WO2017194894A1 - Puce de co-culture cellulaire et son procede de fabrication - Google Patents

Puce de co-culture cellulaire et son procede de fabrication Download PDF

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Publication number
WO2017194894A1
WO2017194894A1 PCT/FR2017/051148 FR2017051148W WO2017194894A1 WO 2017194894 A1 WO2017194894 A1 WO 2017194894A1 FR 2017051148 W FR2017051148 W FR 2017051148W WO 2017194894 A1 WO2017194894 A1 WO 2017194894A1
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WO
WIPO (PCT)
Prior art keywords
support
value
perforation
diameter
face
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
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PCT/FR2017/051148
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English (en)
French (fr)
Inventor
Donald Keith Martin
Nathalie Picollet-D'hahan
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universite Grenoble Alpes
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
Original Assignee
Commissariat a lEnergie Atomique CEA
Universite Grenoble Alpes
Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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Application filed by Commissariat a lEnergie Atomique CEA, Universite Grenoble Alpes, Commissariat a lEnergie Atomique et aux Energies Alternatives CEA filed Critical Commissariat a lEnergie Atomique CEA
Priority to CN201780043057.1A priority Critical patent/CN109699180B/zh
Priority to US16/300,504 priority patent/US11857958B2/en
Priority to EP17727301.8A priority patent/EP3455342B1/fr
Priority to JP2018559754A priority patent/JP6983180B2/ja
Publication of WO2017194894A1 publication Critical patent/WO2017194894A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502707—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by the manufacture of the container or its components
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/16—Microfluidic devices; Capillary tubes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • C12M25/04—Membranes; Filters in combination with well or multiwell plates, i.e. culture inserts

Definitions

  • the present invention relates to a microfluidic cell culture chip comprising several parts, each denoted by the term “module”, and capable of being assembled in pairs.
  • the present invention also relates to a method of manufacturing said chip, as well as its uses in the field of diagnosis.
  • This organomimetic device based on a microfluidic system, comprises a PDMS porous membrane, positioned at the interface of two microfluidic channels for the circulation of fluids along said membrane, and on which is cultured a layer of intestinal epithelial cells on least one side.
  • the coupling of the membrane to support members causes a stretching movement of the membrane in at least one dimension, and thus enables the tissue motion to be reproduced in vivo.
  • This chip based on a microfluidic system, comprises a porous PDMS membrane, positioned at the interface of two microfluidic channels for the circulation of fluids along said membrane, and on which are cultured intestinal epithelial cells Caco-2 which spontaneously form villi beyond two days of culture in the presence of a constant flow of medium of 30 ⁇ 71 ⁇ .
  • PDMS porous PDMS membrane
  • inertia, porosity PDMS
  • PDMS membranes may have intrinsic intrinsic transport, mechanical and structural properties that are different from those of the natural basement membrane of the tissue. By recreating this type of interface, the authors favor the reproduction of the microarchitecture of the organ.
  • these structures are solid, that is to say, not hollowed out, and therefore do not allow the collection of effluents or biofluids secreted by the cells in co-culture on these structures.
  • the detection of known markers is by immunofluorescence after fixation of the cells and labeling with the aid of an antibody, which therefore involves lysis of the cells for analysis.
  • Biol, 5, 1130 in which they compare the Transwell® 2D culture model with a culture model of microvilli-mimicking 3D structures of the cytochrome P450 drug-metabolizing enzyme activity and show that In 2D the activity remains unchanged during the culture, whereas in 3D this activity increases during the formation of microvilli and then stabilizes.
  • the topography of the cell medium has a significant impact on the behavior of cells, so it is important to reproduce as precisely as possible the in vivo conditions of cells, in order to develop reliable models of cell culture.
  • the inventors have developed a microfluidic cell co-culture chip, mimicking organ microvilli with an acinar / tubullary structure and the luminal microenvironment.
  • the microfluidic chip according to the invention comprises a porous biomaterial membrane which comprises folds thus forming 3D hollow protuberances on which adherent cells are grown on either side of said membrane at the level of the protuberances.
  • the coupling of this membrane to a microfluidic system makes it possible to recover cell secretions during their culture in order to analyze their composition.
  • the chip according to the invention makes it possible to carry out these detections in the secretions of the cells. cells grown on the protuberance, which are recovered by the microfluidic system.
  • the chip according to the invention thus makes it possible to collect secretions on living cells and consequently to collect the secretions at different times during the culture, or even over several days for carrying out kinetic studies, while maintaining the viability and the functionality of cells over time.
  • secretions consist of all the molecules that can be secreted in the cell culture medium such as peptides, proteins, amino acids, miRNAs, DNA, RNA.
  • the analysis profile of these secretions makes it possible to obtain the secretome, that is to say the qualitative and quantitative profile of the secretory components.
  • the present invention relates to a microfluidic cell culture chip that contains a central module (104) comprising:
  • a support consisting of a nonabsorbable membrane (1), comprising an upper face (2) and a lower face (3), perforated by at least one perforation (4),
  • a 3D nanostructured porous membrane (5) comprising an upper face (6) and a lower face (7), and comprising at least one protuberance (8), said at least one protuberance comprising an outer face (9) and a internal face (10) and forming a relief structure on the side of the upper face (6) of the 3D nanostructured membrane (5) (FIG.
