Method for diagnosing genitourinary cancers

Abstract

An in vitro method for diagnosing a urological cancer comprising the comparison of a secretome of isolated cells from a urine sample from a patient to be diagnosed with respect: either to a reference secretome obtained from secretions of healthy isolated cells from a urine sample from a healthy person, or to a reference secretome obtained from secretions of healthy cells which are derivatives of standard cell line cultures, characteristic of a determined urological organ, the secretome and the reference secretome being constituted of all the components forming the respective secretions thereof.

Claims

1. An in vitro method for diagnosing a urological cancer comprising: obtaining a urine sample from a patient being diagnosed; isolating uroepithelial cells from the urine sample; culturing the isolated uroepithelial cells on a first surface of a prepared membrane, wherein the membrane is a nanostructured porous membrane; culturing a set of stromal cells on a second surface of the prepared membrane; collecting a sample secretome from the cultured uroepithelial cells; generating a sample overall print corresponding to the sample secretome; comparing the sample overall print with a reference overall print; and generating a report including a result of the comparison of the sample overall print and the reference overall print to a medical professional involved in diagnosing the patient.

2. The in vitro method for diagnosing a urological cancer according to claim 1, wherein: the report comprises deviations detected in the sample overall print from values corresponding to the reference overall print comprising an elevated concentration of at least one of a plurality of expected components; a depressed concentration of at least one of a plurality of expected components; an absence of at least one of a plurality of expected components; and a concentration of an unexpected component.

3. The in vitro method for diagnosing a urological cancer according to claim 2, further comprising diagnosing the patient as presenting a suspected urological cancer when a deviation in at least one expected component is determined by the medical professional to be of a magnitude associated with an occurrence of a urological cancer.

4. The in vitro method for diagnosing a urological cancer according to claim 3, further comprising: diagnosing a suspected kidney cancer when the deviation is associated with a component found in a first reference overall print of a first secretome produced by normal kidney uroepithelial cells; diagnosing a suspected bladder cancer when the deviation is associated with a component found in a second reference overall print of a second secretome produced by normal bladder uroepithelial cells; and diagnosing a suspected prostate cancer when the deviation is associated with a component found in a third reference overall print of a third secretome produced by normal prostate uroepithelial cells.

5. The in vitro method for diagnosing a urological cancer according to claim 1, wherein: the prepared membrane comprises a two-dimensional (2D) culture medium comprising a cell layer of confluent epithelial cells surmounted on a cell layer of confluent fibroblasts.

6. The in vitro method for diagnosing a urological cancer according to claim 5, wherein: the prepared membrane comprises a three-dimensional (3D) culture medium defining a concavity with the first surface being an outer surface, and further wherein, the cell layer of confluent epithelial cells surmounted on the cell layer of confluent fibroblasts being arranged on the first surface.

7. The in vitro method for diagnosing a urological cancer according to claim 4, wherein: the step of preparing the sample overall print is done by mass spectrometry and conducted prior to any separation of the components of the sample secretome, to obtain a mass spectrum of the secretome of the isolated cells from the sample.

8. The in vitro method for diagnosing a urological cancer according to claim 2, wherein the plurality of expected components present in the sample secretome comprise proteins, comprising PSA, PCA3, KLK15, SPINK1, PRSS3, cathepsin D, Apolipoprotein A-I, and PLK2, peptides, amino acids, and nucleic biomarkers comprising DNA, RNA, miRNA, and RNAi comprising miR-141, miR-375, fusion transcripts TMPRSS2-ERG, genes coding for SFPR1, BNC1, γ-glutamyl hydrolase (GGH), diazepam binding inhibitor (DBI), and transcription factor E2F3.

9. The in vitro method for diagnosing a urological cancer according to claim 8, wherein: a plurality of expected components reflected in a reference overall print corresponding to a reference secretome produced by normal prostate uroepithelial cells, comprise proteins PSA, PCA3, KLK15, SPINK1, and PRSS3, the RNA of fusion TMPRSS2-ERG, and the miRNAs miR-141 and miR-375.

10. The in vitro method for diagnosing a urological cancer according to claim 8, wherein: a plurality of expected components reflected in a reference overall print corresponding to a reference secretome produced by normal kidney uroepithelial cells, comprise the cathepsin D protein, and genes coding for SFPR1 and BNC1.

