Cell co-culture chip and process for the production thereof

Abstract

A microfluidic cell culture chip which contains a central module comprising a central unit, which contains a support consisting of a non-resorbable membrane, a 3D nanostructured porous membrane, comprising at least one protuberance, and the 3D nanostructured porous membrane and that at least one protuberance being composed of materials suitable for the culture of two distinct cell types; a base, and the central unit being integrated in the base, and forming a whole with the base.

Claims

1. A microfluidic cell culture chip comprising a central module that comprises: a central unit, which contains a support consisting of a non-resorbable membrane, said support comprising an upper face and a lower face and being perforated by at least one perforation, a 3D nanostructured porous membrane, juxtaposed to the non-resorbable membrane, comprising an upper face and a lower face, and comprising at least one protuberance, said at least one protuberance being hollow, comprising an outer face and an inner face and forming a relief structure on a side of the upper face of the 3D nanostructured porous membrane, said lower face of said 3D nanostructured porous membrane being positioned and secured to said upper face of said support, or said upper face of the 3D nanostructured porous membrane being positioned and secured to said lower face of said support, and said 3D nanostructured porous membrane and the at least one protuberance being composed of materials, suitable for a culture of two distinct cell types; and a base, said central unit being integrated in said base and forming a whole with said base.

2. The microfluidic cell culture chip according to claim 1, wherein said lower face of said 3D nanostructured porous membrane is positioned and secured to said upper face of said support.

3. A method for producing the microfluidic cell culture chip according to claim 2, wherein a production of said central unit comprises: a step of extruding a resorbable polymer solution, through the at least one perforation of said support to form at least one 3D nanostructure on the side of said upper face of said support, followed by a step of polymerising said resorbable polymer solution to make rigid said at least one 3D nanostructure, said at least one 3D nanostructure forming a resorbable polymer mould on the side of said upper face of said support after polymerisation, followed by a step of applying at least one continuous layer of at least one polyelectrolyte covering the upper face of said support and a surface of said resorbable polymer mould, to constitute the 3D nanostructure porous membrane, followed by a step of dissolving said resorbable polymer mould to obtain said lower face of said 3D nanostructured porous membrane positioned and secured to said upper face of said support, an entirety of said inner face of the at least one protuberance facing said at least one perforation.

4. The microfluidic cell culture chip according to claim 1, wherein said upper face of the 3D nanostructured porous membrane is positioned and secured to said lower face of said support.

5. A method for producing the microfluidic cell culture chip according to claim 4, wherein a production of the central module comprises: a step of assembling a support part, consisting of a side frame, an open upper face and a solid lower face comprising a cut in a form of the central unit, and a mould, in shapes and dimensions of said support part, comprising at least one moulded 3D nanostructure on the upper face, a step of pouring a resorbable polymer solution on said upper face of said mould, followed by a step of polymerising said resorbable polymer solution to make rigid said resorbable polymer solution and form a resorbable polymer matrix comprising at least one negative mould of said at least one 3D nanostructure, followed by a step of removing said mould, to obtain said resorbable polymer matrix comprising at least one negative mould of said at least one 3D nanostructure formed in said cut of the solid lower face of said support part, followed by a step of assembling said support part, with a perforated part, comprising a support consisting of a non-resorbable membrane perforated by at least one perforation integrated in a base, said perforated part being in the shapes and dimensions of said support part, and the number of the at least one perforation of said perforated part being identical to the number of moulded 3D nanostructures in said mould used in the step of assembling said support part and said mould, such that said at least one perforation of said support, is aligned with said at least one negative mould of said at least one 3D nanostructure, followed by a step of applying at least one continuous layer of at least one polyelectrolyte on the continuous surface consisting of the lower face of said support and the lower face of said resorbable polymer matrix comprising at least one negative mould of said at least one 3D nanostructure at the said at least one perforation of said support, to constitute the 3D nanostructured porous membrane comprising the at least one protuberance, followed by a step of dissolving said resorbable polymer matrix comprising at least one negative mould of said at least one 3D nanostructure, and a step of removing the support part to obtain said perforated part comprising on the lower face thereof, the 3D nanostructured porous membrane, and on the side of the upper face thereof, said at least one protuberance.

6. The microfluidic cell culture chip according to claim 1, wherein the 3D nanostructured porous membrane includes at least one layer of a polyelectrolyte selected from the group consisting of poly(sodium 4-styrenesulphonate) (PSS), poly(ethyleneimine), poly(diallyldimethylammonium chloride), poly(acrylamide-co-diallyldimethylammonium chloride), diallyldimethylammonium chloride, poly(allylamine hydrochloride) (PAH), polyanetholesulfonic acid, polyacrylic acid, poly(styrene-alt-maleic acid), polyvinyl sulphate, polyvinylsulfonic acid, poly(2-acrylamido-2-methyl-1-propanesulfonic acid), poly(2-acrylamido-2-methyl-1-propanesulfonic acid-co-acrylonitrile), poly(4-styrenesulfonic acid), poly(4-styrenesulfonic-acid-co-maleic acid), and hydrated 4-styrenesulfonic sodium salt, and wherein when said 3D nanostructured porous membrane includes at least two successive layers, a layer consisting of a positively charged polyelectrolyte alternates with a layer consisting of a negatively charged polyelectrolyte.

7. The microfluidic cell culture chip according to claim 1, wherein said at least one protuberance is in a shape of a hollow dome having a circular base.

8. The microfluidic cell culture chip according to claim 7, wherein said at least one perforation of the support has a circular-shaped section at the upper face of the support, which corresponds to an upper section of the at least one perforation, having a diameter d1, which corresponds to an upper diameter d1 of the at least one perforation and a first axis passing through the centre of said upper section of the at least one perforation and perpendicular to said support, and a circular-shaped section at the lower face of the support, which corresponds to a lower section of the at least one perforation, having a diameter d2, which corresponds to a lower diameter d2 of the at least one perforation and a second axis passing through the centre of said lower section of the at least one perforation and perpendicular to said support, wherein said first axis coincides with said second axis, the diameter d1 being a value greater than or equal to 10 m and less than or equal to 500 m, and the diameter d2 being a value greater than or equal to 10 m and less than or equal to 500 m, wherein the value of the diameter d2 is greater than or equal to the value of the diameter d1, wherein said circular base of said at least one protuberance has a diameter d3, said diameter d3 of the circular base of the at least one protuberance being a value greater than or equal to 10 m and less than or equal to 500 m, and wherein said at least one protuberance has a height of 1 to 600 m.

9. The microfluidic cell culture chip according to claim 8, wherein said inner face of said at least one protuberance at least partially faces said at least one perforation of said support.

10. The microfluidic cell culture chip according to claim 9, wherein an entirety of said inner face of said at least one protuberance faces said at least one perforation of said support, and when the value of the upper diameter d1 of said at least one perforation is greater than or equal to the value of the diameter d3 of said at least one protuberance, and a fourth axis, passing through the centre of said circular base and perpendicular to said support, and a first axis, passing through the centre of the upper section of said perforation and perpendicular to said support, coincide with each other; or when the value of the upper diameter d1 of said at least one perforation is greater than the value of the diameter d3 of said at least one protuberance, and the fourth axis, passing through the centre of said circular base and perpendicular to said support, and the first axis, passing through the centre of the upper section of said perforation and perpendicular to said support, are distinct from one another by a distance less than or equal to a value obtained by a formula: (value of d1value of d3)/2.

