Microfluidic chip for growing cells

Abstract

The invention relates to a microfluidic chip (1) for growing cells comprising a reservoir (2) capable of containing a solution, at least two separate chambers (3) capable of being supplied with the solution from the reservoir and capable of receiving one or more cells, at least two separate afferent channels (4) each connecting an outlet (5) of the reservoir, from among at least two separates outlets (5) of the reservoir, to a chamber from among the at least two separate chambers, and at least two separate efferent channels (6) each connecting a chamber from among the at least two separate chambers to a drain (7), the drain allowing the solution to be discharged from each of the at least two chambers.

Description

[0001] DESCRIPTION

[0002] Microfluidic chip for cell growth

[0003] Technical field

[0004] The present invention belongs to the field of microfluidic chips and, in particular, to the field of organs on chips and cancers on chips (OCSP).

[0005] This type of OSCP chip allows the reconstruction of tissues and / or organs. It is a tool for in vitro modeling of human organs and human pathologies, particularly cancer cells.

[0006] These chips are designed to mimic and reproduce in vivo conditions and environments, such as different cellular and extracellular signals, e.g., molecular, structural and physical, for a given system or organ.

[0007] State of the prior art

[0008] The state of the art is known for classic two-dimensional cell culture or growth models. Efforts are being made to find alternatives to these models, which are poorly representative of the complexity of human tissues and organs.

[0009] OCSP devices exist in the state of the art. However, their widespread use is still limited due to the lack of standardized devices and the absence of viable and inexpensive commercial solutions.

[0010] The present invention aims to overcome, at least in part, the drawbacks of the state-of-the-art devices.

[0011] An aim of the invention is, in addition, to propose a microfluidic chip:

[0012] - simpler to manufacture, and / or

[0013] - easier to handle, and / or

[0014] - with low production costs, and / or

[0015] - allowing parallel testing on the same chip, and / or

[0016] - allowing the simulation of a large number of cells or tumors on the same chip, and / or

[0017] - to faithfully model in vitro human organs and / or pathologies, and / or

[0018] - allowing to reproduce or imitate:

[0019] • the conditions encountered in the body and / or in an organism and / or in a biological environment, and / or

[0020] • the conditions of circulation of the fluid in the blood capillaries, and / or

[0021] • the environment of cells in the body and / or in an organism and / or in a biological environment.

[0022] Presentation of the invention

[0023] For this purpose, a microfluidic chip for cell growth is proposed. The microfluidic chip according to the invention comprises:

[0024] - a tank capable of containing a solution,

[0025] - at least two separate chambers capable of being supplied with solution from the reservoir and capable of receiving one or more cells, preferably assembled in 2D or 3D spheroid or organoids,

[0026] - at least two distinct afferent channels, preferably a series of distinct afferent channels, each connecting an outlet of the reservoir, among at least two distinct outlets of the reservoir, to a chamber among the at least two distinct chambers,

[0027] - at least two distinct efferent channels each connecting one chamber among the at least two distinct chambers to an evacuation, said evacuation being capable of allowing the evacuation of solution from each of the at least two chambers.

[0028] Preferably, the microfluidic chip according to the invention is capable of and / or arranged to allow the growth of cells and / or organs.

[0029] Preferably, in the present application, it is understood by "at least two", two or more.

[0030] Preferably, in the present application, "distinct" chamber inlets, chamber outlets, afferent channels or efferent channels are understood to mean independent, individual or different inlets, outlets or channels.

[0031] Preferably, the at least two distinct afferent channels are located upstream of the at least two chambers relative to a direction in which the solution is intended to flow or circulate in the microfluidic chip.

[0032] Preferably, the at least two distinct efferent channels are located downstream of the at least two chambers relative to a direction in which the solution is intended to flow or circulate in the microfluidic chip.

[0033] Preferably, the at least two outlets of the reservoir are, preferably each, arranged in distinct areas of the reservoir. Preferably, the at least two distinct afferent channels are each connected or linked to a distinct area of ​​the reservoir.

[0034] Preferably, the at least two distinct efferent channels are each connected or linked to a distinct area of ​​the reservoir.

[0035] According to the invention, preferably, a single reservoir can supply several chambers with different or distinct solutions. More preferably, a single reservoir can supply each of the chambers with different or distinct solutions. This allows, among other things, to parallelize the tests and assays. This allows, among other things, to obtain more reliable models. This allows, among other things, to test at least two concentrations of drugs on the same chip. This allows, among other things, to directly test the cytotoxicity of drugs in the form of molecules or materials such as, for example, liposomes, inorganic nanoparticles, organic / inorganic nanoparticles, drugs formulated inside a liposomal structure.

[0036] In the present application, different or distinct solutions may be understood to mean solutions of different composition or nature. A solution feeding one of the chambers may comprise one or more solutes and at least a portion of said solute(s) are different from at least a portion of the solute(s) of at least one other of the solutions feeding another of the chambers. A solution feeding one of the chambers may comprise one or more solutes and at least a portion of said solute(s) are in a different concentration from at least a portion of the solute(s) of at least one other of the solutions feeding another of the chambers. Preferably, the solute(s) of a solution feeding one of the chambers may be identical to the solute(s) of one, several or each of the solutions feeding the other chamber(s).

