroids formed as described above with a diameter substantially equal to 130 ± 20 μm is introduced into the study chambers 30a and 30b using a pipette in a fixed position 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 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 the pressure in the deformation chambers are compared to determine the percentage change in area of ​​the spheroids representing the deformation of the spheroids by the deformation of the upper wall.Figure 13 illustrates the average area change as a function of pressure in the spheroid deformation chambers in the lateral position in the study chambers (bottom curve) and in the 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 the lateral position of the latter.

[0242] Example 2: Analysis of the global behavior of spheroids during 1' of a cycle within the framework of a study chamber in the shape of a cylinder with a circular base.

[0243] A device according to Figure 14 is manufactured. A monolithic polydimethylsiloxane (PDMS) substrate 25 is manufactured from a mold manufactured by 3D printing, the mold having reliefs corresponding to the inverted trace of the study chambers 30 and deformation chambers 40, as well as conduits 34 and 42. The substrate 25 thus manufactured is placed on a microscope plate 28, the side of the substrate comprising the imprints of the chambers being in contact with the microscope plate. 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, inlet and outlet conduits of the device are of greater height than the height of the study chambers. The two study chambers 30a and 30b are each bordered on either side by two deformation chambers 40a and 40b connected to the same pressure source by a conduit 42 leading 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.

[0244] A sample of spheroids formed as described above with a diameter substantially equal to 130 ± 20 μm are introduced into the study chambers 30a and 30b using a pipette in a fixed position 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 can be seen in Figure 15, before the application of a sinusoidal pressure cycle at a negative pressure of -300 mbar and with a frequency of 0.5 Hz. Figure 16 illustrates the average change in area of ​​the spheroids over time during the application of the pressure cycle. It appears from Figure 16 that the deformation of the spheroids follows the pressure cycle well over time and therefore that the upper wall deforms well periodically according to the pressure cycle.

[0245] Example 3: Analysis of the deformation of individual cells in spheroids when applied within a study chamber in the form of a cylinder with a circular base.

[0246] In the device of Example 2, samples of spheroids formed as described above with a diameter substantially equal to 130 + 20 μm are introduced into the study chambers 30a and 30b using a pipette in a fixed position in the study chambers. It has been demonstrated that the local deformation of individual cells and their nuclei in the spheroids, fluorescently labeled as described above, can be observed. It is then possible to study the deformation of the nuclei in the cells, the deformation of the cells and / or the behaviors / rearrangements of cells in the spheroids during the deformation of the spheroids.

[0247] Example 4: Analysis of the deformation of individual cells in spheroids when applied within a study chamber in the form of a cylinder with a circular base. In the device of Example 2, two samples of spheroids of different cell combinations and of diameter substantially equal to 130 ± 20 μm are introduced into the study chambers 30a and 30b using a pipette in a fixed position in the study chambers. The first sample is composed of spheroids formed solely of H4-II-EC3 cells and the second sample is composed of spheroids formed from a coculture of H4-II-EC3 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-II-EC3 cells form a shell around the former.The deformation field of the spheroids, in the bright-field image plane, is measured using particle image velocimetry (PIV) software, and the outward radial deformation is calculated. The graphs in Figures 17A to 17C are obtained. Graph 17A represents the radial deformation of a spheroid of the first sample. Graph 17B represents the radial deformation of a spheroid of the second sample. For the first sample containing only H4-II-EC3 cells, the deformation is from the center to the edge and gradually increases. For the second sample containing only the coculture of H4-II-EC3 and NIH-3T3 cells, the spheroid has a static core with little deformation and a lateral region with more deformation.

[0248] The hypothesis that such a difference in behavior was specifically linked to the self-organization of these different cells into a core-envelope structure, and therefore that either H4-Ü-EC3 cancer cells or NIH-3T3 cells could exhibit different mechanical characteristics, one being more rigid than the other, was tested.

[0249] NIH-3T3 cells were labeled with GFP before mixing them with H4-II-EC3 cells. Observing the cells in the spheroids, it is noted that NIH-3T3 cells move towards the interior of the spheroid forming a nucleus, while cancer cells are arranged at the periphery.