11. The microfluidic cell culture chip according to claim 10, comprising at least two protuberances, wherein a maximum number of the at least two protuberances for a surface of the central unit of 1 cm.sup.2 is equal to a formula $\frac{{\left(d3/2\right)}^2}{\left(l+d3\right)-{\left(d3/2\right)}^2}$ where 1 is a distance between two contiguous protuberances, a value of 1 being from 10 m to 100 m, and where d3 is a diameter of the circular base of one of said at least two protuberances.

12. The microfluidic cell culture chip according to claim 8, wherein the outer face of said at least one protuberance supports at least one adherent cell, and wherein the inner face of said protuberance supports at least one adherent cell.

13. The microfluidic cell culture chip according to claim 12, wherein said outer face of said at least one protuberance supports a first set of adherent cells at a stage of confluence, said first set being a set of stromal cells, and said inner face of said at least one protuberance supports a second set of adherent cells at the stage of the confluence, said second set being a set of epithelial cells.

14. The microfluidic cell culture chip according to claim 8, further comprising a solid lower module, said solid lower module comprising a lower unit which includes an upper face, a lower face, and at least one side face, the lower unit comprising at least one tubular-shaped duct comprising an upper orifice of diameter d4 having a third axis passing through the centre of said upper orifice and perpendicular to said support, and a lower orifice of diameter d5, and a base, said lower unit being integrated in said base, and forming a whole with said base, a value of the diameter d4 of said upper orifice being greater than or equal to a value of the diameter d5 of said lower orifice, and said value of the diameter d4 being greater than or equal to the value of the lower diameter d2 of said at least one perforation, said upper face of said lower unit comprising said upper orifice of the at least one tubular-shaped duct, and said lower orifice leading to, itself or by intermediate means, an outside of said base.

15. The microfluidic cell culture chip according to claim 14, wherein said lower module and said central module are assembled such that the upper orifice of the at least one tubular-shaped duct, of said upper face of said lower unit, opens over the at least one perforation of said support of said central unit, and the lower orifice of the at least one tubular-shaped duct, itself leads to an outside of the chip, or via an intermediate element including a reservoir capable of recovering liquids and making it possible for a conveyance to the outside of the chip via an outlet duct leading to the outside of the base of said lower module, wherein said upper face of said lower unit and said lower face of said support of said central unit have an identical shape and an identical surface to one another, and wherein said lower unit and said central unit are assembled by attachment elements situated respectively on each of the bases of the lower module and of the central module, so as to assemble, in a sealed manner, said upper face of said lower unit and said lower face of said support of said central unit.

16. The microfluidic cell culture chip according to claim 14, wherein said upper orifice of said at least one tubular-shaped duct leads to one single perforation of said support, and wherein an entirety of said perforation of said support faces said upper orifice of said at least one tubular-shaped duct, when the value of the lower diameter d2 of said perforation is equal to the value of the diameter d4 of said upper orifice of said at least one tubular-shaped duct, and the second axis, passing through the centre of a lower section of the perforation and perpendicular to said support, and the third axis, passing through the centre of said upper orifice of said at least one tubular-shaped duct and perpendicular to said support, are coincided with each other, or when the value of the lower diameter d2 of said perforation is less than the value of the diameter d4 of said upper orifice of said at least one tubular-shaped duct, and the second axis, passing through the centre of said lower section of the perforation and perpendicular to said support, and the third axis, passing through the centre of said upper orifice of said at least one tubular-shaped duct and perpendicular to said support, are distinct from one another by a distance less than or equal to a value obtained by a formula: (value of d4value of d2)/2.

17. The microfluidic cell culture chip according to claim 14, wherein said lower module comprises at least two ducts, each of the at least two ducts having an upper orifice and a lower orifice, and wherein each of the upper orifices of the at least two ducts leads to the at least one perforation, and the lower orifices of the at least two ducts lead to an outside of the chip, respectively to at least two distinct sites, or the lower orifices of the at least two ducts lead to one same intermediate element including a reservoir capable of recovering liquids and making it possible for a conveyance to the outside of the chip via an outlet duct leading to the outside of the base of said lower module.

18. The microfluidic cell culture chip according to claim 14, further comprising an upper module comprising: an upper unit, which includes a solid surface defining an open volume, edges of the solid surface being capable of being in contact with said central unit of said central module and delimiting the solid surface defining said open volume; and a base, said upper unit being integrated in said base, and forming a whole with said base.

19. The microfluidic cell culture chip according to claim 18, wherein said upper unit of said upper module and said central unit of said central module are assembled, such that said solid surface defining the open volume of said upper unit is positioned above said upper face of the 3D nanostructured porous membrane of said central unit, to form a closed space, delimited by said solid surface of said upper unit and said upper face of the 3D nanostructured porous membrane, said solid surface and said upper face having an identical shape and an identical surface to one another, said upper unit of said upper module and said central unit of said central module being assembled by attachment elements situated on each of the bases of the upper module and of the central module, so as to assemble, in a sealed manner, said solid surface defining the open volume of said upper unit and said upper face of the 3D nanostructured porous membrane of said central unit, and said solid surface defining the open volume of said upper unit has two orifices respectively leading to an inlet/outlet duct, making it possible for said closed space to communicate with an outside of said chip via said inlet/outlet ducts which lead to the outside of the base.

20. The microfluidic cell culture chip according to claim 18, wherein said upper, central, and lower modules are assembled such that said solid surface defining the open volume of said upper unit of said upper module is positioned above said upper face of the 3D nanostructured porous membrane of said central unit of said central module, and the upper orifice of the at least one tubular-shaped duct, of said upper face of said lower unit of said lower module, opens on the at least one perforation of said support of said central unit, said solid surface defining the open volume of said upper unit and said upper face of the 3D nanostructured porous membrane of said central unit having an identical shape and an identical surface to one another, said upper face of said lower unit and said lower face of said support of said central unit having an identical shape and an identical surface to one another, and said upper module, said central module, and said lower module being assembled by attachment elements situated on each of the respective bases.

Description

DETAILED DESCRIPTION

Examples of Circular-Shaped Parts I, H and G

(1) According to a specific embodiment, said support part (I), said mould (H1, H2) and said perforated part (G1, G2) are circular-shaped, making it possible in particular to be adapted to the cell culture boxes with a diameter of 35 mm.

(2) FIG. 67 presents a specific embodiment of the process wherein said mould (H1) and said perforated part (G1) respectively comprise 100 moulded 3D nanostructures and 100 perforations.

(3) Such a process makes it possible to obtain a central unit with 100 protuberances.

(4) FIG. 68 presents a specific embodiment of the process wherein said mould (H2) and said perforated part (G2) respectively comprise 9 moulded 3D nanostructures and 9 perforations.

(5) Such a process makes it possible to obtain a central unit with 9 protuberances.

(6) These examples of numbers of perforations and moulded 3D nanostructures, are not limiting.

(7) Selecting the mould and the perforated part depends on the desired number of protuberances for the 3D nanostructured membrane of the central unit.

(8) In this specific embodiment, the central module consisting of the perforated part G with the protuberances obtained from the process detailed above, can be placed on a cell culture chamber, such as a cell culture box with a diameter of 35 mm containing the culture medium, by way of a part F, as shown in FIG. 71.