11. The in vitro method for diagnosing a urological cancer according to claim 8, wherein: a plurality of expected components reflected in a reference overall print corresponding to a reference secretome produced by normal bladder uroepithelial cells comprise proteins Apolipoprotein A-I and PLK2, and the genes coding for Gamma-glutamyl hydrolase (GGH), diazepam binding inhibitor (DBI), and transcription factor E2F3.

12. The in vitro method for diagnosing a urological cancer according to claim 1, wherein: the reference overall print includes at least one spectrum selected from a group consisting of a first reference overall print of a first secretome produced by normal kidney uroepithelial cells, a second reference overall print of a second secretome produced by normal bladder uroepithelial cells, a third reference overall print of a third secretome produced by normal prostate uroepithelial cells, and a fourth reference overall print of a fourth secretome produced by a population of uroepithelial cells found in a normal urine sample.

13. An in vitro method for evaluating urological health comprising: obtaining a urine sample from a patient being evaluated; isolating uroepithelial cells from the urine sample; culturing the isolated uroepithelial cells on a first surface of a prepared membrane, wherein the membrane is a nanostructured porous membrane defining a concavity, wherein the first surface is one from among an outer surface or an inner surface; culturing a set of stromal cells on a second surface of the prepared membrane, wherein the second surface is the other from among the outer surface and the inner surface; collecting a sample secretome from the cultured uroepithelial cells; preparing a sample overall print corresponding to the collected sample secretome; comparing the sample overall print with a reference overall print; and providing a result of the comparison of the sample overall print and the reference overall print to a medical professional involved in evaluating the urological health of the patient.

14. The in vitro method for evaluating urological health according to claim 13, wherein: a matrix-assisted laser desorption/ionization (MALDI) ion source and a time-of-flight (TOF) mass analyser are used in preparing the sample overall print.

Description

DETAILED DESCRIPTION

(1) 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.

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

(3) 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.

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

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

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

(7) 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.

(8) 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.

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

(10) 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.

(11) 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.

(12) 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.

(13) 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 μg/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.

(43) I—Example of Using the Chip for a Co-Culture

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

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

(46) 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.

(47) 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.

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

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

(50) 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.

(51) 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.

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

(53) 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.

(54) 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.

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

(56) 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.

(57) The Matrigel® matrix from Corning® also contains heparan sulphate proteoglycans (perlecan), of transforming growth factor β (TGF-β), 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.

(58) 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.

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

(60) 4.1. Introduction of Epithelial Cells

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

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

(63) 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.

(64) 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.

(65) 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.

(66) 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.

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

(68) 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.

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

(70) 4.2. Introduction of Cells on the Outer Face of the Protuberances

(71) 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.

(72) 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.

(73) 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.

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

(75) 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.

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

(77) 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 μl 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.

(78) 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 μg/mL of bovine pituitary extract.

(79) 4.3. Examples of Epithelial Cells and Stromal Cells

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

(81) 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.).

(82) 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).

(83) 4.4. Interchangeability of Cultures on the Inner and Outer Faces of the Protuberances

(84) 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.

(85) 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.

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

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

(88) 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.

(89) 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).

(90) 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.

(91) 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.

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

(93) 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.

(94) 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.

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

(96) 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.

(97) II—Example of Using the Chip for the Diagnosis

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

(99) 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.

(100) 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.

(101) 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.

(102) 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.

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

(104) 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.

(105) 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.

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

(107) 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.

(108) 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.

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

(110) 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.

(111) 3. Recovery of Secretions

(112) 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.

(113) 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.

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

(115) 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.

(116) 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.

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

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

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

(120) 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.

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

(122) 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.

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

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

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

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

(127) For this, around 50 μl 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.

(128) 100 μL 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 μL of substrate (TMB) are added, giving rise to a substrate enzyme colorimetric reaction.

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

(130) III—Example of Using the Chip for Screening Molecules

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

(132) IV—Example of Using the Chip to Determine the Effect of a Treatment of Urological Cancers in a Patient

(133) 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.

(134) 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.

(135) 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.

(136) V—Discrimination of Non-Cancerous Cells from Cancerous Cells by the Analysis of the MALDI-TOF Secretome

(137) Three Cell Types Used (lines): PNT2: Hormone-sensitive healthy prostatic epithelium (healthy line) LNCaP: Tumoral prostatic epithelium: hormone-sensitive (primary tumour) (cancerous line) PC3: Metastasis, secondary tumour, hormone-resistant (cancerous line)

(138) Reagents: RPMI 1640 GlutaMAX™ (Thermofisher Scientific) medium, comprising L-Alanyl-Glutamine, L-Arginine, D-Glucose (Dextrose) FCS (foetal calf serum) (PAN Biotech, Cat No: P30-3302, Batch No: P150205)

(139) Cell Culture

(140) The cells of each cell line are cultured in the RPMI 1640 GlutaMAX™ medium in 48-well plates (2D cell culture) at the rate of 50000 cells/well in a volume of 100 μl, for 48 hours with or without FCS.