  • said lower face (7) of said 3D nanostructured porous membrane (5) being positioned integrally on said upper face (2) of said support (1), or said upper face (6) of the 3D nanostructured membrane (5) being positioned integrally on said lower face (3) of said support (1), and said 3D nanostructured porous membrane (5) and the at least one protuberance (8) being composed of materials suitable for culturing two distinct types of cells;
  • non-absorbable membrane means that the membrane can not be removed by either a physical process or a chemical process in an aqueous solvent.
  • perforation which refers to the support, denotes a recess passing through the support from one side, that is to say an opening through the support between the lower face and the upper face, thus allowing communication between these two faces.
  • This perforation of said support creates an empty space through said support, and is characterized by a section at the upper face of said support and a section at the lower face of said support.
  • protuberance which refers to the nanostructured porous membrane in 3D designates a protruding projection forming a relief structure, on the side of the upper face of the porous nanostructured membrane in 3D.
  • porous which refers to the nanostructured porous membrane in 3D means that said membrane comprises continuous pores interconnected with each other, with a diameter of between 2 and 10 nm, extending from the upper face to the lower face. of said membrane. These pores allow exchanges of gas through the membrane, as well as small molecules contained in the culture medium (growth factors, serum proteins, nutrients ensuring the viability of teks cells than Ca 2+ , K + , Na ions + ). These pores also make it possible to pass pharmacological molecules such as inhibitors or anticancer drugs, small ARs (for example interfering RNAs), hormones (for example dihydrotestosterone).
  • 3D nanostructured which refers to the porous membrane designates the presence of at least one relief structure, in 3 dimensions, on the upper face of said porous membrane, the scale of said structure being order of the nanometer.
  • integral which designates the nature of the bond between said support and said membrane, means that chemical bonds as well as hydrophobic and electrostatic interactions are established between said support and said membrane in order to bind said membrane and said support. in a cohesive way by a waterproof connection.
  • base designates the solid part in which said unit is integrated.
  • the invention relates to a microfluidic cell culture chip which contains a central module (104) comprising:
  • a support consisting of a nonabsorbable membrane (1), comprising an upper face (2) and a lower face (3), perforated by at least one perforation (4),
  • a 3D nanostructured porous membrane (5) comprising an upper face (6) and a lower face (7), and comprising at least one protuberance (8), said at least one protuberance comprising an outer face (9) and a internal face (10) and forming a relief structure on the side of the upper face (6) of the 3D nanostructured membrane (5) (FIG. 40), said lower face (7) of said 3D nanostructured porous membrane (5) being positioned integrally on said upper face (2) of said support (1), and said 3D nanostructured porous membrane (5) and the at least one protuberance (8) being composed of materials suitable for the cultivation of two types of cells distinct;
  • the invention relates to a microfluidic cell culture chip which contains a central module (104) comprising:
  • a support consisting of a nonabsorbable membrane (1), comprising an upper face (2) and a lower face (3), perforated by at least one perforation (4),
  • a 3D nanostructured porous membrane (5) comprising an upper face (6) and a lower face (7), and comprising at least one protuberance (8), said at least one protuberance comprising an outer face (9) and a internal face (10) and forming a relief structure on the side of the upper face (6) of the nanostructured membrane in 3D (5), said upper face (6) of the nanostructured membrane in 3D (5) being positioned integrally on said lower face (3) of said support (1), and said 3D nanostructured porous membrane (5) and the at least one protuberance (8) being composed of materials suitable for culturing two distinct types of cells;
  • the invention relates to a microfluidic cell culture chip in which said at least one protuberance (8) of the nanostructured porous membrane of said microfluidic cell culture chip is hollow-domed and has a circular base. (13).
  • the term "domed" which refers to the shape of the protuberance means that said protuberance has a rounded arch-shaped structure with a circular base, forming a convexity on the side of the upper face of said support.
  • said protrusion may have a rounded arch-shaped structure with an oval base, forming a convexity on the side of the upper face of said support.
  • the expression "hollow dome-shaped” signifies that the inner face of said protuberance delimits an empty volume because said protuberance corresponds to a recess of said nanostructured porous membrane in 3D on the side of the lower face thereof, said recess forming and a concavity on the side of the underside of said membrane, and therefore an empty volume.
  • circular base which refers to the hollow dome, designates the empty bottom surface on which the domed protuberance rests and delimited by the internal face of said protuberance.
  • the invention relates to a microfluidic cell culture chip wherein said at least one perforation of the support is characterized by a circular section at the upper face of said support and a circular section at the face. lower of said support.
  • the invention relates to a microfluidic cell culture chip in which the at least one perforation (4) of the support (1) has a section (11) at the level of the upper face (2) of the support. (1) (upper section of the perforation) having an axis (x) passing through the center of said upper section (11) of the perforation (4) perpendicular to said support (1), and a section (12) at the level of the lower face (3) of the support (1) (lower section of the perforation) having an axis (w) passing through the center of said lower section (12) of the perforation (4) and perpendicular to said support (1), and wherein said axes (x) and (w) coincide.
  • the invention relates to a microfluidic cell culture chip in which the diameter d2 of the circular section of said perforation at the lower face of the support is greater than or equal to the diameter d1 of the circular section. said perforation at the upper face of the support.
  • the invention relates to a microfluidic cell culture chip in which the at least one perforation (4) of the support (1) has a section (11) of circular shape at the level of the upper face of the support.