(9) According to an embodiment of the invention, the central module obtained by the process described above, is placed on a lower module such as described in the present invention, comprising at least one duct to collect secretions from the at least one protuberance.

(10) The lower module is of identical shape and identical dimensions to said central module.

(11) The lower module is assembled to the central module in a reproducible and specific alignment which is guided by the flat section of the parts and the pin for aligning the part of the lower module which is inserted in the hole for aligning the central module.

(12) The lower module comprises a number of ducts, identical to the number of perforations, and therefore protuberances of the central module, such that the assembly of said central module with said lower module makes it possible to align the ducts with the perforations and therefore the protuberances, to collect the secretions from the cells via a microfluidic system.

(13) In this other specific embodiment according to the invention, the lower module is replaced by the part F. This part F, used as a support of the central module on the culture box such as represented in FIGS. 67, 68 and 71 comprises square openings making it possible for the circulation of the culture medium to give nutrients to the growth cells on the inner face of said at least one protuberance.

Examples of Square-Shaped Parts I, H and G

(14) According to a specific embodiment, said support part (i), said mould (h1, h2) and said perforated part (g1, g2) are square-shaped.

(15) FIG. 72 presents a specific embodiment of the process wherein said mould (h1) and said perforated part (g1) respectively comprise 100 moulded 3D nanostructures and 100 perforations.

(16) Such a process makes it possible to obtain a central unit with 100 protuberances.

(17) FIG. 73 presents a specific embodiment of the process wherein said mould (h2) and said perforated part (g2) respectively comprise 9 moulded 3D nanostructures and 9 perforations.

(18) Such a process makes it possible to obtain a central unit with 9 protuberances.

(19) These examples of numbers of perforations and moulded 3D nanostructures, are not limiting.

(20) Selecting the mould and the perforated part depends on the desired number of protuberances for the 3D nanostructured membrane of the central unit.

(21) The resorbable polymeric solution is preferably made with chitosan, agarose or alginate.

(22) According to a specific embodiment, the invention relates to a process for producing a microfluidic cell culture chip, wherein said resorbable polymer solution (22) is chitosan.

(23) When chitosan is used, the resorbable mould can be prepared by dissolving 2% chitosan in 2% acetic acid for one night, then by diluting 1.5% chitosan with ethanol. The chitosan solution is then polymerised in a 5M hot bath of NaOH: ethanol at a ratio 1:1.

(24) According to a specific embodiment, the invention relates to a process for producing a microfluidic cell culture chip, wherein the step of polymerising said resorbable polymer solution (22), said resorbable polymer solution (22) being chitosan, is made by an incubation with a 2% acetic acid solution.

(25) When chitosan is used as a resorbable polymeric material, the dissolution is done by an incubation overnight with a 2% acetic acid solution, according to a protocol that is well known to a person skilled in the art.

(26) According to a specific embodiment, the invention relates to a process for producing a microfluidic cell culture chip, wherein said resorbable polymer solution (22) is agarose.

(27) When agarose is used, the resorbable mould can be prepared by heating and by dissolving 40 g/ml of agarose in PBS (phosphate buffered saline). Agarose is polymerised by placing the solution obtained at a temperature below the gelation point thereof.

(28) According to a specific embodiment, the invention relates to a process for producing a microfluidic cell culture chip, wherein the step of polymerising said resorbable polymer solution (22), said resorbable polymer solution (22) being agarose, is carried out by an incubation at a temperature greater than the gelation temperature of agarose.

(29) When agarose is used as a resorbable polymeric material, the dissolution is done by a slow heating from ambient temperature to a temperature of 70 C., for 120 minutes, then by letting the temperature of the agarose return to ambient temperature over one night.

(30) This heating can be done in a water bath. It is important that the temperature slowly increases to minimise thermal convection currents which could damage the 3D nanostructured porous membrane.

(31) Variants of this heating protocol, well known to a person skilled in the art, include the addition of DMSO in the water of the water bath to modify the gelation properties of agarose.

(32) According to a specific embodiment, the invention relates to a process for producing a microfluidic cell culture chip, wherein said resorbable polymer solution (22) is alginate.

(33) According to a specific embodiment, the invention relates to a process for producing a microfluidic cell culture chip, wherein the step of polymerising said resorbable polymer solution, said resorbable polymer solution (22) being alginate, is carried out by an incubation overnight with a solution with no Ca.sup.2+ a Ca.sup.2+ ion binding agent added, such as EDTA or EGTA.

(34) The polyelectrolyte multilayer film comprises, as variable parameters: the number of layers, the thickness of each of the layers, the charge of the polyelectrolyte(s) used.

(35) By varying the number of layers, the roughness, the thickness and the rigidity of the final multilayer film can be modified.

(36) Preferably, the film is composed of 15 layers, 2 nm thick, of polyelectrolytes.

(37) By varying the number of layers or the type of charge for the polyelectrolytes used, the hydrophobicity of the final multilayer film can also be modified.

(38) The extrusion of the 3D nanostructure can be subject to the following defects, due to the pumping system used for the extrusion: translation defect, when there is a translation of the protuberance with respect to leaving the site provided, directly aligned with the perforation of the support, extrusion defect, when there are defects in the shape of the protuberance, like for example a thickening of the base or other defects which will be known to a person skilled in the art.

(39) A protuberance thus formed from 3D nanostructures with a translation defect or an extrusion defect can continue exercising the technical function thereof provided initially within the device, however, as the protuberance thus formed has a less optimal shape, the performance thereof within the device is also less optimal. However, the device can continue to exercise the function thereof provided, but with a reduced performance.

(40) The protuberance can have different changes such as: a tilt with respect to an axis (y), passing through the centre of said opening and which is perpendicular to said support, a variation of the height thereof, a translation with respect to the perforation, due to the translation of the 3D nanostructured porous membrane on the support.

(41) These changes are due to the process for preparing the central module, and in particular at the phase of extruding the polymeric solution through the perforations of said support.

(42) Certain changes are also driven directly during the use of the protuberance in the device.

IExample of Using the Chip for a Co-Culture

(43) 1. Conditions for Maintaining Line Cultures of Prostate Epithelial Cells and Stromal Cells

(44) The culture medium used for all experiments is a Keratinocyte Serum Free Medium (KSFM) (Life Technologies, Carlsbad, CA, Ref. 17005-075) supplemented by 5 ng/mL of epidermal growth factor (EGF) and 50 g/mL of bovine pituitary extract.

(45) The lines of prostate epithelial cells and stromal cells are maintained in the medium are cultured in an atmosphere at 37 C. and 5% CO.sub.2.

(46) The subculturing of the cells in a fresh medium is done every three days for epithelial cells and every two days for stromal cells. For this, the cells are washed with a phosphate buffered saline solution from Dulbecco (D-PBS) without calcium and without magnesium (Life Technologies, Ref. 14190), then incubated with 1 mL of Trypsine-EDTA at 0.25 mg/mL, at 37 C., (Lonza, Basel, CH, Ref. CC-5012) for around 7 minutes.

(47) For all experiments, the culture medium of the cells has been supplemented each day with the fresh culture medium.

(48) 2. Preparing Cells Before the Introduction in the Central Unit

(49) A chemical separation of the cells is done by an incubation of 5 minutes at 37 C. with 1 ml of trypsin-EDTA at 0.25 mg/ml (Life Technologies, Ref. 25300-054) in the PBS medium without calcium and without magnesium.

