Microfluidic device for studying and / or manipulating study medium and / or at least one microobject

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) and having: at least one upper wall (32), at least one lower wall (34), and at least two side walls (33) at least partially formed by a base (25); at least one substrate deformation chamber (40, 40a, 40b) formed at least partially by the substrate (25) and fluidically independent of the study chamber (30, 30a, 30b) and extending at least partially inside the support (20) along one of the side walls (33) of the study chamber (30), the microfluidic device is configured such that deformation of the deformation chamber (40, 40a, 40b) results in application of a mechanical stress to at least one portion of the upper wall (32) in the study chamber (30).

Description

TECHNICAL FIELD

[0001] The present invention relates to a microfluidic device for investigating and / or manipulating investigation media and / or micro-objects in an investigation chamber having at least one elastically deformable upper wall. The present invention also relates to a method for investigating and / or manipulating at least one micro-object by means of the microfluidic device.

[0002] A variety of methods for mechanically manipulating and characterizing micro-objects, in particular single cells or cell aggregates, are currently available. These methods are used, inter alia, in the field of mechanobiology. For example, these methods are used to characterize the mechanical properties of objects or to analyze the reaction of objects to mechanical stimuli. BACKGROUND

[0003] Methods known in the art are, for example, micropipette aspiration, atomic force microscopy and compression using parallel plates that are mechanically moved relative to each other. Many devices have been used for two-dimensional investigations. Such devices can only characterize one object at a time and are complex to use. Furthermore, optical analysis, in particular in the case of two parallel plates, can be complex.

[0004] In addition, a variety of types of microfluidic devices are currently available for performing various operations, in particular monitoring or manipulating small volumes of fluid, typically in the order of microliters or less, and / or small-sized objects in the fluid, typically in the order of millimeters or less.

[0005] A microfluidic device is known in particular from the article by Y.-J. Liu et al., "Confinement and Low Adhesion Induce Fast Amoeboid Migration of Slow Mesenchymal Cells", Cell, Volume 160, Issue 4, Pages 659-672, February 2015, doi:10.1016 / j.cell.2015.01.007, which comprises a chamber having a central compression region with a rigid upper wall and a peripheral region that can be pressurized or depressurized in order to control the lifting of the rigid wall in the central region. In particular, when the rigid wall is in a lower position, this device makes it possible to confine cells in the compartment formed. This device is described as being intended only for investigating single cells and requires good dispersion of the cells in the central region. Furthermore, the fluidic connectivity between the central region and the peripheral region excludes the use of such a device in the presence of fluid flow in the chamber or multiplexing of multiple chambers.

[0006] A microfluidic device is known from international patent application WO2020084148. This microfluidic device includes: a chamber for receiving a substance to be mechanically stimulated; and a row of compression chambers separated from the receiving chamber by an elastic membrane. The compression chambers are independently pressurized and interconnected by chamber separators. In this device, the receiving chamber deforms directly due to the deformation of the elastic membrane caused by the compression chamber. The positive pressure in the compression chamber causes a decrease in the volume of the compression chamber through deformation of the elastic membrane toward the receiving chamber, and vice versa. Because of the thin elastic membrane between the receiving and compression chambers, this device is complex to manufacture and fragile. Furthermore, the achievable compression range is limited. Moreover, the deformation is confined to the region near the elastic membrane and cannot be transmitted to objects further away in the microchannel.

[0007] Therefore, there is a need for a microfluidic device that is easy to manufacture, relatively inexpensive, easy to use, and highly versatile in application, capable of applying mechanical action to objects, especially for determining the mechanical properties of objects or their response to mechanical stimuli.

[0008] What is particularly needed is a device that can be used to study complex and heterogeneous biological materials, such as cancerous tissue, which are known for their unique mechanical properties and may in particular consist of multiple cell types or biopsy samples taken from fibrous tissue that exhibit anisotropy without mechanical response.

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

[0010] There is also a particular need for devices suitable for high-throughput and / or multiplexing practices.

[0011] The purpose of this invention is to meet these needs. Summary of the Invention

[0012] This invention addresses this need using a microfluidic device, the microfluidic device comprising:

[0013] - A support member, the support member comprising a base made of an elastically deformable material;

[0014] - A research chamber extending inside the support member, the research chamber having: at least one upper wall, the upper wall being at least partially, preferably entirely, formed by the base; at least one lower wall; and at least two side walls;

[0015] - At least one support deformation chamber, said support deformation chamber being at least partially formed by a substrate and fluidly independent of the study chamber, and extending at least partially within the support along one of the sidewalls of the study chamber.

[0016] The device is configured such that deformation of the deformation chamber results in mechanical stress being applied to at least a portion of the upper wall of the study chamber within the study chamber, particularly through deformation of the upper wall.

[0017] Spatial concepts such as “upper” and “lateral” should be understood relative to each other. Of course, it should be understood that the device can be oriented in any direction, and that there exists a device orientation that gives it the orientation characteristics described above.

[0018] "Elastically deformable material" refers to a substrate that has the ability to deform reversibly when a force is applied. The Young's modulus of the substrate is preferably less than or equal to 1 GPa, more preferably less than or equal to 0.1 GPa.

[0019] The fact that the study chambers and deformation chambers are arranged at least partially side-by-side facilitates manufacturing and limits costs. Simple molds can be used. There is no need to use thin films that could make the structure fragile or complicate its manufacturing.

[0020] In this invention, the deformation of the deformable chamber is made possible because it is at least partially formed by an elastically deformable substrate. This deformation of the deformable chamber generates local deformation stresses within the substrate volume, particularly at the upper wall of the study chamber formed by the substrate, and causes local stresses to be applied to the upper wall of the study chamber. Therefore, the stress in the upper wall of the study chamber is indirectly obtained through the deformation of the deformable chamber and depends on the local deformation stresses in the substrate generated by the deformation of the deformable chamber.

[0021] This device enables the study and / or manipulation of fluids, gels, and / or microobjects within a research chamber by applying mechanical action to the upper wall of the chamber. It is convenient and cost-effective due to the simple deformation of the chamber. Furthermore, the achievable size of the research chamber allows for the simultaneous containment of a large number of sample objects, enabling the simultaneous study or manipulation of multiple microobjects, whether they are two-dimensional single-cell types or three-dimensional aggregate types.

[0022] Lateral positioning also makes it easy to observe the study chamber from above or below perpendicular to the upper wall and to observe the effects of stress on fluids, gels and / or micro-objects along the axis, especially by microscopy, and the size of the device is particularly well-suited for this purpose.

[0023] The device's structure makes it highly adaptable to a variety of applications, particularly through the simple selection of the shape, structure, and size of both the study chamber and the deformation chamber; through precise control of the stress generated by the upper wall; particularly through control of the pressure applied to the deformation chamber; and through adjustment of the study medium to directly or indirectly stimulate fluids, gels, and / or microobjects; and the device's ease of use.

[0024] In this context, the term "pressure" specifically refers to gauge pressure, that is, pressure relative to atmospheric pressure. Therefore, -600 mbar indicates that the pressure in the pressure chamber is 600 mbar lower than ambient pressure, for example, 600 mbar lower than atmospheric pressure (1.01321 bar). Ambient pressure is typically atmospheric pressure. However, the device can operate in a pressurized environment, such as in a high-pressure chamber, where the ambient pressure is, for example, approximately twice atmospheric pressure (2.02642 bar).

[0025] In this context, the term "pressure" can refer to both positive and negative pressure. "Positive pressure" refers to pressure above ambient pressure. Similarly, "negative pressure" refers to pressure below ambient pressure. Those skilled in the art will understand that the absolute value of the pressure reduction cannot exceed ambient pressure. They will also understand that different pressures are selected for different materials; that is, the greater the hardness of a material, the higher the pressure required to achieve the desired deformation (and vice versa). For example, for materials with hardness comparable to polydimethylsiloxane (PDMS), the pressure (relative to ambient pressure) can be selected in the range of +1000 mbar to -1000 mbar, and more preferably in the range of -100 mbar to -800 mbar.

[0026] This device enables the application of mechanical stimuli to the research medium and / or one or more micro-objects. It can also enable the generation of mechanical stimuli, such as one or more sequences of mechanical stimuli.

[0027] Mechanical stress

[0028] In some embodiments of the invention, the device is configured such that deformation of the deformation chamber causes deformation of the upper wall of the study chamber in the direction of increasing or decreasing volume of the study chamber. The mechanical stress applied by the upper wall is either compressive stress that decreases the volume of the study chamber or expansion stress that increases the volume of the study chamber. The deformation of the upper wall results in the application of mechanical stress within the study chamber.

[0029] The device is preferably configured such that, specifically by applying negative pressure within the deformation chamber, the upper wall of the study chamber deforms in a direction that reduces the volume of the study chamber, particularly its height, thereby reducing the volume of the deformation chamber; and is configured such that, specifically by applying positive pressure within the deformation chamber, the upper wall of the study chamber deforms in a direction that increases the volume of the study chamber, particularly its height, thereby increasing the volume of the deformation chamber. When the deformation chamber deforms, the upper wall may have a curved shape in at least one cross section, preferably in at least two mutually orthogonal cross sections. This curved shape may be a shape that bulges towards the study chamber when the volume of the deformation chamber decreases, or a shape that concaves towards the study chamber when the volume of the deformation chamber increases. This deformation is beneficial because the volume of the study chamber is reduced by applying negative pressure, which is easier to maintain in a microfluidic device, while applying positive pressure is more likely to cause support failure, particularly at the junction between layers (especially between a rigid plate and a substrate). As mentioned in the prior art above, direct deformation through the deformation chamber will work in the opposite direction because, in this case, the increase in the volume of the deformation chamber causes the study chamber to deform in the direction that reduces the volume of the study chamber.

[0030] The device is preferably configured such that the sidewalls of the study chamber deform by no more than 5%, preferably no more than 3%, and even more preferably almost no deformation. This allows deformation to occur only in the height direction, which is easier to control and more effective because, as explained above, the direction of lateral deformation is opposite to the direction of deformation of the upper wall.

[0031] The device is preferably configured such that, under conditions where the pressure inside the deformation chamber is between -100 mbar and -800 mbar, the amount of deformation of the upper wall of the study chamber toward the interior of the study chamber is greater than or equal to 10% of the height of the study chamber, more preferably greater than or equal to 20%, more preferably greater than or equal to 40%, and even more preferably greater than or equal to 60%.