[0037] In the present application, different or distinct solutions supplying the chambers may be understood to mean solutions of identical composition. More preferably, a solution supplying one of the chambers:

[0038] - has a composition identical to at least one other of the solutions supplying another of the chambers,

[0039] - comprises one or more solutes in concentrations different from the solutes of said at least one other of the solutions.

[0040] In the present application, one, several or each of the distinct or different solutions supplying the chambers may: - be of identical composition, and

[0041] - understand one or more solutes in different concentrations.

[0042] Preferably, the microfluidic chip according to the invention is intended to be used in the field of organs on chips and / or cancers on chips (OCSP).

[0043] An organoid is understood to be a three-dimensional multicellular structure that reproduces the microanatomy of an organ in vitro.

[0044] Preferably, at least a portion of each of the at least two afferent channels forms a sinusoid or a serpentine.

[0045] Preferably, the total of the at least two afferent channels forms a sinusoid.

[0046] Preferably, at least a portion, more preferably all, of each of the at least two afferent channels forms a coil.

[0047] The coils preferably have the effect of mimicking or reproducing or imitating the circulation of fluid in blood capillaries. This makes it possible, among other things, to reproduce the phenomena observed in blood capillaries such as, by way of non-limiting example, the phenomenon of opsonization. The phenomenon of opsonization can be defined as the formation of a protein crown. This makes it possible, among other things, to increase the length of the path of the solution in the afferent channels. This makes it possible, among other things, to increase the length of the path of the solution in the afferent channels by limiting the associated increase in size and / or the bulk of the microfluidic chip

[0048] Preferably, the sinusoidal portion or serpentine of one, several or each of the at least two afferent channels comprises at least three periods.

[0049] A "sinusoidal part comprising at least three periods" can be understood to mean a serpentine comprising at least six bends.

[0050] Preferably, an afferent channel having a sinusoidal portion comprising at least three periods has a sufficient length to obtain satisfactory homogenization of the solution circulating in said afferent channel.

[0051] The tank may include at least two separate inlets capable of allowing the tank to be supplied with solution.

[0052] Preferably, the at least two inlets are, preferably each, arranged in separate areas of the reservoir. The inlets of the reservoir are adapted to allow the solution to be supplied to the reservoir.

[0053] Preferably, the reservoir is capable of being supplied with solution.

[0054] Preferably, distinct or different solutions are understood to mean solutions of the same composition but of different concentrations.

[0055] Preferably, the reservoir is capable of being supplied with several liquid solutions.

[0056] Preferably, the reservoir is capable of allowing several liquid solutions to pass through it.

[0057] Preferably, the reservoir is capable of being supplied with solution.

[0058] Preferably, the tank is capable of being supplied with several identical or different solutions.

[0059] Preferably, the at least two inlets of the tank are included in a part of the tank opposite a part of the tank on which the at least two outlets of the tank are included.

[0060] Preferably, the at least two inlets of the tank are located or arranged on or along a portion of the tank.

[0061] Preferably, the at least two inlets of the tank are included in a portion of a wall of the tank.

[0062] Preferably, the at least two inlets of the tank are included in a part of the tank opposite a part of a part of a wall of the tank on which the at least two outlets of the tank are included.

[0063] Preferably, the at least two outlets of the tank are located or arranged on or along a portion of the tank.

[0064] Preferably, the at least two outlets of the tank are included in a portion of a wall of the tank.

[0065] The at least two inputs, preferably each of the at least two inputs, and the at least two outputs, preferably each of the at least two outputs, may be included in the same plane, called the axial plane.

[0066] Preferably, the axial plane is intersecting each of the inlets and each of the outlets of the reservoir. Preferably, the reservoir extends mainly along an axis, called the dilution axis, which extends mainly perpendicular to the main direction in which the solution is intended to circulate in the chip from the reservoir to the outlet.

[0067] Preferably, the tank is oblong in shape.

[0068] Preferably, a length of the reservoir extends along the dilution axis.

[0069] The tank length and / or dilution axis may be curved.

[0070] Preferably, the length of the tank and / or the dilution axis is rectilinear.

[0071] Preferably, the dilution axis is included in the axial plane.

[0072] The direction in which the solution is intended to flow into the tank extends from one, several, or each of the inlets to one, several, or each of the outlets.

[0073] Preferably, the direction in which the solution is intended to flow through the chip is the average or main direction in which the solution flows from the reservoir to the drain.

[0074] Preferably, the at least two inputs are distributed along an axis parallel to the dilution axis, preferably so as to form a succession or a chain or a sequence, and the at least two outputs are distributed along an axis parallel to the dilution axis, preferably so as to form a succession or a chain or a sequence.