  • (1) upper section of the perforation
  • a section (12) of circular shape at the lower face (3) of the support (1) (lower section of the perforation) having a diameter d2 (lower diameter d2 of the perforation) and an axis (w ) passing through the center of said lower section of the perforation and perpendicular to said support (1)
  • the diameter d1 being of a value greater than or equal to 10 ⁇ at a value less than or equal to 500 ⁇ , preferably of a value of 150 ⁇
  • the diameter d2 being of a value greater
  • the invention relates to a microfluidic cell culture chip in which the diameter d2 of the circular section of said perforation at the lower face of the support is greater than the diameter d1 of the circular section of said perforation at the top of the support.
  • the invention relates to a microfluidic cell culture chip in which the diameter d2 of the circular section of said perforation at the lower face of the support is greater than the diameter d1 of the circular section of said perforation at the upper side of the support, such that the ratio (value of dl / value of d2) is from a value of 0.3 to a value less than 1.
  • the invention relates to a microfluidic cell culture chip in which the diameter d2 of the circular section of said perforation at the lower face of the support is greater than the diameter d1 of the circular section of said perforation at the upper side of the support, and said perforation of said support is truncated cone-shaped.
  • the invention relates to a microfluidic cell culture chip in which the value of the upper diameter dl of said at least one perforation (4) is smaller than the value of the lower diameter d2 of said at least one perforation (4), and said perforation (4) is truncated cone-shaped ( Figure 2).
  • the invention relates to a microfluidic cell culture chip in which the diameter d2 of the circular section of said perforation at the lower face of the support is equal to the diameter d1 of the circular section of said perforation at the top of the support.
  • the invention relates to a microfluidic cell culture chip in which the diameter d2 of the circular section of said perforation at the lower face of the support is equal to the diameter d1 of the circular section of said perforation at the upper face of the support, and said perforation of said support is cylindrical.
  • the perforation of said support is therefore of regular shape, that is to say that the diameters of all of its sections are of equal length, and that said sections are of circular shape.
  • the invention relates to a microfluidic cell culture chip in which the value of the upper diameter d1 of said at least one perforation (4) is equal to the value of the lower diameter d2 of said at least one perforation (4), and said perforation (4) is cylindrical ( Figure 1).
  • the invention relates to a microfluidic cell culture chip in which said protuberance (8) has a circular base (13) having a diameter d3 (diameter d3 of the circular base of the protuberance), said diameter d3 of the protrusion (8) being of a value of 10 ⁇ to a value of 500 ⁇ .
  • the invention relates to a microfluidic cell culture chip in which the value of the diameter d3 of said at least one protuberance (8) is equal to the value of the upper diameter d1 of said at least one perforation (4). ) ( Figures 1 and 2).
  • the invention relates to a microfluidic cell culture chip in which the value of the diameter d3 of said at least one protuberance (8) is equal to the value of the greater diameter d1 of said at least one perforation and is equal to the value of the lower diameter d2 of the at least one perforation (4) ( Figures 1 and 3).
  • the invention relates to a microfluidic cell culture chip in which the value of the diameter d3 of said at least one protuberance (8) is greater than the value of the upper diameter d1 of said at least one perforation (4). ) ( Figures 4, 5 and 6).
  • the invention relates to a microfluidic cell culture chip in which the value of the diameter d3 of said at least one protuberance (8) is smaller than the value of the greater diameter d1 of said at least one perforation (4). ) ( Figures 7, 8, 9, 10 and 11). According to a particular embodiment, the invention relates to a microfluidic cell culture chip in which said inner face (10) of said at least one protuberance (8) is wholly or partly facing said at least one perforation ( 4) of said support (1).
  • the nanostructured membrane in 3D comprising at least one protuberance and the support perforated by at least one perforation are positioned relative to one another so that the inner face of said protuberance is wholly or partly facing the circular section. said perforation at the upper face of the support.
  • the invention relates to a microfluidic cell culture chip in which said inner face (10) of said at least one protuberance (8) is entirely facing said at least one perforation (4) of said support (1):
  • the invention relates to a microfluidic cell culture chip in which said inner face (10) of said at least one protuberance (8) is partially opposite said at least one perforation (4) of said support (1):
  • the invention relates to a microfluidic cell culture chip in which the protuberance (4) has an oblique deformation, said oblique deformation being defined by the angle ⁇ formed between
  • the invention relates to a microfluidic cell culture chip in which the angle formed between the axis (y) and the axis (z) is between 0 and 45 °.
  • the invention relates to a microfluidic cell culture chip in which the angle formed between the axis (y) and the axis (z) is between 0 and 60 °.
  • the angle formed between the axis (y) and the axis (z) is greater than 45 °, the inclination of the protuberance partially blocks the recovery of secretions.
  • the theoretical maximum value that the angle ⁇ can take is 90 °, but in this embodiment, the protuberance is completely folded down along the upper face of said nanostructured membrane in 3D, and the recovery of secretions is blocked.
  • the invention relates to a microfluidic cell culture chip in which the outer face (9) of said protuberance (8) supports at least one cell adhered to said outer face, and in which the inner face ( 10) of said protuberance (8) supports at least one cell adhering to said inner face (10), the at least two cells belonging to two different types of cells.
  • adherent cells refers to any type of cells whose growth requires adhesion to a support and for which detachment from said support requires mechanical or enzymatic treatment (for example with trypsin).
  • differentiated cell types is used to refer to cell types of different nature, function, or tissue origin.