[0032] In some embodiments of the invention, the device is configured to apply force, preferably a compressive force, to the contents of the study chamber, particularly to the study medium and / or one or more microobjects. This force can stimulate the study medium and / or one or more microobjects.

[0033] Control Unit

[0034] The device preferably includes a unit for controlling the deformation of the deformation chamber, particularly by applying positive or negative pressure within the deformation chamber, said unit being fluidly connected to the deformation chamber via a channel. The unit for controlling deformation can be configured to apply negative pressure within the deformation chamber of the substrate. The control unit can be a programmable pressure source.

[0035] The channel connecting the control unit to the deformation chamber can extend at least partially within the substrate.

[0036] The deformable chamber is filled with fluid. In the case of a deformable chamber with only a single inlet, the amount of fluid in the deformable chamber is controlled by a control unit. Alternatively, for a deformable chamber that includes a fluid inlet and a fluid outlet, an inlet and / or outlet flow controller is configured.

[0037] The deformation chamber is preferably filled with gas, and the gas pressure in the deformation chamber is preferably controlled by a control unit, which is a controller for controlling the gas pressure in the deformation chamber. Alternatively, the deformation chamber is filled with liquid, and the control unit controls the amount of liquid in the deformation chamber, the pressure in the deformation chamber depending on the amount of liquid present in the deformation chamber or the inlet and outlet flow rates of the deformation chamber.

[0038] Study chamber

[0039] The device may include: a channel for feeding a research chamber, the channel specifically opening to the outside, for feeding a research medium into the research chamber; and / or an outlet channel for the research chamber for extracting the research medium from the research chamber.

[0040] The study chamber may include one or more capillary traps on one of the inner walls, particularly on the upper or lower wall, the capillary traps being convex or concave in shape.

[0041] The device may include an additional chamber located upstream and / or downstream of the research chamber and fluidly connected to the research chamber. The height of the additional chamber may be greater than the height of the research chamber.

[0042] Under uncompressed conditions in a deformable chamber, the chamber can have a roughly polygonal cross-section, and in particular, a rectangular cross-section.

[0043] The ratio of the maximum width at the base of the study chamber to its height is preferably greater than or equal to 5, more preferably greater than or equal to 8, and even more preferably greater than or equal to 10.

[0044] The study chamber can have a constant cross-sectional height.

[0045] The height of the study chamber can remain constant along its length. Alternatively, the height of the study chamber can also vary. This variation along the length allows for the classification of micro-objects based on their size, or the guidance and facilitation of the movement of micro-objects from one end of the study chamber to the other.

[0046] The height of the study chamber is preferably less than or equal to 5 mm, more preferably less than or equal to 1 mm, even more preferably less than or equal to 500 μm, and even more preferably less than or equal to 200 μm.

[0047] The width of the study chamber is preferably less than or equal to 5 cm, more preferably less than or equal to 3 cm, even more preferably less than or equal to 1 cm, and even more preferably less than or equal to 3 mm.

[0048] The width of the study chamber can remain constant along its entire length or vary. Variation in width allows for guiding micro-objects within the study chamber or classifying micro-objects based on their size.

[0049] Deformation chamber

[0050] The deformation chamber preferably extends within the support member over at least a portion of the height of the study chamber, facing a plane defined by one of the sidewalls of the study chamber. The deformation chamber may extend facing one of the sidewalls at a height greater than or equal to 30%, more preferably 50%, even more preferably 70%, and still more preferably 80% of the maximum height of the deformation chamber. The deformation chamber may extend facing one of the sidewalls of the study chamber over its entire height. The majority of the deformation chamber faces one of the sidewalls, which facilitates the fabrication and improvement of deformation as described above. In practice, extending too far above the study chamber will affect the desired deformation described above.

[0051] The deformable chamber preferably extends within the substrate at a height greater than or equal to the height of the chamber under study, with the height ratio preferably greater than or equal to 5, and more preferably greater than or equal to 10. Such a height ratio allows the upper wall of the chamber under study to have good deformability.

[0052] The deformable chamber preferably extends along and on the upper wall of the study chamber and the width of the deformable chamber is less than or equal to 50%, more preferably 40%, even more preferably 30%, or even more preferably 20% of the width of the study chamber, or has no such extension.

[0053] The minimum thickness of the base between the study chamber and the deformation chamber is preferably greater than or equal to the height of the study chamber. More preferably, the minimum thickness of the base between the study chamber and the deformation chamber is less than or equal to 100 times the height of the study chamber, more preferably less than or equal to 50 times the height of the study chamber, even more preferably less than or equal to 40 times the height of the study chamber, and even more preferably less than or equal to 20 times the height of the study chamber. Such a thickness allows for limiting the direct deformation of the study chamber by the deformation chamber, which could produce effects contrary to the intended outcome, as explained above.

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

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

[0056] The deformable chamber can be roughly inverted trapezoidal, with the base of the deformable chamber formed by the short side of the trapezoid.

[0057] The deformation chamber preferably contains air. Alternatively, the deformation chamber may contain liquid, and the deformation of the deformation chamber may depend on the liquid pressure within the deformation chamber, as explained above.

[0058] Support components

[0059] The substrate is preferably at least partially transparent, and more preferably completely transparent.

[0060] The support may include a rigid base that contacts the substrate. The rigid base is preferably at least partially transparent, and more preferably completely transparent.

[0061] The base can be made of glass or transparent plastic material. A rigid base can be planar. Alternatively, the base can be formed from the pores of a multi-cell culture plate.

[0062] In one embodiment of the invention, the rigid base forms at least one wall of the deformable chamber. In an embodiment of the invention that is not mutually exclusive with the foregoing embodiments, the rigid base forms at least one wall of the study chamber. In an embodiment of the invention that is not mutually exclusive with the foregoing embodiments, the rigid base forms at least one wall of both the deformable chamber and the study chamber.

[0063] The deformable and research chambers can be entirely defined by a substrate and a rigid base. Preferably, the deformable and research chambers are formed in the substrate and closed at their bottom by a flat, rigid base. This structure allows for the easy fabrication of microfluidic devices by first manufacturing the substrate and the chambers within it, particularly by molding, 3D printing, or any other technique, and then attaching the substrate to the rigid base to close the chambers.

[0064] Alternatively, the deformation chamber and the research chamber are partially formed by recessed surfaces in the substrate and the rigid base. In other alternatives, the deformation chamber and the research chamber may be formed entirely in the substrate.

[0065] The substrate includes an elastomer, particularly selected from PDMS, Flexdym™, latex, rubber, and any other elastomer and mixtures thereof. The substrate may include a crosslinking agent.

[0066] The substrate can be homogeneous throughout its entire volume. Alternatively, the substrate may include regions of varying density or depressions in its mass, particularly at the upper wall. This allows for control over the deformation of the substrate, and thus the deformation of the upper wall, to achieve a predetermined deformation.

[0067] Analysis System

[0068] Microfluidic devices may include an analysis system, or be functionally coupled to an analysis system, configured to analyze the microfluidic device, particularly a study chamber, and especially a medium or one or more micro-objects within the chamber. The analysis system may be functionally coupled to a unit that controls the deformation of a deformable chamber, and in particular, the analysis system may provide an analysis signal to a control unit. The control unit can direct the deformation of one or more deformable chambers based on changes in the analysis signal.

[0069] The analytical system within the study chamber may include an optical imaging system, particularly an optical microscope or electron microscope, or a confocal imaging system, or a system for measuring electrical properties, particularly the impedance, conductivity, or electrical activity of cellular units during mechanical stimulation of the study medium. The analytical system is preferably positioned above or below the study chamber and configured to acquire images of the fluid, gel, and / or one or more microobjects within the study chamber. The analytical system may be configured to detect static or dynamic information regarding the fluid, gel, and / or one or more microobjects within the study chamber.

[0070] Multiple deformable chambers

[0071] In some embodiments of the invention, the device includes at least two deformable chambers of a substrate, the deformable chambers being at least partially formed by the substrate and independent of the study chamber, and each deformable chamber extending at least partially along the sidewall of the study chamber.

[0072] In some embodiments of the present invention, two deformable chambers are provided, one on each side of the study chamber.

[0073] The two deformable chambers of the support can be configured to extend on the upper wall of the study chamber by less than 90%, more preferably less than 80%, even more preferably less than 70%, and even more preferably less than 50%, and even more preferably not overlap with the upper wall of the study chamber at all.

[0074] In some embodiments of the invention, two deformable chambers extend along the study chamber.

[0075] In some embodiments of the invention, two deformation chambers extend along the study chamber and are spaced apart by a certain distance, such that the stress application fields of the two deformation chambers on the substrate are independent of each other in a certain region of the upper wall of the study chamber. This allows non-uniform and controllable stress to be generated along the study chamber via the upper wall.

[0076] In some embodiments of the invention, two deformable chambers extend along the study chamber and are spaced apart from each other such that the stress fields of the two deformable chambers in the substrate at least partially intersect at some point on the upper wall of the study chamber.

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

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

[0079] In some embodiments of the invention, the two deformation chambers can be controlled independently of each other regarding deformation, specifically, the two deformation chambers are connected to two different deformation control units, or are controlled in different ways by a single deformation control unit. Such deformation chambers can be positioned along the same study chamber. Such deformation chambers enable control of the pressure and / or deformation acting on the upper wall during complex movements.

[0080] The device may include multiple study chambers, which are independent of each other, or preferably fluidly connected in series or parallel. The stress induced by the upper wall, particularly the deformation of the upper wall, in each study chamber is generated by the same one or more deformation chambers, or by different deformation chambers that are fluidly interconnected or not interconnected. In the case of different deformation chambers, the different deformation chambers may be identical. Alternatively, the different deformation chambers may have different shapes to produce different upper wall deformations under the same pressure conditions.

[0081] Those skilled in the art will understand from the above description that there are multiple possibilities for the combination and arrangement of the deformable chamber and the research chamber, and will know how to adapt the structure of the microfluidic device to the intended application.

[0082] Microfluidic devices can be symmetrical about the midplane of the support.