[0075] Preferably:

[0076] - each of the at least two inlets is intersected by a separate plane, called the inlet plane, each of the inlet planes is perpendicular to the axial plane and, preferably to the dilution axis, said inlet planes form a succession of planes parallel to each other,

[0077] - each of the at least two outlets is intersected by a separate plane, called the outlet plane, each of the outlet planes is perpendicular to the axial plane and, preferably to the dilution axis, said outlet planes form a succession of planes parallel to each other, the at least two inlet planes and the at least two outlet planes are parallel to each other. Preferably, at least one inlet plane is between two outlet planes and / or at least one outlet plane is between two inlet planes.

[0078] Preferably, for a room considered:

[0079] - one end of the afferent channel to which the chamber in question is connected is tangent to the chamber in question in order to preferably imitate or reproduce the stresses, in particular the shear forces, exerted on the cells in the organism, and

[0080] - one end of the efferent canal to which the chamber in question is connected is tangent to the chamber in question in order to preferably imitate or reproduce the stresses, in particular the shear forces, exerted on the cells in the organism.

[0081] Preferably, one, several or each of the chambers comprises an annular channel at the periphery, or bordering or forming a periphery, of the or each of the chambers.

[0082] According to the invention, the annular channel bordering the periphery of the chamber(s) or each of the chambers preferably has the effect of imitating or reproducing the conditions in the organism. More preferably, the annular channel bordering the periphery of the chamber(s) or each of the chambers has the effect of distributing the solution around the cell(s).

[0083] Preferably, the end of the afferent canal to which the chamber in question is connected and the end of the efferent canal to which the chamber in question is connected form or constitute a part, preferably at least a part, of the annular canal of the chamber in question.

[0084] According to the invention, there is also provided a method of cell growth on a microfluidic chip, preferably but not exclusively on the microfluidic chip according to the invention, comprising the steps of:

[0085] - supply at least two separate chambers of the microfluidic chip, each of the at least two separate chambers being supplied with a separate solution, called a daughter solution, coming from a reservoir of the microfluidic chip,

[0086] - conveying each of the distinct daughter solutions, from an outlet of the reservoir, among at least two distinct outlets of the reservoir, into a distinct afferent channel, among at least two distinct afferent channels of the microfluidic chip, each preferably being connected to a distinct zone of the reservoir of the microfluidic chip,

[0087] - evacuating the solutions from the at least two chambers via at least two distinct efferent channels of the microfluidic chip, said at least two distinct efferent channels each connecting a chamber, among the at least two distinct chambers, to an evacuation.

[0088] Preferably, the step of supplying each of the at least two distinct chambers of the microfluidic chip with a distinct daughter solution can also be described as a step of supplying, in parallel and / or simultaneously, several distinct chambers, preferably arranged in parallel, of the same microfluidic chip with a distinct solution.

[0089] Preferably, cell growth occurs in the afferent ducts and / or in the at least two distinct chambers.

[0090] Preferably, the method comprises a step of supplying the reservoir with at least two separate solutions, called stock solutions, each being injected into the reservoir via a separate inlet of the reservoir among at least two separate inlets of the reservoir.

[0091] Preferably, the method comprises the step of mixing in the reservoir, in a controlled manner, two mother solutions, from among the at least two distinct mother solutions, so as to obtain a mixture, preferably a desired or sought-after mixture, of the two mother solutions at an outlet of the reservoir, from among the at least two distinct outlets of the reservoir; the mixture obtained constituting a daughter solution which is distinct from at least one other of the at least two distinct daughter solutions.

[0092] Preferably, the desired or sought mixture is obtained by controlling one or more conditions of the mixture.

[0093] The mixing conditions can be:

[0094] - a concentration of the mother solutions, and / or

[0095] - a flow rate of the mother solutions, and / or

[0096] - a reservoir geometry and / or a reservoir volume, and / or

[0097] - an arrangement, preferably relative to the tank outlets, tank inlets, and / or

[0098] - an arrangement, preferably relative to the tank inlets, of the tank outlets.

[0099] Preferably, the step of mixing the two stock solutions so as to obtain a mixture of the two stock solutions consists of diluting, in the reservoir of the microfluidic chip, at least one of the stock solutions to obtain at least one of the daughter solutions.

[0100] Preferably, the method comprises a step of diluting, in the reservoir of the microfluidic chip, one, preferably several, more preferably each, of the stock solutions to obtain respectively one, several or each of the daughter solutions.

[0101] At least one, preferably more than one, more preferably of each, of the stock solutions may comprise one or more solutes in different concentrations from the solute(s) of at least one, preferably more than one, more preferably of each, of the daughter solutions.

[0102] One, preferably several, more preferably each, of the daughter solutions may consist of a dilution of one of the mother solutions.

[0103] Preferably, at least one inlet of the reservoir supplies the reservoir with mother solution different or distinct from at least one other mother solution supplied to the reservoir by one or more other of the inlets of the reservoir.