(50) Independently, a microfluidic chip according to the invention is sterilised by making a 70% ethanol (volume/volume) solution circulate through the ducts, then by drying all of the microfluidic system in a furnace at a temperature of between 35 C. and 45 C. for at least 30 minutes, then by exposing it to a U.V. radiation, and to ozone for 40 minutes.

(51) 3. Preparing the 3D Nanostructured Porous Membrane of the Central Unit

(52) The 3D nanostructured porous membrane consists of successive layers of polyelectrolytes alternating a positively charged polyelectrolyte layer and a negatively charged polyelectrolyte layer. According to the production process, this same membrane consists of protuberances.

(53) The outer face and the inner face of the protuberances, consisting of the polyelectrolyte porous membrane, are covered by an extracellular matrix (ECM) preparation composed of Matrigel and/or collagen, fibronectin or hyaluronic acid.

(54) The Matrigel matrix used here is a commercial product produced by the company Corning@.

(55) It is a reconstituted basal membrane preparation, which is extracted from Engelbreth-Holm-Swarm (EHS) mouse sarcoma, a tumour rich in extracellular matrix proteins. Once isolated, this material is composed of around 60% laminin, 30% collagen IV, and 8% entactin. Entactin is a bridging molecule which interacts with laminin and collagen IV, and contributes to the structural organisation of these molecules of the extracellular matrix.

(56) The Matrigel matrix from Corning also contains heparan sulphate proteoglycans (perlecan), of transforming growth factor (TGF-), of epidermal growth factor, of insulin type growth factor, of fibroblast growth factor, a plasminogen tissue activator and other growth factors which are naturally present in the EHS tumour. It also contains residual matrix metalloproteinases derived from tumour cells.

(57) Matrigel can be used by itself to functionalise the porous membrane, at a concentration of 6 mg/ml, or as a mixture with type I collagen at a concentration of between 0.75 and 2.5 mg/ml.

(58) 4. Introducing Two Cell and Cell Co-Culture Types in the Central Unit

(59) 4.1. Introduction of Epithelial Cells

(60) Initially, the epithelial cells are introduced to form a cell joining layer, i.e. a cell culture at the stage of the confluence.

(61) According to a specific embodiment, the epithelial cells are introduced on the inner faces of the protuberances of the central unit.

(62) Three hours are required to obtain the adhesion of the cells, and 24 hours for the formation of a layer for joining cells, i.e. at a stage of cell confluence. These adherent and proliferative cells secrete their own extracellular matrix and thus establish a basal layer playing the role of a barrier.

(63) The inner face of the protuberances of the central unit is thus covered by a dense single layer of epithelial cells, which is used as a physiological support for the growth and differentiation of human cells, isolated from the patient's urine.

(64) The introduction of cells on the inner face of the protuberances can be done, according to 3 methods: returning the central module in order to have the lower sections of the perforations towards the top and manually pipette a cell suspension. returning the central module in order to have the lower sections of the perforations towards the top and use a robot for handling fluids to introduce a cell suspension. assembling the upper module, the central module and the lower module, and fill the central unit on the side of the inner face of the protuberances using microfluidic ducts of the lower unit. In the case of this pre-assembly of the three modules, the cells are therefore introduced via the ducts of the lower module. This method of introducing cells after a pre-assembly of the three modules is preferred to the two other methods, as it prevents any bacterial contamination, because the previously sterilised system is kept closed.

(65) According to a specific embodiment, the epithelial cells are introduced at a concentration of 3.10.sup.6 cells/mL in the central unit, either directly via the perforations (1.sup.st and 2.sup.nd method) with a syringe, or via the ducts of the lower unit (3.sup.rd method) by using a fluid system, automated and controlled by pressure and flow (Fluigent) or a syringe pump.

(66) Using a syringe pump with an adjustable flow is preferred, in order to provide a smooth and controlled introduction of cells.

(67) A stable and continuous flow is delivered by using pressure pumps (Fluigent, France). Pressurised containers containing the culture medium are kept in a chamber at a controlled temperature and CO.sub.2 level. The flow is adjusted to around 5-10 mL/hour (10 mbar) and the adhesion and the proliferation of the cells is observed over time. All the samples are kept in an incubator, humidified at 37 C. and 5% CO.sub.2.

(68) In a specific embodiment, the central unit comprises protuberances of a height of 350 m with a circular base of 150 m in diameter. The area of the inner surface of the protuberance is thus 329700 m.sup.2, on which around 50 epithelial cells are counted at the confluence stage (joining cell layer), that is around one cell every 66 m.sup.2.

(69) 4.2. Introduction of Cells on the Outer Face of the Protuberances

(70) Secondly, once the layer of joining (or confluent) epithelial cells formed on the inner face of the protuberances, the stromal cells are dispensed on the porous membrane at the outer and inner faces of the protuberances.

(71) The introduction of cells on the inner face of the protuberances can be done according to two methods: returning the central module in order to have the tops of the protuberances towards the top and manually pipette a cell suspension. assembling the upper module, the central module and the lower module and fill the central unit on the side of the inner face of the protuberances using the microfluidic ducts of the lower unit. In the case of this pre-assembly of the three modules, the cells are therefore introduced via the inlet/outlet ducts of the upper unit. This method of introducing cells after a pre-assembly of the three modules, is preferred to the other methods, as it prevents any bacterial contamination because the previously sterilised system is kept closed.

(72) According to a specific embodiment, the stromal cells are introduced via the inlet/outlet ducts of the upper module at a concentration of 3.10.sup.6 cells/mL in the central unit directly using a syringe (1.sup.st method), that is via the ducts of the upper module (2.sup.nd method) by using a fluid system, automated and controlled by pressure and flow (Fluigent) or a syringe pump.

(73) The stromal cells adhere very quickly (less than one hour).

(74) It is not necessary that the stromal cells form a layer of confluent (or joining) cells, the simple adhesion thereof on the outer face in this example is enough.

(75) Generally, the ratio between the epithelial cells and the stromal cells is 1:2.

(76) Thus, according to a specific embodiment, for a co-culture on a surface of 0.7 cm.sup.2, the porous membrane at the outer and inner faces of the protuberances is functionalised with 90 l of a Matrigel solution diluted to 6 mg/ml, then seeded to obtain, in the end, 7000 epithelial cells/cm.sup.2 and 14000 stromal cells (fibroblasts)/cm.sup.2.

(77) The culture medium, introduced via the inlet/outlet ducts of the upper module and via the ducts of the lower module to supply the cell cultures, is identical on either side of the protuberances, and consists of the KSFM culture medium supplemented by 5 ng/mL of epidermal growth factor (EGF) and by 50 g/mL of bovine pituitary extract.

(78) 4.3. Examples of Epithelial Cells and Stromal Cells

(79) These epithelial cells can be non-tumorigenic commercial cell lines (prostate or bladder or kidney) or commercial primary cultures.

(80) These stromal cells can be: either fibroblasts (commercial primary cultures or lines), or mesenchymal cells (commercial cultures or lines), or other stromal cells (endothelial, etc.).

(81) The two cell types used to form these cellular single layers, are called neutral or healthy, they are non-tumorigenic and only play the role of a basal layer. These neutral cells form, at the stage of the confluence, a highly contiguous layer of cells on the inner and outer face of the protuberances, establishing tight seals, that it is possible to characterise by immunofluorescence and imaging (see E-cadherin part 5 marking).