[0083] Research medium

[0084] The study chamber can contain a study medium in a fluid state (particularly a liquid or gel state). In the case of a gel, its Young's modulus is preferably lower than that of the substrate. Such a gel allows deformation of the upper wall to be transferred to one or more micro-objects within the gel.

[0085] The gel can be a hydrogel, particularly selected from the group consisting of agarose, collagen, agar gum, Matrigel™, gelatin, so-called “crosslinked” gels (e.g., polyethylene glycol (PEG)) or other types of hydrogels.

[0086] In some embodiments of the present invention, the research chamber contains a research medium that is different from a gel, particularly a non-hydrogel research medium.

[0087] The gel can be homogeneous within the study chamber. Alternatively, the gel density varies throughout the volume of the study chamber, and in particular, it can exhibit a density gradient from the center of the study chamber to the sidewalls.

[0088] The gel can completely fill the study chamber, or alternatively, it can consist of individual gel islands, which are randomly or precisely positioned within the plane of the study chamber.

[0089] The device can be configured such that deformation of the upper wall of the study chamber causes the study medium to flow from the inlet to the outlet within the study chamber, thus functioning similarly to a diaphragm pump. In this configuration, the study chamber can have a generally frustoconical portion, with the deformable chamber extending along a sidewall on the larger base of the frustoconical shape. The maximum deformation of the upper wall caused by the deformation of the chamber is closer to the larger base than the smaller base. Therefore, the study medium is pumped from the larger base to the smaller base.

[0090] micro objects

[0091] The device can contain at least one micro-object, or better yet, multiple micro-objects, within a research chamber, especially within a research medium.

[0092] Preferably, one or more micro-objects are deformable.

[0093] In some embodiments of the present invention, the height of at least one micro-object is less than 100 µm. In some embodiments of the present invention, the height of at least one micro-object is greater than 100 µm.

[0094] In some embodiments, the height of at least one micro-object is between 100µm and 1000µm, for example, between 100µm and 900µm, for example, between 100µm and 800µm, for example, between 100µm and 700µm, for example, between 100µm and 600µm, for example, between 100µm and 500µm, for example, between 100µm and 400µm, for example, between 100µm and 300µm, or for example, between 100µm and 200µm.

[0095] In some embodiments of the present invention, the height of one or more micro-objects is greater than or equal to the height of the research chamber.

[0096] In some embodiments of the invention, the height of one or more micro-objects is less than the height of the study chamber. In this case, the study medium preferably comprises a gelling agent.

[0097] One or more micro-objects may be selected from droplets, gel-like micro-units (particularly including hydrogels and possibly in the form of gel-like droplets) and / or any biological material, such as cellular units, particularly cells or cell aggregates, such as spheroids or organoids, or cells or cell aggregates derived from patient tissue biopsies, or any type of medium.

[0098] Microdroplets or gel-like microunits can contain biological material, such as cellular units, particularly cells, cell aggregates (e.g., spheroids or organoids), or biopsy tissue from a patient. In cases where multiple gel-like microunits are present in a study chamber, the gel-like microunits can have different stiffnesses.

[0099] In a particular embodiment of the invention, one or more micro-objects comprise biological material and / or a medium for biological material, or are composed of biological material and / or a medium for biological material, such as any medium containing one or more nutrients (e.g., proteins, peptides, amino acids and / or any carbon source).

[0100] The term "biological material" can be used to refer to any cell type, including embryonic cells or non-embryonic cells, as well as any type of extract, lysate, or component of one or more cells, particularly those derived from humans, animals, or plants.

[0101] The term “medium” can be used to refer to any physiologically acceptable medium for any one or more cell types, particularly any medium that includes at least one carbon source and energy source, especially nutrients such as peptides, polypeptides and / or amino acids, carbohydrates, essential minerals and metals, and buffers.

[0102] In some embodiments of the present invention, the biological material comprises or contains living cells, quiescent cells, and / or cultured cells.

[0103] The biological material may include or consist of one or more cell types selected from eukaryotic cells, prokaryotic cells, or viruses or viral particles. This list is not exhaustive.

[0104] In some specific embodiments of the present invention, the biological material may include or be composed of eukaryotic cells, such as mammalian cells, particularly human or non-human mammalian cells, such as cells from humans, mice, rats, dogs, cats, cattle, pigs, chickens, goats, horses, yeast, etc.

[0105] In some specific embodiments of the present invention, the biological material may include or be composed of non-eukaryotic cells, such as prokaryotic cells, such as bacteria or archaea.

[0106] In some specific embodiments of the present invention, the biological material may include or consist of viruses or virus particles, such as enveloped or non-enveloped viruses, RNA viruses, DNA viruses, or any other virus in the Baltimore classification, such as viruses belonging to groups I, II, III, IV, V, VI, or VII of that classification.

[0107] In some specific embodiments of the present invention, the biological material may include or consist of transfected cells with one or more gene modifications, and / or the expression of one or more of their nucleic acids is regulated.

[0108] In some specific embodiments of the present invention, the biological material may include or be composed of one or more somatic cells or progenitor cells, such as one or more pluripotent cells or pluripotent cells, such as embryonic cells or cells obtained from an embryo.

[0109] In some specific embodiments of the present invention, the cells may be differentiated, in the process of differentiation, or in an undifferentiated state.

[0110] In some embodiments of the present invention, the cells may be cancer cells or precancerous cells, or cells obtained from cancerous tissue, or cells from different sources.

[0111] In some embodiments of the invention, the cells (e.g., cancer cells) may be derived from one or more tissues or organs from the following list: adrenal glands, bladder, blood, bones, bone marrow, brain, cartilage, uterus, cervix, endometrium, cornea, esophagus, gastrointestinal tract, nerves, liver, lungs, lymphatic tissue, muscles, heart, pancreas, pituitary gland, prostate, testes, kidneys, salivary glands, skin, thyroid gland, immune system, epithelium, endothelium, mesothelium.

[0112] In some specific embodiments of the present invention, the cells may be selected from blood cells and / or immune system cells, particularly from the following list, which includes: red blood cells, platelets, T lymphocytes, B lymphocytes, white blood cells, dendritic cells, and macrophages.

[0113] In some embodiments of the present invention, the cells may be obtained from patients or individuals suffering from one or more diseases.

[0114] In some embodiments of the present invention, the biological material comprises or consists of three-dimensional aggregates of cells.

[0115] In some embodiments of the present invention, the biological substance and / or medium may include one or more compounds of interest, such as one or more active pharmaceutical preparations, such as one or more antibodies or antibody fragments.

[0116] In some embodiments of the present invention, the biological material includes or consists of one or more spherical bodies.

[0117] In some embodiments of the present invention, the biological material includes or is composed of one or more organoids.

[0118] The term "sphere" can refer to an aggregate of any cell type, such as an aggregate composed of a single cell type or multiple cell types, which is capable of three-dimensional growth and can interact with one or more cell types and / or a three-dimensional matrix (e.g., Matrigel™) or any suitable type of medium.

[0119] The term "organoid" can refer to any cellular structure obtained by amplifying one or more specific cell types in a given tissue, and that the cellular structure is capable of self-organization and / or differentiation, either wholly or partially.

[0120] Research or manipulation methods

[0121] The present invention also relates to a method for studying or manipulating at least one research medium or a microobject using the microfluidic device described above, the method comprising: introducing the research medium and / or microobject into a research chamber or one of the research chambers; and generating stress in the research chamber caused by the upper wall of the research chamber, preferably by deformation of the upper wall, particularly by deformation along a direction that reduces the height of the research chamber, by applying pressure, particularly negative pressure, in one or more deformable chambers, the stress generated by the upper wall in the research chamber exerting mechanical stimulation on the research medium or microobject, or thereby generating movement of the research medium or microobject in the research chamber.

[0122] Mechanical stimulation can be compression, downward pressure, and / or mechanical force.

[0123] In some embodiments of the present invention, the method includes studying the motion of one or more micro-objects contained in a study medium within a study chamber.

[0124] In some embodiments of the invention, the method includes the movement of a study medium (particularly a fluid state study medium) within a study chamber. Deformation of the upper wall can generate stress on the fluid study medium within the study chamber, thereby causing a motion field within the study medium contained in the study chamber. This motion field can be unidirectional, resulting in a uniform fluid flow in one direction, or it can be a more dispersed flow, depending on the intended application.

[0125] The pressure applied to the deformation chamber is preferably between +1000 mbar and -1000 mbar, and more preferably between 0 mbar and -800 mbar.

[0126] During the application of pressure within the deformable chamber, the upper wall may exhibit a curved shape in at least one cross-section, preferably in at least two mutually orthogonal cross-sections. This curved shape may be convex, particularly parabolic, along the direction of the chamber under study.

[0127] During the application of pressure in the deformation chamber, the sidewalls of the chamber preferably deform by no more than 10%, more preferably by no more than 5%, and even more preferably by no significant deformation at all.

[0128] This method may include introducing one or more micro-objects into a study chamber, particularly into a fluid medium contained within the study chamber. The micro-objects can be introduced into the study chamber using a pipette that uses a fluid, particularly a liquid, to introduce the micro-objects.

[0129] The study medium may be a fluid that circulates at least when one or more micro-objects are introduced into the device and / or when pressure is applied in one or more deformable chambers. One or more micro-objects may be introduced into the study chamber along with the study medium.

[0130] This method may include introducing one or more chemical or physiological elements into the research medium, particularly introducing a hardening agent, especially a gelling agent, after the introduction of one or more micro-objects.

[0131] This method may include the steps of introducing a hardener into a study chamber and hardening the hardener. Hardening the hardener may include one or more of the following: polymerization, heating, denaturation, irradiation (e.g., using radiation and / or light, particularly ultraviolet light), gelation, physical crosslinking, such as (reversible) hardening via ionic interactions, and enzymatic crosslinking. Those skilled in the art will clearly understand which hardening methods are suitable for a particular hardener.

[0132] The study medium may include a gelling agent, particularly a hydrogel, which may be introduced into the study chamber along with one or more microobjects, or subsequently introduced into the study medium contained within the study chamber. The method may include gelling the study medium prior to deformation of the upper wall. The Young's modulus of the gelled study medium is preferably lower than that of the substrate. This particularly enables the one or more microobjects to remain stationary. Gelation of the study medium allows deformation of the upper wall to be transferred to one or more microobjects within the gel by compressing the gel. In this case, the method may include observing the movement of one or more microobjects within the gel during or after deformation of the upper wall.