[0104] Preferably, the method comprises the step of homogenizing the mixture obtained in the afferent channel connecting one of the chambers to the outlet at which the mixture of the two mother solutions is obtained.

[0105] Preferably, the mixing of the two mother solutions is carried out along an interface included in or extending along a plane, called the outlet plane, which is:

[0106] • perpendicular to a plane, called an axial plane, in which the at least two inlets and at least two outlets of the tank are included, preferably the axial plane is intersecting the inlets and outlets of the tank,

[0107] • secant at the outlet at which the mixture of the two mother solutions is obtained.

[0108] Preferably, the output plane is between two distinct planes, called input planes, which are: • perpendicular to the axial plane,

[0109] • each intersecting at a different inlet among the at least two inlets of the reservoir. Preferably, the microfluidic chip according to the invention is capable of, more preferably is arranged to, more preferably is specially designed to, implement the method according to the invention.

[0110] Any characteristic of the microfluidic chip according to the invention can be directly transposed to the process and vice versa.

[0111] Description of figures

[0112] Other advantages and particularities of the invention will appear on reading the detailed description of implementations and embodiments which are in no way limiting, and the following appended drawings: [Fig. 1] FIGURE 1 is a schematic representation of a longitudinal section in the axial plane of a microfluidic chip according to an embodiment of the invention,

[0113] [Fig. 2] FIGURE 2 is a schematic representation of a simulation illustrating the steps of conveying daughter solutions into the afferent channels of a microfluidic chip according to one embodiment and of mixing mother solutions in the reservoir of the microfluidic chip according to the embodiment,

[0114] [Fig. 3] FIGURE 3 is a schematic representation of a simulation illustrating the steps of conveying daughter solutions into the afferent channels of a microfluidic chip according to one embodiment and of mixing mother solutions in the reservoir of the microfluidic chip according to the embodiment.

[0115] Description of the embodiments

[0116] The embodiments described below being in no way limiting, it will be possible in particular to consider variants of the invention comprising only a selection of the described characteristics, isolated from the other described characteristics (even if this selection is isolated within a sentence comprising these other characteristics), if this selection of characteristics is sufficient to confer a technical advantage or to differentiate the invention compared to the state of the prior art. This selection comprises at least one characteristic, preferably functional without structural details, or with only a part of the structural details if this part only is sufficient to confer a technical advantage or to differentiate the invention compared to the state of the prior art.

[0117] With reference to FIGURES 1 to 3, an embodiment of a microfluidic chip 1, called chip 1, for the growth of cells according to the invention is presented. The chip 1 comprises a reservoir 2 capable of containing a solution. The reservoir 2 comprises at least two inlets 8 capable of allowing the reservoir 2 to be supplied with solution. The chip 1 comprises two separate inlets 8 in FIGURE 2 and four separate inlets 8 in FIGURES 1 and 3. The solution is, for example, a culture medium. The chip 1 comprises five separate chambers 3 capable of being supplied with solution from the reservoir 2 and capable of receiving one or more cells. The chambers 3 are cylindrical in shape and have a diameter of 10 mm in the axial plane 9, that is to say in the sectional plane of FIGURE 1, depending on the embodiment. The chambers 3 have a thickness of 1.5 mm. The thickness extends perpendicular to the axial plane 9.The thickness extends perpendicular to the direction 11 in which the solution is intended to circulate in the chip 1 from the reservoir 2 to the outlet 7. The chip 1 comprises five distinct afferent channels 4. Each of the afferent channels 4 connects an outlet 5 of the reservoir to a chamber 3. The number of afferent channels 4 is equal to the number of chambers 3. The chip 1 comprises five distinct efferent channels 6. Each efferent channel 6 connects a chamber 3 to an outlet 7. The axial plane 9 intersects the inlets 8 and the outlets 5 of the reservoir 2. According to the embodiment, for each inlet 8 and for each outlet 5, an axis of revolution of an inlet 8 and an axis of revolution of an outlet 5 is included in the axial plane 9. The outlet 7 makes it possible to collect the solution coming from each of the five chambers 3. The afferent 4 and efferent 6 channels have a width of 1 mm. The width extends in the axial plane 9 and perpendicular to the direction.

[0118] 11. The afferent ducts 4 and efferent ducts 6 have a thickness of 1.5 mm.

[0119] If the number of inputs 8 is denoted n and the number of afferent channels 4 is denoted p, the reservoir 2 preferably comprises a number of inputs 8 n which is less than p and which, according to the embodiment, is equal to p-1.

[0120] According to the embodiment presented, the microfluidic chip 1 is made up of two layers of structured poly(dimethylsiloxane) (PDMS) bonded together. The structuring of the PDMS layers consists of patterns etched in the PDMS to form the different elements of the chip 1, including, among others, the reservoir 2, the chambers 3, the afferent channels 4, the outlets 5, the efferent channels 6 and the evacuation 7. For example, the afferent channels 4 and efferent channels 6 have a thickness of 1.5 mm.