  • the invention relates to a microfluidic cell culture chip in which said cell adhering to said outer face is a stromal cell, and said cell adhering to said inner face is an epithelial cell.
  • the invention relates to a microfluidic cell culture chip in which said at least one cell adhering to said outer face (9) is a stromal cell, more particularly a fibroblast, and said at least one adherent cell.
  • said inner face (10) is an epithelial cell.
  • the invention relates to a microfluidic cell culture chip in which said cell adhering to said outer face is an epithelial cell, and said cell adhering to said inner face is a stromal cell.
  • the epithelial cells may be commercial non-tumorigenic prostate, bladder or kidney cell lines, or commercial primary cultures.
  • the stromal cells may be fibroblasts (commercial primary cultures or lineages), mesenchymal cells (commercial cultures or lineages), or other stromal cells such as endothelial cells.
  • the two types of cells grown on the inner and outer faces of said protuberance are called “neutral” or healthy, that is to say they are non-tumorigenic.
  • the invention relates to a microfluidic cell culture chip in which the outer face (9) of said protuberance (8) supports a first set of adherent cells at the confluence stage, and in which the face internal (10) of said protuberance (8) supports a second set of adherent cells at the confluence stage, the cells of the first and second set of adherent cells belonging to two distinct cell types ( Figure 13).
  • the invention relates to a microfluidic cell culture chip in which the outer face (9) of said protuberance (8) supports a first set of adherent cells advantageously at the confluence stage, said first set being a set of stromal cells, more particularly of fibroblasts, and in which the inner face (10) of said protuberance (8) supports a second set of adherent cells at the confluence stage, said second set being a set of epithelial cells (FIG. 52).
  • the invention relates to a micro fluidic cell culture chip in which the outer face (9) of said protuberance (8) supports a first set of adherent cells, said first set being a set of stromal cells, more particularly of fibroblasts, and wherein the inner face (10) of said protuberance (8) supports a second set of adherent cells at the confluence stage, said second set being a set of epithelial cells.
  • the invention relates to a microfluidic cell culture chip in which the outer face and the inner face of the protuberances are covered with an extracellular matrix preparation (ECM) (Matrigel®, collagen, fibronectin, hyaluronic acid), before the introduction of the cells on these surfaces, before cell culture.
  • ECM extracellular matrix preparation
  • said ECM preparation is composed of Matrigel® or a Matrigel® / collagen mixture.
  • the invention relates to a microfluidic cell culture chip in which said 3D nanostructured porous membrane has a thickness of 2 to 300 nm, more particularly 30 nm.
  • the invention relates to a microfluidic cell culture chip in which said 3D nanostructured porous membrane has a thickness of 2 to 300 nm.
  • the invention relates to a microfluidic cell culture chip in which said nanostructured porous membrane in 3D has a thickness of 30 nm.
  • the protrusion having a section at its base of circular shape the surface of this section can be calculated according to the radius.
  • the invention relates to a microfluidic cell culture chip in which said hollow dome-shaped protuberance has a height of 1 to 600 ⁇ , more particularly from 1 to 300 ⁇ , more particularly from 50 to 300 ⁇ , more particularly 50 ⁇ and a surface delimited by the base of the dome from 78.5 ⁇ 2 to 200,000 ⁇ 2 , more particularly from 2,000 to 70,000 ⁇ 2 , more particularly from 17,500 ⁇ 2 , and even more particularly from 7850 ⁇ 2 .
  • the term "height” refers to the distance between the center of the top of the protuberance and the center of the opening of diameter d3.
  • the invention relates to a microfluidic cell culture chip in which said support comprises at least two perforations of identical shape and dimensions, and said porous nanostructured membrane in 3D comprises at least two protuberances of shape and shape. identical dimensions, the number of perforations being equal to the number of protuberances.
  • said membrane comprises at least two protuberances, it is appropriate to define the distance between these at least two protuberances.
  • distance between two contiguous protuberances must be understood as the distance between two points situated respectively on the circumference formed by the intersection of the outer face of said protuberance and the upper face of said nanostructured membrane. in 3D of two contiguous protuberances, and forming the shortest distance among the set of possible distances between the pairs of points respectively located on said circumferences of two contiguous protuberances ( Figure 32).
  • said membrane When said membrane has at least two protuberances, they must be separated by a suitable distance.
  • the invention relates to a microfluidic cell culture chip in which the distance between two contiguous protuberances is of a value 1, said value 1 being from 10 to 100 ⁇ .
  • the invention relates to a microfluidic cell culture chip in which the distance between two contiguous protuberances is of a value 1, said value 1 being from 10 to 100 ⁇ , more particularly from 50 to 100 ⁇ .
  • the invention relates to a microfluidic cell culture chip in which the distance between two contiguous protuberances is of a value 1, said value 1 being 50 to 100 ⁇ .
  • the invention relates to a microfluidic cell culture chip in which said support comprises at least two perforations, and said nanostructured porous membrane comprises at least two protuberances such that the ratio varies from 1 to 1 dl . , 1 being the value of the distance between two protuberances n d2 (dl + l) ' 1
  • the diameter dl is greater than or equal to 10 ⁇ to a value less than or equal to 500 ⁇
  • the diameter d2 is greater than or equal to 10 ⁇ and less than or equal to 500 ⁇ .
  • the lower diameter d2 of said perforation at the lower face of the support is always greater than or equal to the upper diameter d1 of said perforation at the upper face of the support.