[0133] One or more micro-objects may be selected from microdroplets, gelled hydrogel microunits, or any of the aforementioned biological substances. The sample may be a cell sample. The cell sample may be a sample taken from a patient biopsy.

[0134] In cases where multiple gel-like microunits exist within a study chamber, these gel-like microunits can have different hardness levels.

[0135] The device may include a study chamber whose height varies continuously along its length or width, particularly decreasing continuously, or varying in discrete steps, and the method may include introducing multiple deformable micro-objects of different heights (all greater than or equal to the minimum height of the study chamber) into the study chamber, the method including automatically classifying the micro-objects in the study chamber according to their height, and the deformation of the upper wall of the study chamber applying different forces to the micro-objects according to their positions in the study chamber.

[0136] This method can be a mechanical deformation process of one or more micro-objects. The compression of one or more micro-objects by the upper wall during deformation can directly cause deformation of the micro-objects or multiple micro-objects in the study chamber. In this case, the height of the object or multiple objects is preferably greater than or equal to the height of the study chamber. Alternatively, the deformation of one or more micro-objects in the study chamber can be indirect, particularly via the study medium (preferably a gel, especially a hydrogel as described above).

[0137] The method may include fixing one or more objects in place before the upper wall deforms, particularly by compressing the object between the upper and lower walls of the study chamber before deformation, or by using one or more capillary traps in the study chamber, or by gelling the study medium.

[0138] This method may include applying a deformation cycle to one or each deformation chamber to produce a deformation cycle on the upper wall, particularly in the direction of decreasing the height of the chamber under study, and thereby applying a series of stimuli to one or more objects. The term "deformation cycle of one or each deformation chamber" refers to applying a series of successive deformations and / or relaxations to one or each compression chamber according to a predetermined pressure map for each of the deformation chambers. In some embodiments of the invention, the pressure cycle may include repeatedly applying a series of the same pressures, particularly negative pressures, and relaxations over a predetermined time period. In some embodiments of the invention, the pressure cycle may include a series of predetermined different pressures, particularly negative pressures, and / or relaxations over a predetermined time period. Such deformation cycles enable the study medium and / or object or multiple objects to be stimulated by the same or different sequences of stimuli.

[0139] This method can detect the behavior of one or more micro-objects in the study medium during and / or after 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, including detecting the overall motion of one or more objects, the local motion of some of the one or more micro-objects (especially the motion of one or more nuclei of a single or multiple cell unit), the overall deformation of one or more micro-objects (especially instantaneous radial deformation), and / or the local deformation of some or all of the micro-objects (especially the deformation of one or more nuclei of a single or multiple cell unit).

[0140] This method may include gelling the medium before or after upper wall deformation or after one or more cycles of upper wall deformation. The method may also include cyclically deforming one or more objects, gelling a liquid in a study chamber, and detecting the movement of one or more micro-objects in the study chamber.

[0141] The method may include detecting (particularly optically detecting) the lateral deformation (particularly instantaneous radial deformation) and / or local deformation of one or more micro-objects (particularly cell aggregates or cell nuclei of single cells) in response to deformation of the upper wall of the study chamber.

[0142] The method may include comparing the overall or local motion and / or local or overall deformation of one or more detected micro-objects with the overall or local motion and / or overall or local deformation of a real reference sample, or with the results of a simulation performed at the same location in the study chamber.

[0143] This method may include determining the rheological and / or biological properties of one or more micro-objects or portions thereof by determining the deformation of one or more micro-objects or portions thereof and comparing it with the deformation of a reference object with known rheological and / or biological properties, particularly with the deformation of a study medium containing one or more micro-objects or portions thereof, whose rheological properties are known. For example, the method may include: introducing gel-like micro-units of known varying hardness, each of said gel-like micro-units containing a cell unit to be studied; and determining the rheological properties of the cell unit by determining the deformation of the cell unit within the gel-like micro-unit and comparing it with the deformation of a hydrogel of the gel-like micro-unit with known rheological properties at the same location.

[0144] This method may include deforming the study medium and / or pumping the study medium into a study chamber. Deformation of the upper wall of the study chamber can induce flow of the study medium from the inlet to the outlet within the study chamber, thus acting as a membrane pump. This deformation and / or pumping of the study medium can be performed with or without microparticles in the study medium.

[0145] In certain specific embodiments of the method, the present invention also relates to a method for studying or manipulating, particularly a method for studying or manipulating biological substances, the method comprising the following steps:

[0146] a) Obtain the microfluidic device as described above, wherein the microfluidic device contains at least one biological substance in a research chamber;

[0147] b) Apply pressure, especially negative pressure, to the deformation chamber to induce deformation of the upper wall of the study chamber;

[0148] c) Detect the biological material in the research chamber;

[0149] Preferably, the biological material is contacted with one or more adhesives or compounds of interest before, during, or after the detection step. Attached Figure Description

[0150] [ Figure 1 An example of a microfluidic device according to the present invention is illustrated schematically.

[0151] [ Figure 2 ]Schematic illustration Figure 1 The cross-section of the microfluidic device shown is shown.

[0152] [ Figure 3 This schematically illustrates the process of applying negative pressure in the deformation chamber. Figure 1 The cross-section of the microfluidic device shown is shown.

[0153] [ Figure 4A [This is a cross-section of a variant microfluidic device.]

[0154] [ Figure 4B [This is a cross-section of a variant microfluidic device.]

[0155] [ Figure 4C [This is a cross-section of a variant microfluidic device.]

[0156] [ Figure 5A [This is a cross-section of the variant microfluidic device during the application of negative pressure in the deformable chamber.]

[0157] [ Figure 5B [This is a cross-section of the variant microfluidic device during the application of negative pressure in the deformable chamber.]

[0158] [ Figure 6 [This is a cross-section of a variant microfluidic device.]

[0159] [ Figure 7A ]yes Figure 1 The diagram shows a longitudinal section of the research chamber of the microfluidic device, which contains the research medium and microobjects.

[0160] [ Figure 7B [This is a longitudinal section of the research chamber of the variant microfluidic device, which contains the research medium and microobjects.]

[0161] [ Figure 7C [This is a longitudinal section of the research chamber for the variant microfluidic device.]

[0162] [ Figure 8 [This is a cross-section of a variant microfluidic device.]

[0163] [ Figure 9 [Showing] Figure 8 The diagram shows a cross-section of the device when a negative pressure is applied in the deformation chamber.

[0164] [ Figure 10A This shows a cross-section of the stress field in the substrate when a negative pressure is applied in a variant microfluidic device.

[0165] [ Figure 10B The diagram shows how the deformation at the center of the study chamber changes with the negative pressure applied in the deformation chamber.

[0166] [ Figure 11A This shows a cross-section of the stress field in the substrate when positive pressure is applied in a variant microfluidic device.

[0167] [ Figure 11B [This is a graph showing the deformation of the upper wall at the center of the study chamber as a result of the applied positive pressure in the deformation chamber.]

[0168] [ Figure 12 This illustrates a variant microfluidic device according to the present invention.

[0169] [ Figure 13 [This is a graph showing the area of ​​a micro-object viewed from above at the center of the study chamber and at a lateral position within the study chamber as a function of the negative pressure applied in the deformation chamber.]

[0170] [ Figure 14 This illustrates a variant microfluidic device according to the present invention.

[0171] [ Figure 15 These are microscope images of a study chamber containing the study medium and micro-objects.

[0172] [ Figure 16 [This is a graph showing the change in the area of ​​a micro-object over time as viewed from above during a sinusoidal pressure cycle.]

[0173] [ Figure 17A The image shows the radial deformation of cells in the spheroid of the first sample when pressure is applied in the deformation chamber.

[0174] [Figure 17B The image shows the radial deformation of cells in the spheroid of the second sample when pressure is applied in the deformation chamber.

[0175] [ Figure 18 This illustrates a variant microfluidic device according to the present invention.

[0176] [ Figure 19 This illustrates a variant microfluidic device according to the present invention.

[0177] [ Figure 20 This shows a cross-section of a portion of a research chamber containing a hydrogel research medium and microobjects located within the research medium.

[0178] [ Figure 21 The image shows a cross-section of a portion of a research chamber containing a research medium comprising hydrogel units with varying hardness and containing microparticles.

[0179] [ Figure 22 This illustrates a variant microfluidic device according to the present invention.

[0180] [ Figure 23 [Showing] Figure 22 The device is shown in a cross-section taken along line XXIII-XXIII. The study chamber contains a hydrogel study medium and micro-objects located within the study medium.

[0181] [ Figure 24 The illustration shows a variant microfluidic device according to the present invention. Detailed Implementation

[0182] Figure 1 and Figure 2 A microfluidic device 10 according to the invention is shown, which includes a support 20, within which are fluidly independent research chambers 30 and deformable chambers 40.

[0183] The support member 20 includes a substrate 25 made of an elastically deformable material, such as PDMS. PDMS offers advantages due to its non-toxic, elastic, and transparent properties, as well as its low cost. However, the invention is not limited to PDMS; other elastically deformable materials can also be used. Channels or chambers present on the puce can be obtained through soft lithography and / or bonding processes. Preferably, the Young's modulus of the substrate 25 is less than or equal to 1 GPa, more preferably less than or equal to 0.1 GPa. In the illustrated example, the support member 20 also includes a rigid base 28, particularly a glass plate, which supports the substrate 25. Study chamber 30 and deformable chamber 40 are located within cavities of the substrate 25, the bases of which are closed by the rigid base 28.

[0184] For example Figures 4A to 4CDifferent embodiments are shown. For example, the rigid base can include one or more grooves that partially form one or more study chambers and deformation chambers, such as... Figure 4A and Figure 4B As shown. Alternatively, the substrate 25 may include a cavity completely contained within the substrate 25, with a rigid base limiting deformation of the substrate by means of the bases of both the study chamber 30 and the deformable chamber 40, but without defining the study chamber or the deformable chamber, as... Figure 4C As shown. Another alternative is that the rigid base is not a glass plate. The rigid base can be any rigid support capable of supporting the substrate 25, particularly a porous culture plate or a plate made of another rigid material.