(82) 4.4. Interchangeability of Cultures on the Inner and Outer Faces of the Protuberances

(83) According to a specific embodiment, the epithelial cells are introduced on the inner face of the protuberances and the stromal cells are introduced on the outer face of the protuberances.

(84) However, the co-culture can be established in an interchangeable manner, i.e. the stromal cells can also be introduced on the inner face of the protuberances, and the epithelial cells on the outer face of the protuberances. In both cases, the polyelectrolyte layer located between the two cell types, makes it possible to constitute a porous barrier, using the positively and negatively charged polyelectrolyte mesh thereof.

(85) 5. Visualisation of the Cells in the Central Unit (Proof of Concept of the Co-Culture on the Protuberances)

(86) In order to validate the method of co-culture on the protuberances of the central unit, an immunomarking is carried out.

(87) This immunomarking is therefore carried out on dead cells (attached by PFA) and this visualisation has the sole aim of controlling the co-culture being correctly in place, and that the methodology of introducing cells in correct.

(88) The cells are visualised in the central module by immunomarking. Phalloidin is used to identify cortical actin filaments, which follow the edges of the plasma membrane and, consequently provide a means to delimit the extent of the cell and the membrane thereof. E-cadherin is used to detect the cell-cell junctions. Immunostaining is carried out by introducing E-cadherin with a syringe pump via the ducts at ambient temperature. After the formation of a confluent layer of epithelial cells, around 24 hours after the introduction thereof, they are attached for 20 minutes with 4% Perfluoroalkoxy (PFA) (volume to volume) in a solution composed of 10% sucrose in a cytoskeleton buffer (solution A). The cells are then washed with solution A and permeabilised for 3 minutes with a solution A added with 0.1% Triton TX-100. A washing with a TBS solution is carried out for 10 minutes, followed by a second washing with a PBS solution for 30 minutes. The autofluorescence of the PFA is inactivated by the NH.sub.4Cl contained in the TBS solution. The non-specific sites are blocked by an incubation with a PBS solution with 10% goat serum and 3% BSA. The cells are then incubated with a primary antibody for one hour. The primary antibody used is an anti-E-cadherin antibody (Abcam, Ref. ab1416) diluted to 1/50 in a PBS solution with 0.1% Tween-20 and 1% BSA. The cultures are then washed for 30 minutes with a PBS solution, then incubated with a secondary anti-mouse antibody coupled with the cytochrome Cy3 (Jackson, Ref. 115-162-062), diluted to 1/1000 of Phalloidin FITC (Sigma, Ref. P5282) diluted to 1:1000 in a PBS solution with 0.1% Tween-20 and 1% BSA, for 20 minutes. After a washing of 30 minutes with a PBS solution, the rings are counter-stained with Hoechst colourant (Life Technologies, Ref. H-1399), diluted to 1:7000, for 5 minutes. The cells are then washed for 10 minutes and the Dako fluorescent medium is manually introduced. The binding focal points have been detected by marking by using Vinculine. For counter-marking with Vinculine, the cells are pre-permeabilised for 40 seconds with Triton X-100 and attached with a PBS solution with 4% PFA (v/v), for 20 minutes, then washed once with a PBS solution. To avoid any non-specific antibody adsorption, the cells are incubated with a 0.1% BSA and 10% goat serum solution for one hour. The cells are then incubated for one hour with a primary antibody directed against Vinculine (Sigma, Ref. V9131) diluted to 1:700 in a PBS solution with 0.05% Tween 20 and 5% goat serum, then washed 4 consecutive times for 45 minutes with a PBS Solution The cells are then incubated with an anti-mouse antibody, coupled with the cytochrome Cy5, diluted to 1/500 in a PBS solution with 0.05% Tween 20 and with 5% goat serum (Jackson).

(89) The central module is then washed 4 times for 15 minutes with a PBS solutions. The rings and the actin are stained as described above.

(90) The co-culture is observed by fluorescence microscopy or can be observed by other microscopy methods such as phase contrast microscopy, lensless imaging, confocal microscopy, light sheet microscopy.

(91) The images are captured during the cell culture.

(92) To provide a view of the whole of the total width of the device, cell images are recorded using a lensless sensor. SEM analyses are also carried out.

(93) In a specific embodiment, the fluorescence images of the central module containing the co-culture of cells, are obtained using a Zeiss Axiolmager Z1 microscope with a 20 lens equipped with the right Apotome module for acquisitions with a z-stack field depth, with the shot every 3 mm in the axis z, for a tube, 150 mm in diameter. The images are recorded using a digital AxioCam MRm digital camera mounted on the microscope.

(94) 6. Visualisation of the Cells in the Central Unit in Real Time

(95) The cell cultures in the central unit can be monitored in real time by a phase contract microscope observation which makes it possible to visualise the non-marked and living cells, because of the transparency of the materials consisting of the modules.

IIExample of Using the Chip for the Diagnosis

(96) 1. Introduction of Cells Coming from the Patient

(97) According to a specific embodiment, the epithelial cells are introduced on the inner face of the protuberances and the stromal cells are introduced on the outer face of the protuberances.

(98) Once a single layer of cells obtained on each of the faces, that is after 24 hours, the microfluidic chip, thus provided with cells, can be used for the diagnosis of a patient.

(99) For this, the cells are isolated from a urine sample of a patient of at least 50 ml, in particular from 50 to 100 ml. The isolation is done by centrifuging the urine sample at a low speed, in particular 800 g for 5 minutes, making it possible for the sedimentation of the cells contained in the urine sample. This centrifugation step is well known to a person skilled in the art.

(100) The lower part of sedimented cells is then resuspended in the culture medium and the cell suspension is directly introduced in the microfluidic chip according to the invention, which means that the cells do not require any pre-culture before the introduction thereof in the chip.

(101) The concentration of the cells obtained from the urine sample is or varies by a few hundred cells to several thousand.

(102) The isolated urine cells of the patient can be introduced on the side of the face of the protuberance which supports the culture of epithelial cells, or on the side of the face of the protuberance which supports the culture of stromal cells. In other words, these cultures, being interchangeable on either side of the protuberance, the isolated cells of the urine of the patient can be introduced both on the inner face, and on the outer face of the protuberances.

(103) According to a specific embodiment, the isolated cells of the urine of the patient are introduced on the side of the face of the protuberance which supports the culture of epithelial cells. Thus, they are introduced via the ducts of the lower unit, when the single layer of epithelial cells is formed on the side of the inner face of the protuberances, that is via the inlet/outlet ducts of the upper module when the single layer of epithelial cells is formed on the side of the outer face of the protuberances.

(104) The cells isolated from the urine of the patient are exfoliated uroepithelial (or urothelial) cells, including all bladder, prostate and kidney epithelial cells.

(105) In a specific embodiment, the inner face of the protuberances is covered by a layer, pre-formed of previously cultured epithelial cells, the outer face of the protuberances is covered by a layer, pre-formed of fibroblasts (stromal cells), and the isolated cells are dispensed via the ducts of the lower module.

(106) These isolated cells are inserted in this layer, pre-formed of healthy epithelial cells on the side of the inner face of the protuberances, and which is supported by a layer of healthy fibroblasts.