  • the invention relates to a microfluidic cell culture chip in which said 3D nanostructured porous membrane comprises 1 to 100 protuberances for a surface of the central unit of 1 cm 2 .
  • the invention relates to a microfluidic cell culture chip in which said nanostructured porous membrane in 3D comprises a maximum number of protuberances for a surface of the central unit of 1 cm 2 , equal to the formula ⁇ (d3 / 2) 2
  • the nanostructured porous membrane in 3D is composed of a number of successive layers of polyelectrolytes from 1 to 150, it being understood that the successivity of the layers applies from at least two layers.
  • the number of layers makes it possible to vary the mechanical strength of the protuberance. Indeed, the higher the number of layers, the greater the mechanical strength of the protrusion will be so that it can retain its shape while supporting the cell culture on these external and internal faces and resisting the flow (for example medium of culture) on both sides of it.
  • the invention relates to a microfluidic cell culture chip in which the number of successive layers of polyelectrolytes is 1 to 150, in particular 15.
  • polyelectrolyte to form the 3D nanostructured porous membrane provides a biocompatible and functional substitute for mimicking the basal lamina on which adherent cells attach to the tissues.
  • the nanostructured porous membrane in 3D may consist of a layer of a polyelectolyte.
  • the invention relates to a microfluidic cell culture chip in which the 3D nanostructured porous membrane consists of a layer of a polyelectolyte chosen from 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, polystyrene-al-maleic acid, polyvinyl sulphate, polyvinylsulfonic acid, poly (2-acrylamido-2-methyl-1-yl) acid; propanesulfonic acid), poly (2-acrylamido-2-methyl-1-propanesulfonic acid -co-acrylonitrile), poly-4-styrenesulfonic acid, poly (4-styrenes
  • the nanostructured porous membrane in 3D may consist of at least two successive layers of a polyelectolyte.
  • the at least one protuberance being an integral part of the 3D nanostructured porous membrane, the composition of said 3D nanostructured porous membrane will be identical to the composition of said at least one protuberance.
  • the invention relates to a microfluidic cell culture chip in which the 3D nanostructured porous membrane consists of successive layers of a polyelectrolyte chosen from poly (sodium 4-styrenesulfonate) (PSS), the poly (ethyleneimine), poly (diallyldimethylammonium chloride), poly (acrylamide-co-diallyldimethylammonium chloride), diallyldimethylammonium chloride, poly (allylamine hydrochloride) (PAH), polyanetholesulfonic acid, polyacrylic acid, polystyrene-al-maleic acid, polyvinyl sulphate, polyvinylsulfonic acid, poly (2-acrylamido-2-methyl-1-propanesulphonic acid), poly (2-acrylamido-2- acid) methyl-1-propanesulfonic acid-acrylonitrile), poly-4-styrenesulfonic acid, poly (4-styrenesulphonic acid-co
  • PSS poly
  • said layers may correspond to at least two polyelectrolytes of different nature: at least one negatively charged polyelectrolyte and at least one positively charged polyelectrolyte.
  • the layer-by-layer construction of this membrane must be carried out in such a way that a positively charged polyelectrolyte layer alternates with a negatively charged polyelectrolyte layer.
  • the membrane is thus highly cohesive due to the electrostatic interactions between the positively charged polyelectrolyte layers and the negatively charged polyelectrolyte layers. This makes it possible to obtain a membrane having a Young's modulus of the order of one kiloPascal.
  • the lower layer and the upper layer of the porous membrane may consist, independently of one another, of any one of the polyelectrolytes.
  • the charge of the polyelectrolyte constituting the last layer, and thus constituting the upper face of said membrane has an impact on the hydrophobicity of the membrane.
  • the last layer is constituted by a negatively charged polyelectrolyte
  • the upper face of the membrane is relatively hydrophilic in nature compared to a membrane whose upper face is positively charged.
  • the last layer is constituted by a positively charged polyelectrolyte
  • the upper face of the membrane is relatively hydrophobic in nature compared to a membrane whose upper face is negatively charged.
  • the roughness of the upper face of the membrane is governed by the groups borne by the polyelectrolyte constituting the last layer of the membrane, ie the layer constituting the upper face of said membrane.
  • this polyelectrolyte has an attached benzene ring and a bound sulfate group, which thus provide a rougher surface than a PAH layer, an electrolyte that does not have benzene nucleus attached.
  • the roughness of the nanostructured porous membrane in 3D is of the order of one nanometer.
  • the invention relates to a microfluidic cell culture chip in which the number of successive layers is from 1 to 150, in particular 15, of a positively charged polyelectrolyte and a negatively charged polyelectrolyte, said negatively charged polyelectrolyte layer and said positively charged polyelectrolyte layer being alternating with each other.
  • the invention relates to a microfluidic cell culture chip in which the 3D nanostructured porous membrane consists of successive layers of at least two polyelectrolytes, of which at least one polyelectrolyte is positively charged and at least one The polyelectrolyte is negatively charged, said positively charged polyelectrolyte being selected from: poly (sodium 4-styrenesulphonate), poly (ethyleneimine), poly (diallyldimethylammonium chloride), poly (acrylamide-co-diallyldimethylammonium chloride), chloride diallyldimethylammonium, and said negatively charged polyelectrolyte being selected from poly (allylamine hydrochloride), polyanetholesulfonic acid, polyacrylic acid, polystyrene-maleic acid, polyvinyl sulfate, polyvinylsulfonic acid, poly (2-acrylamido-2-methyl-1-propanesulfonic acid), poly (2-acrylamido
  • said negatively charged polyelectrolyte layer and said positively charged polyelectrolyte layer being alternating with each other.