[0185] In all cases, the study chamber 40 includes at least one upper wall 32, two side walls 33, and a lower wall 34 formed by the base 25, as shown in the figure.

[0186] The study chamber 30 and the deformation chamber 40 are arranged in the support so that they extend laterally relative to each other, at least partially. Figure 1 In the example shown in Figure 4, the deformable chamber 40 has a rectangular cross-section and extends laterally over its entire height H relative to the study chamber 30. However, the cross-sectional shape of the deformable chamber 40 is not limited to a rectangle. Figure 5A and Figure 5B As shown, it can take some other cross-sectional shapes, especially roughly polygonal shapes, particularly roughly convex quadrilaterals, such as roughly trapezoids, where the shorter side forms the upper end (e.g., Figure 5A As shown), or the shorter side forms the bottom (as shown). Figure 5B (As shown). One or more edges in the edge may be rounded, especially if they form acute angles in the substrate, such as... Figure 5B As shown. Furthermore, the deformable chamber 40 can partially extend below the study chamber 30, particularly below the upper wall 32, as shown. Figure 5B As shown.

[0187] The cross-section of the chamber 30 can be approximately polygonal, especially a convex quadrilateral, and particularly a rectangle.

[0188] The maximum width w at the base of the study chamber 30 can be between 200µm and 5cm, more preferably between 300µm and 1cm, for example, substantially equal to 4mm, and the maximum height h of the cross-section of the study chamber 30 can be less than the maximum width w at its base, more preferably less than or equal to 10mm, more preferably less than or equal to 500µm. The maximum height h can be specifically defined based on one or more objects under study located between the upper wall 32 and the lower wall 34 of the study chamber 30. The ratio of the maximum width w to the maximum height h of the study chamber is preferably greater than or equal to 5, more preferably equal to 10. The selection of the cross-sectional dimensions of the study chamber 30 depends particularly on the objects under study in the study chamber and the envisioned research method, as becomes clear when reading the examples given below.

[0189] The maximum width W at the base of the deformable chamber 40 can be between 200µm and 5cm, more preferably between 300µm and 1cm, for example, substantially equal to 5mm. The maximum height H of the cross-section of the study chamber 30 is preferably less than the maximum width W at its base, and preferably less than or equal to 10mm, more preferably less than or equal to 7mm, for example, substantially equal to 3mm. The maximum height H is specifically defined according to the object or objects to be studied in the study chamber 30. The ratio of the maximum width w to the maximum height h of the study chamber is preferably greater than or equal to 5, more preferably equal to 10.

[0190] The height H of the deformable chamber 40 is greater than or equal to the height h of the study chamber 30. Preferably, the height ratio is greater than or equal to 5, and more preferably greater than or equal to 10.

[0191] The deformation chamber 40 and the research chamber 30 are spaced apart by the thickness e of the substrate 25. The minimum substrate thickness e between the research chamber and the deformation chamber is greater than or equal to the height h of the research chamber 30. Preferably, the minimum substrate thickness e between the research chamber and the deformation chamber is less than or equal to 40 times the height h of the research chamber, and more preferably less than or equal to 20 times the height h of the research chamber 30. For example, the substrate thickness is approximately 2 mm.

[0192] Obviously, the above dimensions and shapes can be adjusted according to the object or multiple objects to be studied in the laboratory and the proposed research method, as clearly shown in the examples given below.

[0193] exist Figure 1In the example, the study chamber 30 is connected to a fluid inlet and a fluid outlet at its two longitudinal ends via fluid circulation channels 34. Such channels 34 may extend within the substrate 25 and / or at the junction between the substrate 25 and the rigid base 28. These fluid circulation channels can fluidly connect the study chamber 30 to external fluid devices and / or other fluid devices connected in series with the study chamber 30. Other devices may be located outside or included within the support 20 of this microfluidic device, particularly upstream or downstream of the study chamber 30. The invention is not limited to this connection. Preferably, it is conceivable that the study chamber 30 includes only one fluid inlet or no fluid inlet, particularly for the study of one or more micro-objects enclosed within the study chamber in a static study medium.

[0194] like Figure 1 and Figure 15 As shown, the deformation chamber 40 is preferably fluidly connected to a deformation control unit 45 via a channel 42 extending in the base 25 and / or at the junction between the base 25 and the rigid base 28, and particularly fluidly connected to a unit for controlling the pressure within the deformation chamber. This control unit 45 allows for the application of positive or negative pressure within the deformation chamber 40, causing the chamber to deform, particularly as... Figure 3 As shown. This deformation of the deformable chamber generates stress in the substrate 25, which, through stress transmission within the elastically deformable substrate 25, causes deformation of the upper wall 32 of the study chamber 30. As... Figure 3 , Figure 5A , Figure 5B , Figure 9 and Figure 10A As shown, when a negative pressure exists within the deformable chamber 40, the volume of the study chamber 30 decreases due to the lowering of the center of the upper wall 32. In this case, the upper wall 32 exhibits a shape that curves inward into the study chamber 30 in at least two mutually orthogonal directions. Conversely, as... Figure 11A As shown, under positive pressure, the volume of the study chamber 30 increases due to the rise of the center of the upper wall 32. In this case, the upper wall 32 exhibits a curved shape towards the outside of the study chamber 30. The maximum deformation d of the upper wall 32, especially the maximum deformation at its center, depends particularly on the absolute pressure in the deformable chamber (e.g., ...). Figure 10B and Figure 11B (as shown in the graph) and the parameters of the device, particularly its size, shape, and flexibility, can be easily determined through comparative tests known to those skilled in the art. For example, in the case of an inverted trapezoidal deformable chamber (such as... Figure 5B As shown), the maximum deformation d3 of the upper wall 32 is greater than that of the rectangular deformation chamber (e.g. Figure 2 The deformation d2 shown is greater than that of a rectangular deformation chamber (as shown in the figure), while the deformation of a non-inverted trapezoidal deformation chamber is greater than that of a rectangular deformation chamber (as shown in the figure).Figure 5A The deformation d1 shown is as follows.

[0195] The pressure in the deformable chamber is preferably between -100 mbar and 1000 mbar, and more preferably between -100 mbar and -500 mbar.

[0196] During the application of pressure in the deformation chamber 40, for pressures below -500 mbar in the deformation chamber 40, the deformation of the sidewall 33 of the study chamber 30 does not exceed 5%, preferably not exceeding 3%. Slight buckling of the sidewall may occur due to the deformation of the upper wall 32. During the application of pressure in the deformation chamber 40, the maximum width of the study chamber 30 remains substantially constant, specifically, the change does not exceed 2%, preferably not exceeding 1%. This is particularly due to the distance between the deformation chamber and the study chamber, which makes the direct deformation caused by the base 20 negligible, and also due to the greater width of the study chamber relative to its height.

[0197] For example, when the pressure within the deformation chamber 40 is between -100 mbar and -500 mbar, the amount by which the upper wall 32 of the study chamber 30 deforms into the interior of the study chamber 30 is greater than or equal to 10% of the height of the study chamber, more preferably greater than or equal to 40%, as represented by the deformation height of the upper wall (in micrometers) relative to the pressure applied in the deformation chamber. Figure 10B As shown.

[0198] The cross-sectional height h of the study chamber can remain constant, such as Figure 1 As shown in Figure 4.

[0199] The height h of the study chamber 30 can also remain constant along the entire length of the study chamber, such as... Figure 1 As shown.

[0200] Alternatively, such as Figure 7B As shown, the height h of the study chamber 30 can vary monotonically along its length. This allows for the classification or guidance of the objects under study within the study chamber 30.

[0201] Alternatively, one of the walls of the chamber, particularly the upper wall 30 or the lower wall 33, can be studied in relation to the length of the chamber (e.g., Figure 7C (as shown) or in the width of the study chamber (such as Figure 6The surface (shown) includes recessed or protruding reliefs 38. These reliefs can function as capillary traps, with trapping force varying with the height of the study chamber and controlled by the pressure within the deformable chamber 40. Under minimum negative pressure in the deformable chamber, these reliefs can trap one or more micro-objects, or under minimum positive pressure, they can release one or more micro-objects. Alternatively, the reliefs 38 can also serve as guides for one or more micro-objects within the study chamber 30, especially if they are in the form of ribs or grooves extending along the length of the study chamber 30.

[0202] In this context, guidance can be continuous and its strength can depend on the height of the study chamber 30, or it can occur only when the height of the study chamber is below a threshold height. Guidance can act directly on one or more microobjects, or it can act indirectly, particularly via a liquid study medium or hydrogel.

[0203] The study chamber 30 can be approximately cylindrical in shape. Figure 1 In the example, the study chamber 30 is approximately rectangular prism in shape. However, it may differ; the study chamber 30 can have other shapes, particularly cylindrical with a circular or other shaped bottom (such as...). Figure 14 , Figure 15 and Figure 18 (as shown), or other non-cylindrical shapes.

[0204] Furthermore, the present invention is not limited to a single deformable chamber 40 for deforming the upper wall 32 of the study chamber 30, particularly as Figures 8 to 1 As shown in Figure 0. The device may include a plurality of deformable chambers 40a, 40b, particularly two substantially identical deformable chambers, each of which extends on each side of the study chamber 30. Preferably, the two deformable chambers 40a, 40b are connected to the same pressure control member 45, and the pressure in the two deformable chambers 40a, 40b is exactly the same, such as... Figure 14 As shown. The presence of the two deformation chambers 40a and 40b makes it possible to achieve greater deformation of the upper wall 32, such as... Figure 9 As shown. Alternatively, the two deformable chambers 40a and 40b are not identical, and / or are not under the same pressure conditions during the application of pressure.

[0205] Additionally or supplementally (not shown), the device may include deformation chambers for deforming the upper wall into a more complex deformation profile than a simple curve, particularly deformation chambers distributed along the length, which may be spaced apart or not spaced apart and apply the same or different pressures.

[0206] In some embodiments of the present invention, particularly Figure 12 and Figure 14 In the illustrated embodiment, the device may include multiple research chambers 30a and 30b connected in fluid series, with the outlet of one research chamber 30a fluidly connected to the inlet of the research chamber 30b. Each research chamber may have one or more deformable chambers on its lateral side, which are controlled independently or simultaneously by one or more pressure control units 45.