[0121] The afferent channels 4 form a serpentine or a sinusoid comprising at least three periods, six periods depending on the embodiment. The sinusoidal shape offers two distinct technical effects. A first effect of reproducing the sinuous or tortuous appearance of the blood capillaries. Thus, the conditions of circulation of the fluid in the blood capillaries and, consequently, the different effects likely to occur in vivo, such as, for example, opsonization or even modifications of the flow of the fluid circulating in the blood capillaries, are reproduced. Another effect is to increase the length of the path of the solution in the afferent channels 4 by limiting the associated increase in size and / or the bulk of the chip 1. Increasing the length of the afferent channels 4 aims to approach the in vivo conditions, that is to say the significant distance traveled by the fluid in the blood capillaries.

[0122] The reservoir 2 extends mainly along the dilution axis 10 which extends mainly perpendicular to the direction 11 in which the solution is intended to circulate in the chip 1 from the reservoir 2 to the discharge 7. The inlets 8 are distributed along an axis parallel to the dilution axis 10 and the outlets 5 are distributed along an axis parallel to the dilution axis 10. According to the embodiment, the dilution axis is included in the axial plane 9. In the axial plane 9 and along the dilution axis 10, the reservoir 2 comprises a succession of inlets 8 located on one side of the reservoir 2. In the axial plane 9 and along the dilution axis 10, the reservoir 2 comprises a succession of outlets 5 located on the side of the reservoir 2 which is opposite the side comprising the succession of inlets 8.

[0123] In a direction extending along the dilution axis 10, the reservoir 2 comprises an alternation of inlets 8 and outlets 5. In a direction extending along the dilution axis 10, each inlet 2 is between two successive outlets 5. In a direction extending along the dilution axis 10, the three central outlets 5, that is to say each outlet 5 with the exception of the two end outlets 5 of the succession of outlets 5, are each between two successive inlets 8.

[0124] Each of the inlets 8 is intersected by a separate inlet plane 12. Each of the inlet planes 121, 122, 123, 124 is perpendicular to the axial plane 9. Furthermore, the inlet planes 121, 122, 123 and 124 are perpendicular to the dilution axis 10. The inlet planes 121, 122, 123, 124 form a succession of planes parallel to each other. Each of the outlets 5 is intersected by a separate outlet plane 13. Each of the outlet planes 131, 132, 133, 134, 135 is perpendicular to the axial plane 9. The outlet planes 5 form a succession of planes parallel to each other. The input planes 121, 122, 123, 124 and the output planes 131, 132, 133, 134, 135 are therefore parallel to each other. Each input plane 12 is between two output planes 13. Each of the output planes 132, 133, 134, that is to say each output plane 13 with the exception of the two output planes 131, 135 at the ends of the succession of output planes 13, is between two input planes 12.

[0125] The characteristics of the reservoir 2 described in the three preceding paragraphs have the effect of ensuring the mixing of two stock solutions, preferably distinct, each injected into the reservoir 2, by two successive inlets 12. The mixing of the stock solutions is illustrated in figures 2 and 3 and detailed in the part devoted to the method according to the invention detailed below. The mixing is obtained at the outlet 5 located between the two successive inlets 8, along the dilution axis 10, by which the two stock solutions, preferably distinct, are injected into the reservoir 2. The mixing is obtained at the outlet plane 13 located between the two successive inlet planes 12 comprising the two successive inlets 8, along the dilution axis 10, by which the two stock solutions, preferably distinct, are injected into the reservoir 2.

[0126] According to the non-limiting embodiment presented in Figures 1 and 3, each inlet plane 12 is located equidistant between two outlet planes 13. In addition, each of the outlet planes 132, 133, 134 is located equidistant between two inlet planes 12. According to the non-limiting embodiment presented in Figure 2, however, depending on the desired mixture, it is possible to adapt:

[0127] - the geometry of tank 2 and / or the volume of tank 2, and / or

[0128] - the arrangement of the inlets 8 of the tank 2 and / or the arrangement of the outlets 5 of the tank 2, preferably the arrangement of the inlets 8 of the tank 2 is adapted in relation to the arrangement of the outlets 5 of the tank 2 or vice versa. In addition, the geometry of the tank 2 and / or the volume of the tank 2 and / or the arrangement of the inlets 8 and / or the arrangement of the outlets 5 can be adapted according to:

[0129] - the concentration of the stock solutions, and / or

[0130] - the flow rate of the stock solutions, and / or

[0131] - the type of cells to be cultured, and / or - the number and dimensions of the coils, and / or

[0132] - the size and shape of the growing rooms.

[0133] In order to reproduce the shear forces exerted on the cells in the organism, the chip 1 is arranged so that, for a given chamber 3, the end 14 of the afferent channel 4 to which the given chamber 3 is connected is tangent to the given chamber 3. Advantageously, in order to reproduce the shear forces exerted on the cells in the organism, the chip 1 is arranged so that, for a given chamber 3, the end 15 of the efferent channel 6 to which the given chamber 3 is connected is tangent to the given chamber 3.