  • the invention relates to a microfluidic cell culture chip in which the 3D nanostructured porous membrane consists of successive layers of a positively charged polyelectrolyte and a negatively charged polyelectrolyte, said positively charged polyelectrolyte being poly (sodium 4-styrenesulphonate) (PS S) and said negatively charged polyelectrolyte being poly (allylamine hydrochloride) (PAH), said negatively charged polyelectrolyte layer and said positively charged polyelectrolyte layer being alternately one by report to the other.
  • PS S sodium 4-styrenesulphonate
  • PAH allylamine hydrochloride
  • the invention relates to a microfluidic cell culture chip in which the number of successive layers of poly (sodium 4- styrenesulphonate) (PSS) and / or poly (allylamine hydrochloride) (PAH), is from 1 to 150, in particular from 15.
  • PSS poly(sodium 4- styrenesulphonate)
  • PAH poly (allylamine hydrochloride)
  • the invention relates to a microfluidic cell culture chip in which each polyelectrolyte layer has a thickness of 2 to 300 nm, more particularly about 2 nm.
  • the invention relates to a microfluidic cell culture chip in which the number of successive layers is 15, a positively charged polyelectrolyte and a negatively charged polyelectrolyte, said negatively charged polyelectrolyte layer and said positively charged polyelectrolyte layer being alternating with each other, each layer having a thickness of 2 nm.
  • the invention relates to a microfluidic cell culture chip in which the value of the thickness of all the successive layers of polyelectrolytes is comprised of the value of the thickness of a layer at a value less than half the diameter d3 of said protuberance.
  • the invention relates to a microfluidic cell culture chip according to RX in which the 3D nanostructured porous membrane (5) consists of at least one layer of a polyelectolyte chosen from poly (sodium 4 styrene sulphonate) (PSS), poly (ethyleneimine), poly (diallyldimethylammonium chloride), poly (acrylamide-co-diallyldimethylammonium chloride), diallyldimethylammonium chloride, poly (allylamine hydrochloride) (PAH), polyanetholesulfonic acid, polyacrylic acid, polystyrene-al-maleic acid, polyvinyl sulphate, polyvinylsulfonic acid, poly (2-acrylamido-2-methyl-1-propanesulphonic acid), poly (2-acrylamido-2-methyl-1-propanesulfonic acid-acrylonitrile), poly (4-styrenesulfonic acid), poly (4-styrenesulfonic acid),
  • said 3D nanostructured porous membrane (5) is comprised of at least two successive layers, a layer of a positively charged polyelectrolyte is alternately a layer of negatively charged polyelectrolyte, said 3D nanostructured porous membrane (5) being in particular constituted by 1 to 150 successive layers of polyelectrolyte,
  • said 3D nanostructured porous membrane (5) consisting of 15 successive layers of poly (sodium 4-styrenesulfonate) (PSS) and / or poly (allylamine hydrochloride) (PAH), alternately one of the other.
  • PSS poly (sodium 4-styrenesulfonate)
  • PAH poly (allylamine hydrochloride)
  • the nanostructured porous membrane in 3D is therefore composed of successive layers of polyelectrolytes and thus has as variable parameters:
  • the hydrophobicity of said membrane and said protuberance can also be modified.
  • the invention relates to a microfluidic cell culture chip in which said support has a thickness of 2 ⁇ to 1000 ⁇ , more particularly 20 ⁇ .
  • the invention relates to a microfluidic cell culture chip in which said perforation has a surface delimited by said non-resorbable membrane of 78.5 ⁇ 2 to 200,000 ⁇ 2 , more particularly from 2,000 to 70 000 ⁇ 2 , even more particularly 17 500 ⁇ 2 , and even more particularly 7850 ⁇ 2 .
  • the support comprises at least one perforation. When said support comprises at least two perforations, it is appropriate to define the distance between these at least two perforations.
  • the distance between two contiguous perforations shall be understood as the distance between two points situated respectively on the circumference of each of the upper circular sections of the said contiguous perforations and forming the shortest distance, among all the possible distances between the torques. points respectively the circumference of each of the upper circular sections of said contiguous perforations.
  • said support comprises at least two perforations, these must be separated by an appropriate distance.
  • the invention relates to a microfluidic cell culture chip in which said distance between two contiguous perforations is of a value L, said value L being from 10 to 100 ⁇ , more particularly from 50 to 100 ⁇ ( Figure 32).
  • the invention relates to a microfluidic cell culture chip in which said support comprises 1 to 100 perforations for a surface of the central unit of 1 cm 2 .
  • the invention relates to a microfluidic cell culture chip in which the support is composed of bioimprinted plastic, polycarbonate, tissue culture plastic, glass or SU-8 resin.
  • the SU-8 resin is a novolac epoxy-based negative photosensitive polymer resin having an average epoxide group functionality of about 8.
  • the SU-8 resin is well known to those skilled in the art and commonly used in the manufacture of microsystem.
  • the negative term means that the UV-exposed parts become cross-linked, while the rest of the film remains soluble and can be removed by washing.
  • Bio-printed plastics correspond to plastics known to those skilled in the art that are suitable and compatible with 3D printing equipment (extrusion, temperature constraints).