[0207] Study of media and micro-objects

[0208] In a study or manipulation process, a study medium 50 containing one or more micro-objects 55 may be introduced into a study chamber 30 before or during the application of pressure to one or more deformable chambers 40. The study medium 50 may be introduced using any method commonly used in the field of microfluidics, particularly using pipettes and containers containing the study medium. The study medium and / or micro-objects or multiple micro-objects may remain stationary in the study chamber during the application of pressure, or they may move from the fluid inlet to the fluid outlet.

[0209] The research medium can be a liquid. The research medium may include a hardening agent, particularly a gelling agent, such as a hydrogel. The research medium can be hardened before, during, or after applying pressure in one or more deformation chambers 40. In the case of a hardened research medium, particularly a gel-like research medium, its Young's modulus is preferably lower than that of the substrate. This hardened research medium allows deformation of the upper wall to be transferred to one or more micro-objects within the research medium. The research medium can be homogeneous within the research chamber 30. Alternatively, the research medium can also have a variable density within the volume of the research chamber 30, and can particularly have a density gradient from the center of the research chamber to the sidewalls.

[0210] One or more micro-objects are preferably deformable.

[0211] One or more micro-objects may be selected from microdroplets, gel-like microunits (particularly including hydrogels and possibly in the form of gel-like microdroplets), and / or any biological material, such as cellular units, particularly cells or cell aggregates, such as spheroids or organoids. In cases where multiple gel-like microunits are present in the study chamber, these gel-like microunits may have different stiffnesses. The microdroplets or gel-like microunits may contain biological material, particularly cellular units, such as cells, cell aggregates, especially spheroids or organoids, or obtained from a patient biopsy sample.

[0212] In a particular embodiment of the invention, one or more micro-objects comprise biological material and / or a medium for biological material, or are composed of biological material and / or a medium for biological material, such as any medium containing one or more nutrients (e.g., proteins, polypeptides, amino acids and / or any carbon source).

[0213] The term "biological material" can be used to refer to any type of cell (including embryonic or non-embryonic cells) and any type of extract, lysate or component of one or more cells, particularly those derived from humans, animals or plants.

[0214] The term “medium” can be used to refer to any physiologically acceptable medium for any one or more cell types, particularly any medium that includes at least one carbon and energy source, specific nutrients such as peptides, polypeptides and / or amino acids, carbohydrates, essential minerals and metals, and buffers.

[0215] In some embodiments of the present invention, the biological material comprises or contains living cells, quiescent cells, and / or cultured cells.

[0216] In some embodiments of the present invention, the biological material includes or is composed of nucleic acids or nucleic acid fragments, particularly DNA and / or RNA.

[0217] The biological material may include or consist of one or more types of cells selected from eukaryotic cells, prokaryotic cells, viruses, or viral particles. This list is not exhaustive.

[0218] In some specific embodiments of the present invention, the biological material may include or be composed of eukaryotic cells, such as mammalian cells, particularly human or non-human mammalian cells, such as cells from humans, mice, rats, dogs, cats, cattle, pigs, chickens, goats, horses, yeast, etc.

[0219] In some specific embodiments of the present invention, the biological material may include or be composed of non-eukaryotic cells, such as prokaryotic cells, such as bacteria or archaea.

[0220] In some specific embodiments of the present invention, the biological material may include or consist of viruses or virus particles, such as enveloped or non-enveloped viruses, RNA viruses, DNA viruses, or any other virus in the Baltimore classification, such as viruses belonging to groups I, II, III, IV, V, VI, or VII of that classification.

[0221] In some specific embodiments of the present invention, the biological material may include or consist of transfected cells with one or more gene modifications, and / or the expression of one or more of their nucleic acids is regulated.

[0222] In some specific embodiments of the present invention, the biological material may include or be composed of one or more somatic cells or progenitor cells, such as one or more pluripotent cells or pluripotent cells, such as embryonic cells or cells obtained from an embryo.

[0223] In some specific embodiments of the present invention, the cells may be differentiated, in the process of differentiation, or in an undifferentiated state.

[0224] In some embodiments of the present invention, the cells may be cancer cells or precancerous cells, or cells obtained from cancerous tissue, or cells from different sources.

[0225] In some embodiments of the invention, the cells (e.g., cancer cells) may be derived from one or more tissues or organs from the following list: adrenal glands, bladder, blood, bones, bone marrow, brain, cartilage, uterus, cervix, endometrium, cornea, esophagus, gastrointestinal tract, nerves, liver, lungs, lymphatic tissue, muscles, heart, pancreas, pituitary gland, prostate, testes, kidneys, salivary glands, skin, thyroid gland, immune system, epithelium, endothelium, mesothelium.

[0226] In some specific embodiments of the invention, the cells may be selected from blood cells and / or immune system cells, particularly from the following list: red blood cells, platelets, T lymphocytes, B lymphocytes, white blood cells, dendritic cells, and macrophages.

[0227] In some embodiments of the invention, the cells can be obtained from a patient or individual suffering from one or more diseases. These diseases may include those that alter the mechanical properties of tissues, such as fibrosis, cancer, or any other disease that leads to tissue scarring.

[0228] In some embodiments of the present invention, the biological material includes or consists of a three-dimensional cell culture.

[0229] In some embodiments of the present invention, the biological substance and / or medium may include one or more compounds of interest, such as one or more active pharmaceutical preparations, such as one or more antibodies or antibody fragments.

[0230] In some embodiments of the present invention, the biological material includes or consists of one or more spherical bodies.

[0231] In some embodiments of the present invention, the biological material includes or is composed of one or more organoids.

[0232] The term "sphere" can refer to an aggregate of any type of cell, such as an aggregate of a single cell type or multiple cell types, which is capable of three-dimensional growth in suspension and can interact with one or more cell types and / or a three-dimensional matrix (e.g., Matrigel™) or any suitable type of medium.

[0233] The term "organoid" can refer to any cellular structure obtained by amplifying one or more specific cell types in a given tissue, and that the cellular structure is capable of self-organization and / or differentiation, either wholly or partially.

[0234] like Figure 7A and Figure 7B As shown, the height of one or more micro-objects may be greater than or equal to the height of the study chamber. Alternatively, the height of one or more micro-objects may be less than the height of the study chamber 30. In this case, the study medium preferably comprises a gelling agent, particularly a hydrogel, to gel before, during, or after pressure is applied in one or more deformable chambers.

[0235] Analysis System

[0236] like Figure 14 As shown, the microfluidic device may include an analysis system 60, or functionally coupled to an analysis system, configured to analyze a study chamber 30, particularly a study medium 50 or one or more micro-objects 55 within the study chamber 30. This analysis system may or may not be functionally coupled to a unit for controlling the deformation of a deformation chamber, and in particular, the analysis system may supply an analysis signal to a control unit. The control unit can control the deformation of one or more deformation chambers based on changes in the analysis signal.

[0237] The analytical system 60 in the study chamber 30 can be an optical imaging system, particularly an optical microscope or an electron microscope, or something similar. Figure 14 The confocal imaging system or any other analytical system shown is specifically designed for measuring electrical properties, particularly for studying the impedance, conductivity, or electrical activity of cellular units during mechanical stimulation of a medium. The analytical system 60 is preferably positioned above or below the study chamber and configured to image the liquid, gel, and / or one or more micro-objects within the study chamber. The analytical system 60 can be configured to detect static or dynamic information regarding the fluid, gel, and / or one or more micro-objects within the study chamber.

[0238] method

[0239] In some embodiments of the invention, deformation of the upper wall 32 of the study chamber 30 applies a force to the micro-objects 55 within the study chamber, thereby causing deformation of these micro-objects. Subsequently, specifically using microscopy and image analysis methods, the deformation of one or more micro-objects 55 can be determined. The determined deformation can be a complete deformation of one or more micro-objects, or a localized deformation of only a portion of one or more micro-objects, particularly a localized deformation of one or more cells or nuclei within a cell aggregate. This deformation measurement allows for the determination of the mechanical properties of one or more micro-objects or a portion thereof, particularly their stiffness.

[0240] If the size of one or more micro-objects is smaller than the height h of the study chamber 30, the deformation can be determined directly or indirectly through the study medium. The deformation of the upper wall 32 causes the volume deformation of the study medium in the study chamber 30 (especially when gelled), which in turn causes the deformation of one or more micro-objects.

[0241] The method may include comparing the deformation of a micro-object or a portion thereof with the actual or calculated deformation of a reference sample at the same or equivalent location in the same study chamber in order to infer the properties of one or more micro-objects, particularly deformability and / or biological properties.

[0242] In some embodiments, deformation of the upper wall of the study chamber 30 results in movement of one or more micro-objects or portions of one or more micro-objects within the study chamber, particularly the movement of one or more cells or nuclei within a cell aggregate. This movement may occur during deformation due to the upper wall 32 acting on the study medium containing one or more micro-objects (especially during gelation) or on one or more micro-objects.

[0243] The method may include comparing the motion of one or more micro-objects or portions of one or more micro-objects with the motion of a reference sample in the same study chamber to infer the properties of one or more micro-objects, particularly biological properties.

[0244] In some embodiments of the present invention, the method may include studying the deformation and motion of one or more micro-objects as described above.

[0245] In some embodiments of the invention, the method includes applying pressure cycles in one or more deformable chambers 40, the pressure cycles corresponding to a series of periodic repetitions of pressure / relaxation in the one or more deformable chambers 40 over a predetermined time period. The different pressures in the cycles may be the same or different, and may be spaced apart from each other by the same or different times. This pressure cycle enables the application of cyclic deformation to the upper wall 32 over a specific time period, which produces periodic stimulation to one or more micro-objects 55. The response of one or more micro-objects (especially biological material) to this cyclic stimulation can then be studied (particularly through imaging and image analysis). The method may include studying the correlation between the deformation of one or more micro-objects over time and the applied pressure cycle, or studying the correlation between the motion of one or more micro-objects during the applied pressure cycle and the applied pressure cycle. It is also possible to study the correlation between the motion of one or more micro-objects over time and the applied pressure cycle, or the correlation between the motion of one or more micro-objects during the applied pressure cycle and the applied pressure cycle, or as a response to the stimulation applied by the deformation cycle after the application of the pressure cycle, especially in the case of one or more micro-objects comprising biological material.