[0250] These results therefore suggest that NIH-3T3 cells assemble into a nucleus forming a more rigid structure compared to the lateral part consisting of H4-II-EC3 cells.

[0251] In particular, the quantitative data indicate that the nucleus of NIH-3T3 cells is approximately 30 to 50 times stiffer than the outer layer formed by H4-II-EC3 cells. It is therefore possible to determine the mechanical properties of different three-dimensional cell arrangements of heterogeneous structure and to determine, by comparison with the mechanical properties of known cell arrangements, the biological properties of a cell sample to be studied. This example therefore demonstrates that the device is particularly suitable for the characterization of heterogeneous structures, as well as for the identification of a demarcation between healthy cells and cancer cells.

[0252] Devices according to figures 18 and 19 are manufactured with a multi-well plate as a base. 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 manufactured by 3D printing, the mold having ribs corresponding to the inverted trace of the study chambers 30 and deformation chambers 40, as well as conduits 34 and 42. The substrate 25 thus manufactured is of a size adapted to the wells of a multi-well plate, the side of the substrate comprising the imprints of the chambers being in contact with the bottom of the well.

[0253] The device according to figure 18 comprises a single study chamber which is substantially circular in top view connected to two channels 34 for inlet and outlet of the sample, the channels forming between them an acute angle and a moon-shaped deformation chamber in bottom view partially surrounding the study chamber. The pressure in the deformation chamber is controlled using a control channel 42. At -600 mbar in the deformation chamber 40, the upper wall of the study chamber deforms in its center towards the inside of the study chamber by approximately 43 μm.

[0254] The device according to figure 19 comprises a single study chamber which is substantially circular in top view 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 in kidney shapes in top view. The pressure in the deformation chambers is controlled using a common control channel 42. They are therefore subjected to the same pressure. At -600 mbar in the deformation chambers 40a and 40b, the upper wall 32 of the study chamber deforms in its center towards the inside of the study chamber by approximately 49 μm. of a cell aggregate in a gelled medium. In the device of Example 1, a sample of spheroids of metastatic breast cancer cells of type MDA-MB-231 in a liquid medium are introduced into the study chamber 30 with a diameter greater than the height of the study chamber. The 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 hours in the deformation chambers 40. After the stimulation, the liquid medium is replaced by a basement membrane matrix, called Matrigel™, and the cell migration in the matrix in response to the cyclic stimulation is studied over time for 16 hours. The cancer cells of MDA-MB-231 migrate in the matrix towards the outside, which makes it possible to study the metastatic capacity of the cancer cells in response to an external stimulus.

[0255] Example 7: Stimulation of spheroids in a hydrogel.

[0256] In the device of Example 1, a sample of spheroids in a gelled medium of garose is studied. The spheroids are smaller in dimension than the height of the study chamber. Such a configuration makes it possible to stimulate and apply mechanical stress to the spheroids instead of imposing a deformation. The upper wall will deform by its deformation the hydrogel containing the spheroids, which will laterally displace the spheroids with the hydrogel in the study chamber as illustrated in Figure 20. This makes it possible to apply a shear stress and / or extensions on the spheroids.

[0257] Such deformation dynamics of spheroids in hydrogel could be used to analytically determine the rheological properties of tissues or cell aggregates embedded inside a chamber. Indeed, the amount of deformation of the spheroid can be extracted by comparing it to the deformation of the surrounding hydrogel with known rheological properties: A very soft microtissue will deform strongly while a very stiff microtissue will remain almost undeformed compared 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.

[0258] Example 8: Stimulation of in hydrogel units.

[0259] 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 in the study chamber each having spheroids. This allows for multiplexing the ability to apply different ranges of stresses in a study. As mentioned in Example 7, the hydrogel of known stiffness can be used to analyze the stiffness of the spheroids in relation to the rheological properties of the hydrogel. With hydrogel units, it is possible to have hydrogel units of different rheological properties in the same study chamber. These different hydrogel units with different stiffnesses when compressed can produce different flows within them, thus producing different forces and mechanical stresses in the spheroids embedded within as illustrated in Figure 21.