[0134] In order to reproduce the conditions in the organism in which the fluid supplying the cells is distributed around the cells, the chip 1 is arranged so that each chamber 3 comprises an annular channel 16. The annular channel 16 of a chamber 3 considered is located at the periphery of the chamber 3 considered.

[0135] According to the embodiment, the end 14 of the afferent channel 4 to which a chamber 3 in question is connected and the end 15 of the efferent channel 6 to which the chamber 3 in question is connected form a part of the annular channel 16 of the chamber 3 in question. With reference to FIGURES 1 to 3, an embodiment of a method for cell growth on a microfluidic chip 1 is presented. The method comprises the step of supplying at least two separate chambers 3 of the chip 1 with a separate solution, called a daughter solution. The daughter solutions come from the reservoir 2 of the chip 1. The method comprises the step of conveying each of the daughter solutions into a separate afferent channel 4 from one of the outlets 5 of the reservoir 2 of the chip 1. The method comprises the step of discharging the solutions from the chambers 3 to the discharge 7. Each of the solutions is discharged via a separate efferent channel 6 of the chip 1.The method comprises the step of supplying the reservoir 2 with separate solutions, called stock solutions. Each of the stock solutions is injected into the reservoir 2 via an inlet 8 separate from the reservoir 2.

[0136] The cells growing in chip 1 are, for example, cancer cells. The cells can be grown on the surface of the PDMS after its functionalization with a biological macromolecule (polysaccharide, protein). The cells growing in chip 1 are, for example, manufactured by bioprinting. Bioprinting consists of using a mixture of biocompatible materials and cells via bioprinters to create 3D cellular structures from computer-designed patterns. The cells growing in chip 1 form a tumor when assembled in 3 dimensions. Preferably, the tumor is cylindrical in shape and is located in the center of the chambers 3. Such a configuration will make it possible to maintain, all around the tumor, an annular space equal to or greater than 1 mm in which the solution will circulate.According to the embodiment, the solution is a nutrient medium to which an anticancer drug can be added to test the effectiveness of the system as a preclinical model. The thickness of the biological part will be 1.5 mm.

[0137] With reference to figures 2 and 3, the method comprises the step of mixing in the tank 2, in a controlled manner, two mother solutions so as to obtain a mixture of the two mother solutions at an outlet 5 of the tank 2. The mixture obtained constitutes a daughter solution which is distinct from at least one other of the daughter solutions.

[0138] The mixing of the two mother solutions is carried out along the interface 1321, 1322, 1323 included in the outlet plane 13. The interfaces 1321, 1322, 1323 are included or merged in the outlet planes 132, 133, 134, that is to say in each outlet plane 13 with the exception of the two outlet planes 131, 135 at the ends of the succession of outlet planes 13.

[0139] With reference to Figure 2, a simulation is presented illustrating the mixing of two separate stock solutions, each containing a solute in different concentration, at the interface 1321. The chip 1 in which the method is implemented comprises two inputs 8 and three outputs 5. The parameters used for the simulation are: a solvent with a density of 1000 kg / m 3 and whose viscosity is 10' 3 Pa.s, the solute has a diffusivity in the solvent of 10' 8 m 2 / s. The flow rates of the stock solutions injected at the inlets 8 are such that the liquid velocity in the afferent channels 4 is equal to 0.5 mm / s. The stock solution injected at the level of the inlet 8 intersected by the inlet plane 121 has a solute mass concentration of 1.10' 1 pg / ml and the mother solution injected through inlet 8 intersected by inlet plane 122 has a mass concentration of 0 pg / ml. It is observed that a solute concentration gradient is established at interface 1321. The concentration gradient is established along dilution axis 10. The daughter solution obtained at outlet 5 intersected by outlet plane 132 has a concentration of 5.10' 2 pg / ml. The outlet 5 intersected by the outlet plane 132 is included, along the dilution axis 10, between the inlet 8 intersected by the inlet plane 121 and the inlet 8 intersected by the inlet plane 122.

[0140] It is also noted that the respective solute concentrations at the two end outlets 5 are identical to the respective concentrations of the two stock solutions injected at the end inlets 8. In other words, the concentration at the end outlet 5 intersected by the outlet plane 131 is 1.10' 1 pg / ml and it is equal to the concentration of the stock solution injected into the reservoir 2 through the end inlet 8 intersected by the inlet plane 121. Similarly, the concentration at the end outlet 5 intersected by the outlet plane 133 is 0 pg / ml and it is equal to the concentration of the stock solution injected into the reservoir 2 through the end inlet 8 intersected by the inlet plane 122.

[0141] The step of mixing the two stock solutions so as to obtain a mixture of the two stock solutions may consist of a step of mixing, in the reservoir 2, two types of suspended particles each contained in one of the separate stock solutions. For example, the stock solution may be water, a physiological medium, protein-enriched plasma, a culture medium or blood. For example, the two types of particles may be of a different nature and / or size. Preferably, the desired mixture is obtained by controlling one or more of the mixing conditions detailed above.