[0246] The following section will describe in detail examples of the use of the microfluidic device described above.

[0247] Example

[0248] Cell culture and spheroid formation

[0249] H4-II-EC3, NIH-3T3, and MDA-MB-231 GFP cancer cells were cultured in DMEM medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and / or streptomycin antibiotics.

[0250] Spherical bodies were prepared using U-shaped non-adhesive porous culture plate 96 (Corning catalog number 7007).

[0251] To obtain spheroid cocultures, the two cell types are mixed in different ratios depending on the desired size of the spheroids and the intended application. For example, a mixture of one hundred H4-II-EC3 cells and four hundred NIH-3T3 cells will produce larger spheroids, while a mixture of one hundred cells of each type will produce smaller spheroids. The cells are co-cultured for 72 hours to form spheroids.

[0252] microscope

[0253] Images were acquired using a motorized Nikon Ti2 rotating disk epifluorescence microscope equipped with a 20x objective lens. Illumination was provided by a Lumencor LED light source for epifluorescence imaging, or by an Oxius laser array for confocal imaging. Images were acquired using a Hamamatsu C13440-20CU SCMOS camera. Raw data were acquired using Nikon Elements software (version 5.11.01, Build 1367).

[0254] Fluorescent labeling

[0255] Cells can be labeled using immunofluorescence or standard assay kits. All reagents are introduced into the device using a pipette, without the need for pressure. Actin was labeled using Alexa Fluor™ 488 Phalloidin (Thermofisher, catalog number: A12379) at a 1:200 dilution. Cell nuclei were labeled using NucBlue™ Live ReadyProbes™ Reagent (Hoechst 33342, Invitrogen, catalog number: R37605) according to the manufacturer's instructions. Membranes were labeled using CellBrite® Steady 650 reagent (Biotium, catalog number: 30108) according to the manufacturer's instructions.

[0256] Measurement of spherical dimensions

[0257] The size of the sphere was analyzed from a microscope image using macros in ImageJ (FIKI) software. For each pressure value, the circumference, total area, major axis, and minor axis of the sphere could be measured and recorded.

[0258] Example 1: In the context of a rectangular prism-shaped study chamber, analyze how the overall compression of a sphere changes with its position within the study chamber.

[0259] Manufacturing such as Figure 12The apparatus is shown. A monolithic polydimethylsiloxane (PDMS) substrate 25 is fabricated using a 3D-printed mold, which includes protrusions and recesses corresponding to the negative shapes of the study chamber 30, the deformable chamber 40, and the conduits 34, 42. The substrate 25, fabricated in this manner, is placed on a microscope slide 28, with the substrate side (including the imprinted portions of the chambers) in contact with the microscope slide. The apparatus includes two identical study chambers 30a, 30b, which are elongated and generally rectangular in both cross and longitudinal sections, and are connected in series. Two deformable chambers 40a, 40b are provided on each side of the longitudinal axis of the two study chambers 30a, 30b, which are connected to the same pressure source via conduits 42, the conduits opening into the two deformable chambers. The two pairs of deformable chambers, each corresponding to a study chamber, are not interconnected. In this example, these deformable chambers are subjected to the same pressure. The height of the study chamber is approximately 100 µm.

[0260] Using a pipette fixed in place within the study chamber, spherical samples with a diameter approximately 130 ± 20 µm, as described above, were introduced into study chambers 30a and 30b. Before and after applying different negative pressures in the deformation chamber, the visible area of ​​the spherical samples in the central and lateral positions within the study chamber was measured using microscopy and image analysis (as described above). The results before and after applying pressure in the deformation chamber were compared to determine the percentage change in the spherical area, representing the deformation of the spherical sample due to the deformation of the upper wall. Figure 13 The relationship between the change in the average area of ​​the sphere and the pressure in the deformation chamber is depicted in both the lateral position (bottom curve) and the central position (top curve) within the study chamber. Observations show that the deformation of the sphere depends on the pressure applied in the deformation chamber and the position of the sphere within the study chamber; the deformation of the upper wall 32 is more significant at the center of the study chamber than at its lateral position.

[0261] Example 2: Analyze the overall behavior of a sphere during pressure cycles within a cylindrical research chamber with a circular bottom.

[0262] Manufacturing such as Figure 14The apparatus is shown. A monolithic polydimethylsiloxane (PDMS) substrate 25 was fabricated using a 3D-printed mold, the mold including protrusions and recesses corresponding to the negative shapes of the study chamber 30, the deformable chamber 40, and the conduits 34, 42. The substrate 25, fabricated in this manner, was placed on a microscope slide 28, with the chamber-imprinted substrate side in contact with the microscope slide. The apparatus includes two identical study chambers 30a, 30b, which are circular when viewed from above and interconnected in series by conduits. The connecting conduits at the inlet and outlet of the apparatus are higher than the height of the study chambers. Two deformable chambers 40a, 40b are respectively located on both sides of the study chambers 30a, 30b, the two deformable chambers being connected to the same pressure source via conduits 42, the conduits opening into the two deformable chambers. The two pairs of deformable chambers corresponding to each study chamber are not interconnected. In this example, these deformable chambers are subjected to different pressures to simultaneously study the deformation of a sphere under two different pressures. The height of the study chamber is approximately 100 µm.

[0263] Using a pipette positioned in a fixed position within the study chamber, spherical samples with a diameter approximately 130 ± 20 µm, as described above, were introduced into study chambers 30a and 30b. Figure 15 As shown, the visible area of ​​the sphere at the center of the study chamber was measured by microscopy and image analysis (as described above) before a sinusoidal pressure cycle of 0.5 Hz and -300 mbar was applied. Figure 16 The average change in the area of ​​the sphere is depicted during cycles of applied pressure. From Figure 16 It is evident that the deformation of the sphere tracks the pressure cycle well over time, and the upper wall does indeed undergo periodic deformation according to the pressure cycle.

[0264] Example 3: Analyze the deformation of a single cell in a spherical body during application against the background of a study chamber in the form of a cylindrical body with a circular base.

[0265] In the apparatus of Example 2, a spherical sample with a diameter of approximately 130 ± 20 µm, as described above, was introduced into study chambers 30a and 30b using a pipette positioned in a fixed position within the study chamber. The study showed that localized deformation of individual cells and their nuclei within the fluorescently labeled spherical samples, as described above, could be observed. This allowed for the study of nuclear deformation, cell deformation, and / or cellular behavior / rearrangement within the cells of the spherical samples during deformation.

[0266] Example 4: Analyze the deformation of a single cell in a spherical body during application against the background of a study chamber in the form of a cylindrical body with a circular base.

[0267] In the apparatus of Example 2, two types of spherical samples with different cell combinations and a diameter of approximately 130 ± 20 µm were introduced into study chambers 30a and 30b using a pipette positioned in a fixed position within the study chamber. The first sample consisted of spherical bodies formed solely by H4-II-EC3 cells, while the second sample consisted of spherical bodies formed by a co-culture of H4-II-EC3 and NIH-3T3 cells. Before deformation of the upper wall, NIH-3T3 cells were distributed at the center of the spherical bodies, while H4-II-EC3 cells formed a shell around the NIH-3T3 cells. The deformation field of the spherical bodies in the plane of a bright-field microscope was measured using particle imaging (PIV) software, and the radial outward deformation was calculated. This yielded... Figures 17A to 1 The curve for 7C. Figure 17A This represents the radial deformation of the sphere in the first sample. (Curve) Figure 17B This represents the radial deformation of the spheroids in the second sample. For the first sample, which only includes H4-II-EC3 cells, the deformation occurs from the center towards the edge and gradually increases. For the second sample, which consists of a co-culture of H4-II-EC3 cells and NIH-3T3 cells, the spheroids consist of a relatively stable, slightly deformed core and a more deformed lateral region.

[0268] The hypothesis has been tested that this behavioral difference is specifically associated with the self-organization of these different cells into a nucleus-capsule structure, and therefore cancer H4-II-EC3 cells and NIH-3T3 cells may have different mechanical properties, with one type of cell being more rigid than the other.

[0269] NIH-3T3 cells were labeled with GFP before being mixed with H4-II-EC3 cells. Observation of cells in the spheroids revealed that NIH-3T3 cells migrated into the interior of the spheroids to form nuclei, while cancer cells were arranged on the periphery of the spheroids.

[0270] Therefore, these results indicate that the NIH-3T3 cell aggregates form a more rigid structure compared to the lateral portion composed of H4-II-EC3 cells.

[0271] Specifically, quantitative data indicate that the inner layer of NIH-3T3 cells is approximately 30 to 50 times stiffer than the outer layer formed by H4-II-EC3 cells.

[0272] Therefore, it is possible to determine the mechanical properties of different three-dimensional heterogeneous cell arrangements and, by comparing them with the mechanical properties of known cell arrangements, to determine the biological properties of the cell sample under study. Thus, this example demonstrates that the device is particularly suitable for characterizing heterogeneous structures and identifying the boundary between healthy and cancerous cells.

[0273] Example 5: Microfluidic device for multi-well culture plates.

[0274] Using multi-well culture plates as a base for manufacturing Figure 18 and Figure 19 The device was developed because rectangular structures are not suitable for microfluidic devices. A more suitable structure was developed. A monolithic polydimethylsiloxane (PDMS) substrate 25 was fabricated using a 3D-printed mold, which included ribs corresponding to the negative shapes of the study chamber 30, the deformable chamber 40, and the conduits 34 and 42. The substrate 25, fabricated in this way, was sized to fit the pores of a porous culture plate, with one side of the substrate including the chamber imprint contacting the bottom of the culture well.

[0275] Figure 18 The apparatus includes: a single, generally circular study chamber viewed from above, connected to sample inlet and outlet channels 34 at acute angles to each other; and a crescent-shaped deformable chamber viewed from below, partially surrounding the study chamber. The pressure within the deformable chamber is controlled via a control channel 42. Under a pressure of -600 mbar within the deformable chamber 40, the upper wall of the study chamber deforms approximately 43 μm at its center towards the interior of the study chamber.