(107) 2. Observation of the Proliferation of Cells Coming from the Patient

(108) The proliferation of isolated cells is thus monitored, in order to observe the progression of the proliferation of the isolated cells in the device and to examine if this proliferation results in replacing healthy basal cells and affects the overall secretory profile of the tissue.

(109) 3. Recovery of Secretions

(110) Once the introduction of cells isolated from the urine of the patient is done, the epithelial cells of patients are stimulated by adding 0.1 ng/ml of DHT (Dihydrotestosterone) on the outer or inner face of the protuberance. This stimulation of cells by DHT lasts between 24 hours and 48 hours.

(111) The membrane consisting of the outer and inner faces of the protuberances being porous, this stimulation can be made equally on either side of the protuberances.

(112) The epithelial cells can also be stimulated by adding mibolerone (non-metabolised hormone).

(113) The stimulation of the epithelial cells is thus, made after the binding of two cell types on either side of the protuberances, and after the growth thereof until the confluence stage.

(114) The secretions can be recovered when the isolated cells of the patient bind and are inserted in this layer, pre-formed of healthy epithelial cells on the side of the inner face of the protuberances, and which is supported by a layer of healthy fibroblasts on the side of the outer face of the protuberances. The binding of the isolated cells of the patients lasts around 3 hours and the integration thereof lasts around 6 hours.

(115) The accumulation of a sufficient volume of secretions progressively occurs.

(116) The final recovery of the secretions for the analysis of secretome is carried out after having left at least 12 hours pass.

(117) More specifically, the secretions are recovered at the end of the 24 to 48 hours of stimulation with DHT.

(118) They are then analysed by a device making it possible for the analysis of compounds in the solution. According to a specific embodiment, the secretome is analysed by mass spectrometry.

(119) The secretions can be analysed in line by sensors incorporated in said chip.

(120) It must be noted, that the different modules composing said chip are not affected when the secretions are recovered or when the secretions are continuously analysed by the sensors in line.

(121) Searching for specific markers by immunological methods can also be done in the recovered secretions.

(122) For example, the detection of PSA (prostate-specific antigen), reference biomarker of prostate cancer, can be made.

(123) For a protuberance of a height of 350 m and a circular base with a diameter of 150 m, the volume of secretions recovered at the end of 24 hours is around 2 nL.

(124) The detection and the quantification of PSA is done by an ELISA test.

(125) For this, around 50 l of medium inside several protuberances are collected then deposited in a 96-well plate, placed at 37 C. for 45 minutes. Five successive washes with distilled water are necessary, in order to remove proteins not attached to the anti-PSA primary antibody.

(126) 100 L of free anti-PSA secondary antibody coupled with HRP (Horseradish peroxidase) are then added in each well before 45 minutes of incubation at 37 C. of the ELISA plate. Finally, 100 L of substrate (TMB) are added, giving rise to a substrate enzyme colorimetric reaction.

(127) After 15 minutes at 37 C., the reaction is stopped by adding 100 L of sulphuric acid and the absorbance is detected using an ELISA plate reader at 450 nm.

IIIExample of Using the Chip for Screening Molecules

(128) In a specific embodiment, the microfluidic cell culture chip according to the invention, is used for screening molecules.

IVExample of Using the Chip to Determine the Effect of a Treatment of Urological Cancers in a Patient

(129) In a specific embodiment, the microfluidic cell culture chip according to the invention, is used to determine the effect of a treatment for a urological cancer in a patient suffering from a urological cancer.

(130) In this embodiment, the analysis of the secretome of isolated cells of the urine of the patient, inserted in the culture of epithelial cells on the protuberance, is done before and after the treatment of the patient, and/or during the treatment.

(131) The comparison of the secretome obtained before the treatment with that obtained after the treatment, and/or that obtained during the treatment, makes it possible to determine the effect of the treatment on the urological cancer of which the patient is suffering from.

BRIEF DESCRIPTION OF THE DRAWINGS

(132) 1 support consisting of a non-resorbable membrane (central unit) 2 upper face of the support consisting of a non-resorbable membrane (central unit) 3 lower face of the support consisting of a non-resorbable membrane (central unit) 4 perforation of the support consisting of a non-resorbable membrane (central unit) 5 3D nanostructured porous membrane (central unit) 6 upper face of 3D nanostructured porous membrane (central unit) 7 lower face of 3D nanostructured porous membrane (central unit) 8 protuberance (central unit) 9 outer face of protuberance (central unit) 10 inner face of protuberance (central unit) 11 section of the perforation at the upper face of the support (central unit) 12 section of the perforation at the lower face of the support (central unit) 13 circular base of the protuberance (central unit) 14 duct (lower unit) 15 upper orifice of the duct (lower unit) 16 lower orifice of the duct (lower unit) 17 reservoir (lower unit) 18 duct of the reservoir (lower unit) 19 orifices of the upper unit leading to the inlet/outlet ducts (upper unit) 20 upper orifice of the duct (lower module) 21 lower orifice of the duct (lower module) 22 resorbable polymer 23 3D nanostructure 24 epithelial cell 101 upper module 102 upper unit 103 base of the upper module 104 central module 105 central unit 106 base of the central module 107 lower module 108 lower unit 109 base of the lower module 201 attachment elements 202 inlet/outlet ducts (upper module) 203 chamber (upper unit) 204 attachment elements 205 set of lower orifices of the ducts (lower module) 206 set of upper orifices of the ducts (lower module) 207 set of protuberances (central module) 208 moulded 3D nanostructure 209 upper face of the mould 210 resorbable polymer matrix 211 negative mould of a 3D nanostructure 212 lower face of the matrix F support part of the central module 213 side frame of the support part 214 open upper face of the support part 215 solid lower face of the support part 216 cut of the solid face of the support part 217 alignment pin 218 alignment hole H, H1, H2, h1, h2 mould I, i support part G, G1, G2, g1, g2 perforated part

(133) FIG. 1: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is in whole, facing the perforation.

(134) FIG. 2: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is less than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is in whole, facing the perforation.

(135) FIG. 3: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and to the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is partially facing the perforation.

(136) FIG. 4: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is less than the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is partially facing the perforation.

(137) FIG. 5: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is less than the value of the diameter d2 of the lower section of said perforation, and is less than the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is partially facing the perforation.

(138) FIG. 6: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is less than the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is partially facing the perforation.

(139) FIG. 7: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is in whole, facing the perforation.

(140) FIG. 8: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is in whole, facing the perforation.

(141) FIG. 9: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is less than the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is in whole, facing the perforation.

(142) FIG. 10: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is less than the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is partially facing the perforation.

(143) FIG. 11: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is less than the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is in whole, facing the perforation.

(144) FIG. 12: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the protuberance is tilted along an axis (z) with respect to the vertical axis (y).

(145) FIG. 13: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the inner face of the protuberance is covered by an assembly of a first cell type at the stage of the confluence, and the outer face of the protuberance is covered by an assembly of a second cell type at the stage of the confluence.

(146) FIG. 14: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is in whole, facing the duct.

(147) FIG. 15: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is equal to the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d5 of the lower orifice, and wherein the protuberance is in whole, facing the duct.

(148) FIG. 16: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is greater than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is in whole, facing the duct.

(149) FIG. 17: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is greater than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is in whole, facing the duct.

(150) FIG. 18: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is greater than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is in whole, facing the duct.