[0142] Referring to Figure 3, a simulation is presented illustrating the mixing of four stock solutions at interface 1321. Chip 1 in which the method is implemented corresponds to chip 1 shown in Figure 1 and comprises four inputs 8 and five outputs 5. The parameters used for the simulation are: a solvent with a density of 1000 kg / m 3 and whose viscosity is 10- 3 Pa.s, the solute has a diffusivity in the solvent of 10' 8 m 2 / . The flow rates of the stock solutions injected at the inlets 8 are such that the liquid velocity in the afferent channels 4 is equal to 0.5 mm / s. Only two separate stock solutions, each with a different solute concentration, are used to obtain five daughter solutions, each with a different solute concentration. A stock solution with a solute mass concentration of 100 pg / ml is injected at the inlet 8 intersected by the inlet plane 121 and at the inlet 8 intersected by the inlet plane 123. Another stock solution with a solute mass concentration of 0 pg / ml is injected at the inlet 8 intersected by the inlet plane 122 and at the inlet 8 intersected by the inlet plane 124.

[0143] As in Figure 2, it is observed in Figure 3 that a solute concentration gradient is established at the interfaces 1321, 1322 and 1323. The concentration gradient is established along the dilution axis 10. The three daughter solutions obtained at the outlets 5 intersected by the outlet planes 132, 133, 134 have distinct concentrations. The daughter solution obtained at the outlet 5 intersected by the outlet planes 132 has a concentration of 25 pg / ml, the daughter solution obtained at the outlet 5 intersected by the outlet planes 133 has a concentration of 50 pg / ml and the daughter solution obtained at the outlet 5 intersected by the outlet planes 134 has a concentration of 76 pg / ml. The outlet 5 intersected by the outlet plane 132 is included, along the dilution axis 10, between the inlet 8 intersected by the inlet plane 121 and the inlet 8 intersected by the inlet plane 122.The outlet 5 intersected by the outlet plane 133 is included, along the dilution axis 10, between the inlet 8 intersected by the inlet plane 122 and the inlet 8 intersected by the inlet plane 123. The outlet 5 intersected by the outlet plane 134 is included, along the dilution axis 10, between the inlet 8 intersected by the inlet plane 123 and the inlet 8 intersected by the inlet plane 124. As in Figure 2, it can be seen in Figure 3 that the respective solute concentrations at the two end outlets 5 are identical to the respective concentrations of the two stock solutions injected at the end inlets 8. In other words, the concentration at the end outlet 5 intersected by the outlet plane 131 is 100 pg / ml and is equal to the concentration of the stock solution injected into the reservoir 2 through the end inlet 8 intersected by the inlet plane 121.Similarly, the solute concentration at the end outlet 5 intersected by the outlet plane 135 is 0 pg / ml and is equal to the concentration of the stock solution injected into the reservoir 2 through the end inlet 8 intersected by the inlet plane 122.

[0144] It is noted in Figures 2 and 3 that a solute concentration gradient persists at the outlets 5 intersected by the outlet planes 132, 133, 134 and also in the first sinusoid of the respective afferent channels 4 connected to the outlets 5 in question. For this purpose, the method comprises the step of homogenizing the mixture obtained, i.e. the daughter solution, in the afferent channel 4 connected to the outlet 5 at which the mixture of the two mother solutions is obtained. Advantageously, a circulation of the mixture obtained along at least three periods of the afferent channel 4 makes it possible to obtain a perfectly homogeneous mixture.

[0145] Preferably, the chip 1 according to the invention is suitable, preferably is arranged, to implement the method according to the invention. The method according to the invention is suitable, preferably is designed, to be implemented by the chip 1 according to the invention. Also, any characteristics of the chip 1 according to the invention can be introduced into the method according to the invention and vice versa.

[0146] Of course, the invention is not limited to the examples which have just been described and numerous adjustments can be made to these examples without departing from the scope of the invention.

[0147] Thus, in variants which can be combined with each other of the embodiments described above:

[0148] - the chip 1 comprises at least two distinct chambers 3, and / or - the chip 1 comprises at least two distinct afferent channels 4, and / or

[0149] - chip 1 comprises at least two efferent channels 6, and / or

[0150] - the tank 2 comprises at least two separate inlets 8 capable of allowing the tank 2 to be supplied with solution.

[0151] In addition, the various features, forms, variations and embodiments of the invention may be combined with each other in various combinations to the extent that they are not incompatible or mutually exclusive.

Claims

DEMANDS 1. Microfluidic chip (1) for cell growth, said microfluidic chip comprises: - a reservoir (2) suitable for containing a solution, - at least two separate chambers (3) capable of being supplied with solution from the reservoir and capable of receiving one or more cells, - at least two separate afferent channels (4), each connecting one outlet (5) of the reservoir, from among at least two separate outlets (5) of the reservoir, to one chamber from among the at least two separate chambers, - at least two separate efferent channels (6) each connecting one chamber among the at least two separate chambers to an evacuation (7), said evacuation being suitable for allowing the evacuation of solution from each of the at least two chambers.