  • Tissue culture plastics correspond to plastics, constituting the petri dishes, which comprise a bottom treatment (treatment performed by electric discharge or plasma to make the surface hydrophilic, that is to say with a net negative charge) and allow the fixation and growth of eukaryotic adherent cells (unlike untreated plastic culture dishes for bacteriology).
  • the invention relates to a microfluidic cell culture chip in which the support is composed of bio-printed plastic, polycarbonate, tissue culture plastic, glass or SU-8 resin, and said support is transparent.
  • the invention relates to a microfluidic cell culture chip which contains a solid lower module (107) comprising
  • a base (109), said lower unit (108) being integrated in said base (109), and forming a whole with said base (109), and said upper face of said lower unit comprising said upper orifice (15) from at least one channel, and said lower orifice (16) opening, itself or by intermediate means (17), outside said base (109) ( Figures 41 and 45).
  • the upper orifice of the at least one channel is located within the lower unit, the at least one channel extends through the lower unit and then through the base of the lower module, said unit and said base forming a part in one piece, so that the lower orifice of the at least one channel opens out of said base.
  • the lower orifice of the at least one channel opens either itself outside said base, or opens into an intermediate means such as a reservoir, which opens out of said base.
  • the invention relates to a microfluidic cell culture chip in which said lower module (107) and said central module (104) are assembled such as the upper orifice (15) of the at least one channel (14). ), of said upper face of said lower unit (108), opens on at least one of said perforations (4) of said support (1) of said central unit (105), and the lower orifice (16) of the at least one a channel (14) emerges on the outside of the chip, said upper face of said lower unit (108) and said lower face of said support (3) of said central unit (105) having a shape and a surface identical to each other; .
  • outside the chip should be interpreted as “outside the pedestal”, and vice versa.
  • the upper orifice (15) of the at least one channel (14) of said upper face of said lower unit (108) opens on at least one of said perforations (4) said support (1).
  • This at least one channel makes it possible to recover the secretions of the cells cultured on the internal face of the at least one protuberance.
  • the invention relates to a microfluidic cell culture chip in which said lower module (107) and said central module (104) are assembled such as the upper orifice (15) of the at least one channel ( 14), of said upper face of said lower unit (108), opens on at least one of said perforations (4) of said support (1) of said central unit (105), and the lower opening (16) of the least one channel (14), opens on the outside of the chip, via an intermediate means consisting of a reservoir (17) capable of recovering liquids and allowing routing to the outside of the chip via an outlet channel (18). ) opening out from the base (109) of said lower module (107), said upper face of said lower unit (108) and said lower face of said support of said central unit (105) having a shape and a surface identical to each other .
  • the invention relates to a microfluidic cell culture chip, wherein said lower orifice (16) of the at least one channel (14) opens out of the chip on a device allowing the analysis of compounds in solution.
  • This analysis device can be constituted by any analytical device known to those skilled in the art such as a mass spectrometer, an NMR device, a chromatography device (in the liquid or gaseous phase), a device based on the immuno interactions logic (ELIS A, immunoprecipitation), a PCR or RT-PCR device.
  • a mass spectrometer an NMR device
  • a chromatography device in the liquid or gaseous phase
  • ELIS A immunoprecipitation
  • PCR or RT-PCR device a device based on the immuno interactions logic
  • the secretions of cells are composed of both proteins and peptides but also DNA and RNA.
  • the selected analysis device (s) must make it possible to analyze all of these molecules.
  • the invention relates to a microfluidic cell culture chip, wherein said lower orifice (16) of the at least one channel (14) opens out of the chip on a device allowing the analysis of compounds in solution, said device being a mass spectrometer.
  • the microfluidic chip according to the invention can be coupled directly to a chip integrating a mass spectrometer to perform a real-time and continuous analysis of secretions.
  • the invention relates to a microfluidic cell culture chip in which said lower unit (108) and said central unit (105) are assembled by fixing elements (204) respectively located on each of the bases of the lower module (109) and the central module (106), so as to sealingly assemble said upper face of said lower unit (108) and said lower face of said support (3) of said central unit (105).
  • the invention relates to a microfluidic cell culture chip, wherein the value of the diameter d4 of said upper orifice (15) of said channel (14) is greater than the value of the diameter d5 of said lower orifice (16). ) of said channel (14).
  • the invention relates to a microfluidic cell culture chip, wherein the value of the diameter d4 of said upper orifice (15) of said channel (14) is equal to the value of the diameter d5 of said lower orifice (16). ) of said channel (14), said at least one channel being of cylindrical shape.
  • the invention relates to a microfluidic cell culture chip, wherein said upper orifice (15) of said at least one channel (14) has a diameter d4 and an axis (t) passing through the center of said upper orifice (15) and perpendicular to said support (1), such that the value of the diameter d4 is greater than or equal to the value of the lower diameter d2 of said at least one perforation (4).
  • the invention relates to a microfluidic cell culture chip, wherein said upper orifice (15) of said at least one channel (14) opens onto a single perforation (4) of said support (1).
  • the invention relates to a microfluidic cell culture chip, wherein said at least one perforation (4) of said support (1) is entirely facing said upper orifice (15) of said at least one channel (14):
  • the invention relates to a microfluidic cell culture chip, wherein said at least one perforation (4) of said support (1) is partly opposite said upper orifice (15) of said at least one channel (14):
  • the invention relates to a micro fluidic cell culture chip, wherein said lower module (107) comprises at least two channels (14).