(141) The 100 μl of culture supernatant are then sampled directly in each of the wells (the cells being adherent cells). For each of the culture conditions (three cell lines with and without FCS), 0.8 μl of supernatant are analysed in MALDI-TOF on a CHCA matrix (cyano-4-hydroxycinnamic acid) as a triplicate (that is 3×0.8 μl).

(142) MALDI on a CHCA Matrix:

(143) Cyano-4-hydroxycinnamic acid or CHCA is a derivative of cinnamic acid and is a member of the phenylpropanoid family. It is used as a matrix for the peptides and the nucleotides in MALDI mass spectrometry analyses. The matrix solution is composed of a crystallised molecule (CHCA, for example) and of a counterion source such as trifluoroacetic acid (TFA) to generate ions [M+H].

(144) MALDI Methodology:

(145) The matrix solution is mixed with the sample in a 1:1 ratio, then deposited on a MALDI plate. The solvents are vaporised, only leaving the recrystallised matrix, but with analyte molecules incorporated in MALDI crystals.

(146) Then, when the laser is activated on the matrix crystal in the place where the droplet has dried, the matrix absorbs laser energy. The matrix is thus desorbed and ionised. The matrix transfers the protons to the analyte molecules, thus charging the analyte which can then be analysed in any mass spectrometry with ion acceleration. In the present example, time of flight (TOF) mass spectrometry is used.

(147) Obtaining of MALDI-TOF Spectrums:

(148) The measurements on each of the biological samples per line are taken three times. Each spectrum is standardised with respect to the AUC (total area under the spectrum) then treatment of the spectrums with two methods, “baseline substraction” (matrix effect) and “smoothing”.

(149) The average spectrum over these 3 measurements is represented for each of the lines cultured with FCS (FIG. 75) or without FCS (FIG. 74).

(150) Discrimination of Peaks

(151) Methodology:

(152) Analysis type: “2D Peak Distribution” (ClinPro Tools software)

(153) The 2D peak distribution view displays the distribution of two peaks (x, y) in the spectrums of three class models, i.e. the three cell lines cultured without FCS (FIG. 76) and with FCS (FIG. 77). The ellipses represent the standard deviation of the average of the class of the area of the peaks (N=3 MALDI measurements per cell line). The data is displayed on a two-dimensional plane.

(154) By default, the two first peaks (=best separators) are represented. The x axis represents the values of the area of the peak/intensity with respect to the highest peak according to the p-value thereof, and the y axis represents the values of the area of the peak/intensity for the second highest peak. The measurements of the axis are given in the arbitrary units which are selected automatically to be adapted to the optimal alignment in the plane.

(155) The study of FIGS. 76 and 77 shows that the deprivation of FCS cells facilitates the reading of the MALDI spectrums. This can be explained by the fact that the composition of the FCS is not well known and can vary from one batch to the other. However, the absence of FCS can cause a stress in the cells which thus secrete a lot more proteins. This explains why the discrimination of the 3 lines is more difficult to make on the supernatant of cells cultured with FCS.

(156) The culture in a minimum medium for example with BSA (bovine serum albumin) instead of FCS, preserving the cellular viability and making it possible to obtain best controlled peaks, can be considered.

BRIEF DESCRIPTION OF THE DRAWINGS

(157) 1 support consisting of a non-resorbable membrane (central unit)

(158) 2 upper face of the support consisting of a non-resorbable membrane (central unit)

(159) 3 lower face of the support consisting of a non-resorbable membrane (central unit)

(160) 4 perforation of the support consisting of a non-resorbable membrane (central unit)

(161) 5 3D nanostructured porous membrane (central unit)

(162) 6 upper face of 3D nanostructured porous membrane (central unit)

(163) 7 lower face of 3D nanostructured porous membrane (central unit)

(164) 8 protuberance (central unit)

(165) 9 outer face of protuberance (central unit)

(166) 10 inner face of protuberance (central unit)

(167) 11 section of the perforation at the upper face of the support (central unit)

(168) 12 section of the perforation at the lower face of the support (central unit)