2. Chip according to claim 1, wherein at least a part of each of the at least two afferent channels (4) forms a sinusoid or a serpentine.

3. Chip according to the preceding claim, wherein the sinusoidal portion or the serpentine of the at least two afferent channels (4) comprises at least three periods.

4. Chip according to any one of the preceding claims, wherein the reservoir (2) comprises at least two separate inlets (8) suitable for supplying the reservoir with solution.

5. Chip according to the preceding claim, wherein the at least two inputs (8) of the tank (2) are included in a part of the tank opposite a part of the tank on which are included the at least two outlets (5) of the tank.

6. Chip according to any one of the preceding claims, wherein the at least two inputs (8) and the at least two outputs (5) are contained in a plane (9), called axial plane (9).

7. Chip according to any one of the preceding claims, wherein the reservoir (2) extends mainly along an axis (10), called the dilution axis (10), which extends mainly perpendicularly to a direction (11) along which the solution in the chip is intended to flow from the reservoir to the outlet.

8. Chip according to the preceding claim, wherein the at least two inputs (8) are distributed along an axis parallel to the dilution axis (10) and the at least two outputs (5) are distributed along an axis parallel to the dilution axis.

9. Chip according to claim 6 or according to any one of claims 7 or 8 taken in combination with claim 6, wherein: - each of the at least two entrances (8) is intersected by a distinct plane (12), called an entrance plane, each of the entrance planes (121, 122, 123, 124) is perpendicular to the axial plane (9), said entrance planes form a succession of parallel planes, - each of the at least two outputs (5) is intersected by a distinct plane (13), called output plane, each of the output planes (131, 132, 133, 134, 135) is perpendicular to the axial plane, said output planes form a succession of parallel planes, the at least two input planes and the at least two output planes are parallel to each other.

10. Chip according to the preceding claim, wherein at least one input plane (12) is included between two output planes (13) and / or at least one output plane is included between two input planes.

11. Chip according to any one of the preceding claims, wherein, for a given chamber (3): - one end (14) of the afferent channel (4) to which the chamber in question is connected is tangent to the chamber in question, and - one end (15) of the efferent channel (6) to which the chamber under consideration is connected is tangent to the chamber under consideration.

12. Chip according to any one of the preceding claims, wherein one, several or each of the chambers (3) comprise an annular channel (16) at the periphery of the, the or each of the chambers.

13. Chip according to the preceding claim taken in combination with claim 11, wherein the end (14) of the afferent channel (4) to which the chamber in question is connected and the end (15) of the efferent channel (6) to which the chamber in question is connected form a part of the annular channel (16) of the chamber in question.

14. A cell growth method on a microfluidic chip (1), comprising the steps of: - to supply at least two separate chambers (3) of the microfluidic chip, each of the at least two separate chambers being supplied with a separate solution, called daughter solution, from a reservoir (2) of the microfluidic chip, - convey each of the distinct daughter solutions from an outlet of the tank, among at least two separate outlets (5) of the reservoir, in a separate afferent channel (4), among at least two separate afferent channels, of the microfluidic chip, - evacuate the solutions from the at least two chambers via at least two separate efferent channels (6) of the microfluidic chip, said at least two separate efferent channels each connecting one chamber, among the at least two separate chambers, to an outlet (7).

15. A method according to the preceding claim, comprising a step of supplying the reservoir (2) with at least two separate solutions, referred to as stock solutions, each being injected into the reservoir via a separate inlet (8) of the reservoir from among at least two separate inlets of the reservoir.

16. A method according to the preceding claim, comprising the step of mixing in the reservoir (2), in a controlled manner, two stock solutions, from among the at least two distinct stock solutions, so as to obtain a mixture of the two stock solutions at the level of an outlet (5) of the reservoir, from among the at least two distinct outlets of the reservoir; the mixture obtained constituting a daughter solution which is distinct from at least one other of the at least two distinct daughter solutions.

17. A method according to the preceding claim, comprising the step of homogenizing the mixture obtained in the afferent channel (4) connecting one of the chambers (3) to the outlet (5) at which the mixture of the two stock solutions is obtained.

18. A method according to claim 16 or 17, wherein the mixing of the two stock solutions is carried out along an interface (1321, 1322, 1323) contained in a plane (13), referred to as the outlet plane (13), which is: • perpendicular to a plane (9), called the axial plane (9), in which are included the at least two inlets (8) and the at least two outlets (8) of the reservoir, • secant at the outlet where the mixture of the two stock solutions is obtained.

19. A method according to the preceding claim, wherein the exit plane (13) is contained between two distinct planes (12), called the inlet plane (12), which are: • perpendicular to the axial plane (9), • each secant at a different inlet (8) among the at least two inlets of the reservoir (2).

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