(151) FIG. 19: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is in whole, facing the duct.

(152) FIG. 20: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is greater than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(153) FIG. 21: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is less than the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(154) FIG. 22: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is less than the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(155) FIG. 23: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is less than the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is greater than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(156) FIG. 24: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(157) FIG. 25: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is greater than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(158) FIG. 26: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is greater than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(159) FIG. 27: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is greater than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(160) FIG. 28: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is greater than the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(161) FIG. 29: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(162) FIG. 30: Schematic, cross-sectional view of a central unit comprising a protuberance in the 3D nanostructured membrane, and a perforation in the support, and wherein the value of the diameter d1 of the upper section of said perforation is equal to the value of the diameter d2 of the lower section of said perforation, and is greater than the value of the diameter d3 of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a duct, of which the value of the diameter d4 of the upper orifice is equal to the value of the diameter d2 of the lower section of said perforation, and is equal to the value of the diameter d5 of the lower orifice, and wherein the protuberance is partially facing the duct.

(163) FIG. 31: Schematic, cross-sectional view of a central unit comprising two protuberances in the 3D nanostructured membrane, and two perforations in the support, and wherein the value of the diameter d1 of the upper section of each of the perforations is equal to the value of the diameter d2 of the lower section of each of the perforations, and is equal to the value of the diameter d3 of the circular base of each of the protuberances, said central unit being positioned on a lower unit comprising two ducts, of which the value of the diameter d4 of the upper orifice of each of the ducts is equal to the value of the diameter d2 of the lower section of each of the perforations and is equal to the value of the diameter d5 of the lower orifice, and the two lower orifices of the two ducts respectively leading to a reservoir, leading to the outside of the lower module via an outlet duct (protuberance in whole, facing the duct).

(164) FIG. 32: Schematic, cross-sectional view of a central unit comprising two protuberances in the 3D nanostructured membrane, and two perforations in the support, and wherein the value of the diameter d1 of the upper section of each of the perforations is equal to the value of the diameter d2 of the lower section of each of the perforations, and is equal to the value of the diameter d3 of the circular base of each of the protuberances, said central unit being positioned on a lower unit comprising two ducts, of which the value of the diameter d4 of the upper orifice of each of the ducts is equal to the value of the diameter d2 of the lower section of each of the perforations and is equal to the value of the diameter d5 of the lower orifice, and the two lower orifices of the two ducts leading to the outside of the lower module in two distinct sites (protuberance in whole, facing the duct).

(165) FIG. 33: Schematic, cross-sectional view of a central unit comprising two protuberances in the 3D nanostructured membrane, and two perforations in the support, and wherein the value of the diameter d1 of the upper section of each of the perforations is equal to the value of the diameter d2 of the lower section of each of the perforations, and is equal to the value of the diameter d3 of the circular base of each of the protuberances, said central unit being positioned on a lower unit comprising two ducts, of which the value of the diameter d4 of the upper orifice of each of the ducts is equal to the value of the diameter d2 of the lower section of each of the perforations and is equal to the value of the diameter d5 of the lower orifice, and the two ducts are connected to one another such that the two lower orifices of the two ducts lead to the outside of the lower module in the same site (protuberance in whole, facing the duct).

(166) FIG. 34: Schematic, cross-sectional view of a central unit comprising two protuberances in the 3D nanostructured membrane, and two perforations in the support, and wherein the value of the diameter d1 of the upper section of each of the perforations is equal to the value of the diameter d2 of the lower section of each of the perforations, and is equal to the value of the diameter d3 of the circular base of each of the protuberances, said central unit being positioned on a lower unit comprising two ducts, of which the value of the diameter d4 of the upper orifice of each of the ducts is equal to the value of the diameter d2 of the lower section of each of the perforations and is equal to the value of the diameter d5 of the lower orifice, and the two lower orifices of the two ducts respectively leading to a reservoir, each of the reservoirs leading to the outside of the lower module in the same site, via the outlet ducts connected to one another (protuberance in whole, facing the duct).

(167) FIG. 35: Schematic, cross-sectional view of a central unit comprising two protuberances in the 3D nanostructured membrane, and two perforations in the support, and wherein the value of the diameter d1 of the upper section of each of the perforations is equal to the value of the diameter d2 of the lower section of each of the perforations, and is equal to the value of the diameter d3 of the circular base of each of the protuberances, said central unit being positioned on a lower unit comprising two ducts, of which the value of the diameter d4 of the upper orifice of each of the ducts is equal to the value of the diameter d2 of the lower section of each of the perforations and is greater than the value of the diameter d5 of the lower orifice, and the two ducts are connected to one another such that the two lower orifices of the two ducts lead to the same site on a reservoir, which leads to the outside of the lower module via an outlet duct (protuberance in whole, facing the duct).

(168) FIG. 36: Schematic, cross-sectional view of a central unit comprising two protuberances in the 3D nanostructured membrane, and two perforations in the support, and wherein the value of the diameter d1 of the upper section of each of the perforations is equal to the value of the diameter d2 of the lower section of each of the perforations, and is equal to the value of the diameter d3 of the circular base of each of the protuberances, said central unit being positioned on a lower unit comprising two ducts, of which the value of the diameter d4 of the upper orifice of each of the ducts is equal to the value of the diameter d2 of the lower section of each of the perforations and is greater than the value of the diameter d5 of the lower orifice, and the two lower orifices of the two ducts respectively lead to two distinct sites on one same reservoir, which leads to the outside of the lower module via an outlet duct (protuberance in whole, facing the duct).

(169) FIG. 37: Schematic, cross-sectional view of a central unit comprising four protuberances in the 3D nanostructured membrane, and four perforations in the support, and wherein the value of the diameter d1 of the upper section of each of the perforations is equal to the value of the diameter d2 of the lower section of each of the perforations, and is equal to the value of the diameter d3 of the circular base of each of the protuberances, said central unit being positioned on a lower unit comprising four ducts, of which the value of the diameter d4 of the upper orifice of each of the ducts is equal to the value of the diameter d2 of the lower section of each of the perforations and the two lower orifices of a first set of two ducts respectively lead to two distinct sites on a first reservoir, and the two lower orifices of a second set of two ducts respectively lead to two distinct sites on a second reservoir, the first and the second reservoir respectively leading to the outside of the lower module in distinct sites (protuberance in whole, facing the duct).

(170) FIG. 38: Schematic, perspective view of the upper module.

(171) FIG. 39: Schematic, perspective view of the central module.

(172) FIG. 40: Schematic, perspective view taken from above the upper face of the 3D nanostructured porous membrane, of a central unit comprising a set of protuberances.

(173) FIG. 41: Schematic, perspective view of the lower module.

(174) FIG. 42: Schematic, perspective view of the microfluidic chip comprising the assembly of the upper module, of the central module and of the lower module.

(175) FIG. 43: Photo of the upper module (side view of the opening of the chamber).

(176) FIG. 44: Photo of the central module. Top view of the upper face of the 3D nanostructured porous membrane comprising a set of protuberances.

(177) FIG. 45: Photo of the lower module. Top view of the upper face of the lower unit comprising the set of upper orifices of the ducts.

(178) FIG. 46: Photo of the upper module and of the lower module.

(179) FIG. 47: Photo of the disassembled upper module, of the disassembled central module and of the disassembled lower module.