(169) 13 circular base of the protuberance (central unit)

(170) 14 duct (lower unit)

(171) 15 upper orifice of the duct (lower unit)

(172) 16 lower orifice of the duct (lower unit)

(173) 17 reservoir (lower unit)

(174) 18 duct of the reservoir (lower unit)

(175) 19 orifices of the upper unit leading to the inlet/outlet ducts (upper unit)

(176) 20 upper orifice of the duct (lower module)

(177) 21 lower orifice of the duct (lower module)

(178) 22 resorbable polymer

(179) 23 3D nanostructure

(180) 24 epithelial cell

(181) 101 upper module

(182) 102 upper unit

(183) 103 base of the upper module

(184) 104 central module

(185) 105 central unit

(186) 106 base of the central module

(187) 107 lower module

(188) 108 lower unit

(189) 109 base of the lower module

(190) 201 attachment elements

(191) 202 inlet/outlet ducts (upper module)

(192) 203 chamber (upper unit)

(193) 204 attachment elements

(194) 205 set of lower orifices of the ducts (lower module)

(195) 206 set of upper orifices of the ducts (lower module)

(196) 207 set of protuberances (central module)

(197) 208 moulded 3D nanostructure

(198) 209 upper face of the mould

(199) 210 resorbable polymer matrix

(200) 211 negative mould of a 3D nanostructure

(201) 212 lower face of the matrix

(202) F support part of the central module

(203) 213 side frame of the support part

(204) 214 open upper face of the support part

(205) 215 solid lower face of the support part

(206) 216 cut of the solid face of the support part

(207) 217 alignment pin

(208) 218 alignment hole

(209) H, H1, H2, h1, h2 mould

(210) I, i support part

(211) G, G1, G2, g1, g2 perforated part

(212) 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.

(213) 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.

(214) 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.

(215) 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.

(216) 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.

(217) 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.

(218) 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.

(219) 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.

(220) 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.

(221) 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.

(222) 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.

(223) 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).

(224) 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.

(225) 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.

(226) 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.

(227) 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.

(228) 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.

(229) 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.

(230) 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.

(231) 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.

(232) 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.

(233) 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.

(234) 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.

(235) 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.

(236) 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.

(237) 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.

(238) 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.

(239) 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.

(240) 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.

(241) 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.

(242) 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).

(243) 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).

(244) 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).

(245) 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).

(246) 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).

(247) 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).

(248) 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).

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

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

(251) 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.

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

(253) 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.

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

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

(256) 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.

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

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

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

(260) 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.

(261) 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.

(262) 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.

(263) 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.

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

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

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

(267) 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.

(268) 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.

(269) 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,

(270) 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.

(271) 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.

(272) 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.

(273) 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.

(274) 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.

(275) 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.

(276) 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,

(277) 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.

(278) 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.

(279) 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.

(280) 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.

(281) 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.

(282) 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.

(283) 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.

(284) 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.

(285) 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).

(286) 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).

(287) FIG. 74: Maldi analysis of 2D cell culture supernatants (FCS without serum) containing cellular secretions of LNCaP (solid line), PC3 (Dot) and PNT2 (Dot and dash). Each spectrum is standardised with respect to the AUC (total area under the spectrum) then treated by “baseline substraction” and “smoothing”. An average spectrum over 3 measurements and for each line is represented. The spectrums are obtained on a Maldi CHCA (Cyano-4-hydroxycinnamic acid) matrix.

(288) FIG. 75: Maldi analysis of 2D cell culture supernatants (with FCS serum) containing the cellular secretions of LNCaP (solid line), PC3 (Dot) and PNT2 (Dot and dash). Each spectrum is standardised with respect to the AUC (total area under the spectrum) then treated by “baseline substraction” and “smoothing”. An average spectrum over 3 measurements and for each line is represented. The spectrums are obtained on a Maldi CHCA (Cyano-4-hydroxycinnamic acid) matrix.

(289) FIG. 76: Ellipses representing the standard deviation of the area under the 2D peaks of the MALDI spectrums obtained for the secretions of cell lines cultured without FCS. O: PC3 (cancerous line) X: LNCaP line (cancerous line). D: PNT2 line (healthy line).

(290) FIG. 77: Ellipses representing the standard deviation of the area under the 2D peaks of the MALDI spectrums obtained for the secretions of cell lines cultured with FCS. O: PC3 (cancerous line) X: LNCaP line (cancerous line). D: PNT2 line (healthy line).