[0260] Example 9: Microfluidic devices generating a displacement of the study medium and

[0261] A device according to Figure 22 is manufactured according to the same method as in Example 1. The device comprises a study chamber 30 in the shape of a funnel in top view and a deformation chamber 40 extending along the long side of the funnel shape. The deformation chamber 40 has in section substantially the same shape as the deformation chamber of Figure 5B. The deformation of the deformation chamber by the application of pressure in the deformation chamber generates a deformation of the upper wall 32 of the study chamber in the widened part of the funnel only, as illustrated in Figure 23. A hydrogel comprising spheroids is introduced into the study chamber.

[0262] 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 spheroids embedded in the hydrogel, as illustrated in Figure 23. Such deformation produces spatially heterogeneous deformation in the hydrogel. Different device configurations can be designed to achieve different hydrogel deformations. For example, introducing reliefs 65 on the edges in the study chamber as illustrated in Figure 24 generates more bending-type deformations in the hydrogels between reliefs in the study chamber.

[0263] The invention is not limited to the examples and embodiments described above. The characteristics of the different embodiments described above can be combined within additional variants.

Claims

Claims 1. Microfluidic device (10) comprising: - a support (20) comprising 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 in the support (20) at least partially along one of the side walls (33) of the study chamber (30), the device being configured so that the deformation of the deformation chamber (40, 40a, 40b) generates the application of a mechanical stress of at least a portion of the upper wall (32) of the study chamber in the study chamber (30), in particular by deformation of the upper wall (32).

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

3. Device according to any one of the preceding claims, configured so that a reduction in volume of the deformation chamber (40, 40a, 40b), in particular by a 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 the volume, in particular a reduction in the height h, of the study chamber (30) and so that an increase in volume of the deformation chamber (40, 40a, 40b), in particular by a 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 the volume, in particular an increase in the height h, of the study chamber, said deformation of the upper wall (32) generating the application of a mechanical stress in the study chamber (30, 30a, 30b).

4. Device according to any one of the preceding claims, in which 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 still greater than or equal to 10.

5. Device according to any one of the preceding claims, in which 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 still less than or equal to 50 times the height h of the study chamber (30, 30a, 30b), better still less than or equal to 40 times the height h of the study chamber (30, 30a, 30b), even better still 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, in which 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 still, over a height greater than or equal to 70% of its maximum height H, even better still 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 side 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 by 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.

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

10. A device according to any preceding claim, comprising or being operatively coupled to an analysis system (60) configured to analyzing the microfluidic device, in particular the study chamber, in particular 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, in which the study chamber (30, 30a, 30b) comprises a study medium (50) and / or at least one micro-object (55), better still a plurality of micro-objects in the study chamber, in particular in the study medium (50).

12. Device according to claim 11, in which the micro-object(s) (55) may be chosen from microdroplets, gelled micro-units, 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. 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 the introduction of the study medium (50) and / or the micro-object (55) into the or one of the study chambers (30, 30a, 30b), the application of a pressure, in particular negative, in the or at least one deformation chamber (40, 40a, 40b) generating a stress by the upper wall (32) of the study chamber in the study chamber (30, 30a, 30b), preferably a deformation of the upper wall, in particular in the direction of a reduction in the height of the study chamber, the stress by the upper wall in 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 microobject (55) in the study chamber (30, 30a, 30b).

14. Method according to claim 13, in which the study medium (50) comprises a gelling agent, in particular a hydrogel, introduced with the microobject(s) into the study chamber or afterwards into the study medium contained in the study chamber, the study medium (50) having, after gelling, a lower Young's modulus than the substrate (25).

15. Method according to one of claims 13 and 14, comprising the deformation of the study medium and / or the pumping of the study medium into the study chamber (30, 30a, 30b), the 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 membrane pump.

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