(180) FIG. 48: Schematic, cross-sectional view of the support consisting of a non-resorbable membrane, of the central unit, comprising a perforation.

(181) FIG. 49: Schematic, cross-sectional view of the support consisting of a non-resorbable membrane, of the central unit, comprising a perforation, through which a resorbable polymer has been extruded to form a 3D nanostructure on the side of the upper face of said support.

(182) FIG. 50: Schematic, cross-sectional view of the support consisting of a non-resorbable membrane, of the central unit, comprising a perforation, through which a resorbable polymer has been extruded to form a 3D nanostructure on the side of the upper face of said support on which a polyelectrolyte layer has been applied to obtain the 3D nanostructured porous membrane comprising a moulded protuberance on said 3D nanostructure.

(183) FIG. 51: Schematic, cross-sectional view of the support consisting of a non-resorbable membrane and comprising a perforation, of the central unit, on which is positioned secured to the 3D nanostructured membrane comprising a hollow protuberance.

(184) FIG. 52: Photo using a confocal microscope of the inner face of a protuberance supporting a culture of epithelial cells at the stage of the confluence.

(185) FIG. 53: Schematic, cross-sectional view of a mould comprising at least one moulded 3D nanostructure on the side of the upper face thereof.

(186) FIG. 54: Schematic, cross-sectional view of a mould covered with resorbable polymer on the side of the upper face of said mould.

(187) FIG. 55: Schematic, cross-sectional view of a resorbable polymer matrix comprising at least one negative mould of at least one moulded 3D nanostructure.

(188) FIG. 56: Schematic, cross-sectional view of a resorbable polymer matrix comprising at least one negative mould of at least one moulded 3D nanostructure, the lower face of said matrix being covered by a polyelectrolyte layer.

(189) FIG. 57: Schematic, cross-sectional view of a resorbable polymer matrix comprising at least one negative mould of at least one moulded 3D nanostructure assembled with a perforated part comprising a support consisting of a non-resorbable membrane perforated by at least one perforation, said matrix being assembled on the side of the lower face thereof with the upper face of said support, such that the negative mould of the 3D nanostructure is aligned with said perforation of the support.

(190) FIG. 58: Schematic, cross-sectional view of a resorbable polymer matrix comprising at least one negative mould of at least one moulded 3D nanostructure assembled with a perforated part comprising a support consisting of a non-resorbable membrane perforated by at least one perforation, said matrix being assembled on the side of the lower face thereof with the upper face of said support, such that the negative mould of the 3D nanostructure is aligned with said perforation of the support,

(191) wherein the continuous surface constituted by the lower face of said support and the lower face of said resorbable polymer matrix comprising at least one negative mould at said at least one perforation of said support, is covered by a polyelectrolyte layer to form a 3D nanostructured membrane comprising at least one protuberance.

(192) FIG. 59: Schematic, cross-sectional view of the central module corresponding to the perforated part comprising, on the side of the lower face of the support, a 3D nanostructured porous membrane and on the side of the upper face of the support, at least one protuberance in the extension of the at least one perforation of the support of the perforated part.

(193) FIG. 60: Schematic, cross-sectional view of a mould comprising at least one moulded 3D nanostructure on the side of the upper face thereof, assembled on the side of the upper face thereof with a support part comprising a cut in the lower face thereof.

(194) FIG. 61: Schematic, cross-sectional view of a mould comprising at least one moulded 3D nanostructure on the side of the upper face thereof, assembled on the side of the upper face thereof with a support part comprising a cut in the lower face thereof, where said mould is covered by resorbable polymer on the side of the upper face thereof.

(195) FIG. 62: Schematic, cross-sectional view of a resorbable polymer matrix comprising at least one negative mould of at least one moulded 3D nanostructure, said matrix being formed at the cut of the support part.

(196) FIG. 63: Schematic, cross-sectional view of a support part containing at the cut of the solid lower face thereof, a resorbable polymer matrix comprising at least one negative mould of at least one moulded 3D nanostructure, said support part being assembled to a perforated part comprising a support with at least one perforation, such that said negative mould of at least one moulded 3D nanostructure is aligned with said perforation.

(197) FIG. 64: Schematic, cross-sectional view of a support part containing at the cut of the solid lower face thereof, a resorbable polymer matrix comprising at least one negative mould of at least one moulded 3D nanostructure, said support part being assembled to a perforated part comprising a support with at least one perforation, such that said negative mould of at least one moulded 3D nanostructure is aligned with said perforation,

(198) the continuous surface constituted by the lower face of said support and the lower face of said resorbable polymer matrix comprising at least one negative mould at the said at least one perforation of said support, being covered by a polyelectrolyte layer to form a 3D nanostructured membrane comprising at least one protuberance.

(199) FIG. 65: Schematic, cross-sectional view of the support of the perforated part comprising, on the side of the lower face thereof, a 3D nanostructured porous membrane and on the side of the upper face thereof, at least one polyelectrolyte protuberance in the extension of the at least one perforation of the support of the perforated part, and the support part I.

(200) FIG. 66: Schematic, cross-sectional view of the central module corresponding to the perforated part comprising, on the side of the lower face of the support, a 3D nanostructured porous membrane, and on the side of the upper face of the support, at least one protuberance in the extension of the at least one perforation of the support of the perforated part.

(201) FIG. 67: Schematic, perspective view of a circular-shaped support part I, of a circular-shaped mould H and comprising 100 moulded 3D nanostructures (H1), of a circular-shaped perforated part G and comprising 100 perforations (G1) and of a support part F of the central module.

(202) FIG. 68: Schematic, perspective view of a circular-shaped support part I, of a circular-shaped mould H and comprising 9 moulded 3D nanostructures (H2), of a circular-shaped perforated part G and comprising 9 perforations (G2) and of a support part F of the central module.

(203) FIG. 69: Schematic, perspective views of the assembly of a circular-shaped support part I on a circular-shaped mould H and comprising 100 moulded 3D nanostructures (H1), via the alignment pin of I and the alignment hole of H1. A: Top view when the two elements are assembled. B: Top view when the elements are disassembled. C: Profile view when the two elements are assembled. D: Profile view when the two elements are disassembled.

(204) FIG. 70: Schematic, perspective views of the assembly of a circular-shaped support part I on a circular-shaped perforated part G and comprising 100 perforations (G1) via the alignment pin I and the alignment hole of G1. A: Top view when the two elements are assembled. B: Top view when the elements are disassembled. C: Bottom view when the two elements are assembled. D: Bottom view when the two elements are disassembled. E: Profile view when the two assembled elements are returned such that G1 is oriented towards the top and I is oriented towards the bottom.

(205) FIG. 71: Schematic, perspective views of the assembly of a circular-shaped perforated part G and comprising 100 perforations (G1) on a support part F of the central module. A: Bottom view of the two assembled elements. B: Bottom view when the two elements are disassembled. C: Profile view when the two elements are assembled. D: Profile view when the two elements are assembled.

(206) FIG. 72: Schematic, perspective view of a square-shaped support part i, of a square-shaped mould H and comprising 100 moulded 3D nanostructures (h1), of a square-shaped perforated part G comprising 100 perforations (g1).

(207) FIG. 73: Schematic, perspective view of a square-shaped support part i, of a square-shaped mould H and comprising 9 moulded 3D nanostructures (h2), of a square-shaped perforated part G and comprising 9 perforations (g2).