[0276] Figure 19 The apparatus comprises: a single research chamber, generally circular when viewed from above, connected to sample inlet and outlet channels 34, the inlet and outlet channels being opposite each other; and two identical deformable chambers, kidney-shaped when viewed from above and located on either side of the research chamber. The pressure within the deformable chambers is controlled via a common control channel 42. Therefore, the deformable chambers experience the same pressure. Under a pressure of -600 mbar in deformable chambers 40a and 40b, the upper wall 32 of the research chamber deforms at its center toward the interior of the research chamber by a deformation distance of approximately 49 µm.

[0277] Example 6: Cyclic compression of cell aggregates in a gel-like medium.

[0278] In the apparatus of Example 1, a sample of spherical MDA-MB-231 metastatic breast cancer cells in a liquid medium was introduced into study chamber 30, the diameter of which was larger than the height of the study chamber. In a deformable chamber 40, the spherical cells were subjected to approximately sinusoidal pressure cycling at a frequency of 0.5 Hz and a pressure of -350 mbar for 12 hours in the liquid medium. After stimulation, the liquid medium was replaced with Matrigel™ basement membrane matrix, and cell migration in response to the cyclic stimulation was studied within the matrix over 16 hours. MDA-MB-231 cancer cells migrate outward within the matrix, making it possible to study the metastatic ability of cancer cells in response to external stimuli.

[0279] Example 7: Stimulation of spheroids in a hydrogel.

[0280] In the apparatus of Example 1, spherical samples in an agarose gel medium were studied. The height of the spherical samples was less than the height of the study chamber. This configuration allows for stimulation of the spherical samples and the application of mechanical stress, rather than the imposition of deformation. Figure 20 As shown, deformation of the upper wall causes deformation of the hydrogel containing the spherical bodies, which in turn causes lateral displacement of the spherical bodies within the study chamber. This allows shear stress and / or tensile stress to be applied to the spherical bodies.

[0281] The deformation dynamics of spheroids within hydrogels can be used to analyze and determine the rheological properties of tissue or cell aggregates integrated within chambers. In fact, the amount of deformation of the spheroids can be extracted by comparing the deformation of the spheroids with that of the surrounding hydrogel with known rheological properties: very soft micro-tissues will deform significantly, while very stiff micro-tissues will hardly deform compared to the surrounding gel. Therefore, tissues with different hardnesses can be tested by adjusting the hardness of the hydrogel surrounding the tissue. In extreme cases, the hydrogel can be replaced by a purely viscous liquid hydrogel (e.g., glycerol, paraffin oil, or other liquids).

[0282] Example 8: Stimulation of spherical bodies in hydrogel units.

[0283] Using the method described above for incorporating spherical structures within a hydrogel, hydrogel units 65a to 65c, each comprising a spherical structure, can also be placed within a fluid medium in a study chamber. This enables multiplexing of stresses of varying ranges during the study. As described in Example 7, the hardness of the spherical structure relative to the rheological properties of the hydrogel can be analyzed using a hydrogel of known hardness. Hydrogel units with different rheological properties can be placed within the same study chamber. These hydrogel units of varying hardness will generate different flows within them under pressure, resulting in different forces and mechanical stresses within the spherical structures integrated therein, such as… Figure 21 As shown.

[0284] Example 9: Microfluidic devices that generate motion in the research medium and spherical bodies.

[0285] like Figure 22 The device shown is manufactured using the same method as in Example 1. The device includes a funnel-shaped research chamber 30 when viewed from above and a deformable chamber 40 extending along the long side of the funnel. The cross-sectional shape of the deformable chamber 40 is similar to... Figure 5B The shapes of the deformable chambers in them are basically the same. For example... Figure 23As shown, deformation of the upper wall 32 of the study chamber occurs only in the widened portion of the funnel, caused by applying pressure to the deformation chamber. A hydrogel comprising spherical bodies is introduced into the study chamber.

[0286] In this design, the study chamber is pressurized only from one end (the end adjacent to the deformable chamber), which propels the hydrogel and the spherical bodies bound within it forward, such as... Figure 23 As shown. This deformation produces spatial heterogeneity within the hydrogel. Different device configurations can be designed to achieve different hydrogel deformations. For example, as... Figure 24 As shown, introducing a concave-convex portion 65 at the edge of the study chamber will produce more bending-type deformations in the hydrogel between the concave-convex portions in the study chamber.

[0287] This invention is not limited to the examples and embodiments described above. Features of the various embodiments described above can be combined to form other variations.

Claims

1. A microfluidic device (10), the microfluidic device comprising: - Support member (20), the support member comprising a base (25) made of an elastically deformable material; - Research chambers (30, 30a, 30b) extending inside the support (20) and having: at least one upper wall (32) formed at least partially, preferably entirely, by the base (25); at least one lower wall (34); and at least two side walls (33). - At least one base deformation chamber (40, 40a, 40b), said deformation chamber being at least partially formed by said base (25) and fluidly independent of said study chamber (30, 30a, 30b) and extending at least partially within said support (20) along one of the sidewalls (33) of said study chamber (30), The microfluidic device is configured such that deformation of the deformable chambers (40, 40a, 40b) results in mechanical stress being applied to at least a portion of the upper wall (32) of the study chamber (30) in the study chamber, particularly through deformation of the upper wall (32).

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

3. The microfluidic device according to any one of the preceding claims, wherein the microfluidic device is configured such that the volume of the deformable chambers (40, 40a, 40b) decreases, particularly by negative pressure in the deformable chambers (40, 40a, 40b), causing the upper wall (32) of the study chamber to deform in a direction that reduces the volume of the study chamber (30), particularly the height h, and is configured such that the volume of the deformable chambers (40, 40a, 40b) increases, particularly by positive pressure in the deformable chambers (40, 40a, 40b), causing the upper wall (32) of the study chamber to deform in a direction that increases the volume of the study chamber, particularly the height h, and the deformation of the upper wall (32) results in the application of mechanical stress in the study chambers (30, 30a, 30b).

4. The microfluidic device according to any one of the preceding claims, wherein, The deformable chambers (40, 40a, 40b) extend in the substrate (25) at a height H greater than or equal to the height h of the study chambers (30, 30a, 30b), with the height ratio H / h preferably greater than or equal to 5, and more preferably greater than or equal to 10.

5. The microfluidic device according to any one of the preceding claims, wherein, The minimum thickness e of the base between the study chambers (30, 30a, 30b) and the deformable chambers (40, 40a, 40b) is greater than or equal to the height h of the study chambers (30, 30a, 30b), and / or less than or equal to 100 times the height h of the study chambers (30, 30a, 30b), more preferably less than or equal to 50 times the height h of the study chambers (30, 30a, 30b), even more preferably less than or equal to 40 times the height h of the study chambers (30, 30a, 30b), and even more preferably less than or equal to 20 times the height h of the study chambers (30, 30a, 30b).

6. The microfluidic device according to any one of the preceding claims, wherein, The deformable chambers (40, 40a, 40b) extend in the support (20) toward a plane defined by one of the sidewalls (33) of the study chambers (30, 30a, 30b) at a height greater than or equal to 50% of the maximum height H of the deformable chamber, more preferably at a height greater than or equal to 70% of the maximum height H of the deformable chamber, and even more preferably at a height greater than or equal to 80% of the maximum height H of the deformable chamber.

7. The microfluidic device according to any one of the preceding claims, the microfluidic device comprising at least two deformable chambers (40a, 40b) of the substrate, the deformable chambers being formed at least partially by the substrate (25) and independent of the study chambers (30, 30a, 30b), and each of the deformable chambers extending at least partially along the sidewall (33) of the study chamber, particularly extending on any side of the study chambers (30, 30a, 30b).

8. The microfluidic device according to any one of the preceding claims, the microfluidic device comprising a plurality of research chambers (30a, 30b) connected in series in fluid connection, wherein the stress, in particular the deformation of the upper wall (32) in each of the research chambers is generated by the same one or more deformation chambers (40, 40a, 40b) or by different deformation chambers that are fluidly connected or not connected to each other.

9. The microfluidic device according to any one of the preceding claims, the microfluidic device comprising a unit (45) for controlling the deformation of the deformable chambers (40, 40a, 40b), particularly for controlling the pressure in the deformable chambers, the unit being fluidly connected to the deformable chambers (40, 40a, 40b) via a channel (42).

10. The microfluidic device according to any one of the preceding claims, the microfluidic device comprising an analysis system (60) or functionally coupled to the analysis system, the analysis system being configured to analyze the microfluidic device, particularly the study chamber, particularly the study medium or one or more micro-objects in the study chamber (30, 30a, 30b).

11. The microfluidic device according to any one of the preceding claims, wherein, The research chambers (30, 30a, 30b) include a research medium (50) and / or at least one micro-object (55), and more preferably, a plurality of micro-objects are included in the research chambers, particularly in the research medium (50).

12. The microfluidic device according to claim 11, wherein, The micro-object (55) can be selected from: microdroplets; gel-like micro-units, particularly including hydrogels and capable of being in the form of gel-like microdroplets; and / or any biological substance, such as cellular units, especially cells or cell aggregates, such as spheroids or organoids; or any type of medium.

13. A method for studying or manipulating at least one research medium (50) or a microobject (55) using a microfluidic device (10) according to any one of the preceding claims, the method comprising: The study medium (50) and / or the micro-object (55) are introduced into one of the study chambers or the study chambers (30, 30a, 30b); pressure, particularly negative pressure, is applied in one or more deformable chambers (40, 40a, 40b) to generate stress, preferably deformation, of the upper wall (32) of the study chamber, particularly along a direction that reduces the height of the study chamber, in the study chamber (30, 30a, 30b), which exerts mechanical stimulation on the study medium (50) or the micro-object (55), or thereby generates movement of the study medium (50) or the micro-object (55) in the study chamber (30, 30a, 30b).

14. The method according to claim 13, wherein, The study medium (50) includes a gelling agent, particularly a hydrogel, which is introduced into the study chamber along with the microobject, or subsequently introduced into the study medium contained in the study chamber, wherein the gelled study medium (50) has a Young's modulus lower than that of the substrate (25).

15. The method according to claim 13 or 14, the method comprising deforming the research medium and / or pumping the research medium into the research chambers (30, 30a, 30b), wherein the deformation of the upper wall (32) of the research chambers (30, 30a, 30b) specifically generates flow of the research medium (50) from the inlet to the outlet of the research chambers (30, 30a, 30b), thereby acting as a diaphragm pump.

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