  • the invention relates to a microfluidic cell culture chip, in which each of the upper orifices (15) of the at least two channels (14) opens onto a perforation (4) and the at least two lower openings (16) at least two channels (14) open out of the chip respectively at at least two distinct locations ( Figure 32).
  • the invention relates to a microfluidic cell culture chip, in which each of the upper orifices (15) of the at least two channels (14) opens onto a perforation (4) and the at least two lower openings (16) at least two channels (14) open respectively to an intermediate means consisting of a reservoir (17) capable of recovering liquids and allowing routing to the outside of the chip via an outlet channel (18) opening to the outside of the base (109) of said lower module (107) ( Figure 31).
  • the invention relates to a microfluidic cell culture chip, in which each of the upper orifices (15) of the at least two channels (14) opens onto a perforation (4) and the at least two lower openings (16) at least two channels (14) open respectively to an intermediate means constituted by a reservoir (17) capable of recovering liquids and allowing a routing outside the chip via an outlet channel (18) opening outside the base (109) of said lower module (107), said output channels each of said reservoirs being connected together to open out of the chip at the same location ( Figure 34).
  • the invention relates to a microfluidic cell culture chip, in which the at least two channels (14) are connected together to form a network, such that each of the upper orifices (15) of the at least two two channels (14) open on a perforation (4) and the lower orifice (16) of each of the at least two channels (14) open themselves outside the chip at the same location or via an intermediate means constituted a reservoir (17) capable of recovering liquids and allowing a routing outside the chip via an outlet channel (18) opening out of the base (109) of said lower module (107) (FIGS. and 35).
  • the invention relates to a microfluidic cell culture chip, in which each of the upper orifices (15) of the at least two channels (14) opens onto a perforation (4) and a first set of at least two lower openings (16) of the at least two channels (14) open on a first intermediate means constituted by a reservoir (17), and at least one second set of at least two lower openings (16) of the at least two channels (14) open on at least a second intermediate means constituted by a reservoir (17), said first and at least second reservoirs (17) being able to recover liquids and each allowing a routing outside the chip via channels respective outlets (18) opening out of the base (109) of said lower module (107) at different locations (Figure 37).
  • the invention relates to a microfluidic cell culture chip, in which each of the upper orifices (15) of the at least two channels (14) opens onto a perforation (4) and the at least two lower openings (16) of all of the at least two channels (14) open on the same intermediate means constituted by a reservoir (17) adapted to recover liquids and allowing a routing outside the chip via an outlet channel (18) opening out of the base (109) of said lower module (107) ( Figure 36 ).
  • the invention relates to a microfluidic cell culture chip in which said lower module (107) has a height of 100 ⁇ to 2 cm.
  • the invention relates to a microfluidic cell culture chip in which said base (109) of the lower module (107) has a height of 100 ⁇ to 2 cm.
  • the invention relates to a microfluidic cell culture chip in which said lower unit (108) has a height of 100 ⁇ to 2 cm.
  • the invention relates to a microfluidic cell culture chip in which the lower module (107) is composed of printed organic plastic, polycarbonate, tissue culture plastic or SU8.
  • the invention relates to a microfluidic cell culture chip in which the lower module (107) is composed of bio-printed plastic, polycarbonate, tissue culture plastic, glass or SU-8 resin, and said lower module (107) is transparent.
  • the invention relates to a microfluidic cell culture chip in which the base of the lower module (109) is composed of bio printed plastic, polycarbonate, tissue culture plastic or SU8.
  • the invention relates to a microfluidic cell culture chip in which the base of the lower module (109) is composed of bio-printed plastic, polycarbonate, tissue culture plastic, glass or SU-resin. 8, and said base of the lower module (109) is transparent.
  • the invention relates to a microfluidic cell culture chip in which the lower unit (108) is composed of bioimpressed plastic, polycarbonate, tissue culture plastic or SU8.
  • the invention relates to a microfluidic cell culture chip in which the lower unit (108) is composed of bio-printed plastic, polycarbonate, tissue culture plastic, glass or SU-8 resin. , and said lower unit (108) is transparent.
  • the invention relates to a microfluidic cell culture chip which contains an upper module (101) comprising:
  • a solid and hollow upper unit (102), which consists of a solid surface defining an open volume (chamber) (203), and the edges of the solid surface being able to be in contact with said central unit (105) said central module (104) and delimiting the surface of the opening of said open volume;
  • said upper unit (102) being integrated in said base (103), and forming a whole with said base (103).
  • the chamber (203) is integrated in the base (103) of the upper module (101), and is thus surrounded on all its surfaces by said base (103), with the exception of the surface of the open volume of said chamber ( 203).
  • the invention relates to a microfluidic cell culture chip in which said upper unit (102) of said upper module (101) and said central unit (105) of said central module (104) are assembled so that said surface of the opening of said upper unit (102) is positioned above said upper face (6) of the 3D nanostructured porous membrane (5) of said central unit (105), said surface of the opening and said upper face (6) having a shape and a surface identical to each other.
  • the upper unit has an opening on the outside (window), at the level of the solid surface constituting said chamber for inserting a piece of transparent glass compatible with the observation under a microscope, preferably a microscope slide, to observe cell culture on the outer side of the nanostructured porous membrane in 3D and the outer face of the at least one protuberance.
  • the opening is preferably located at a location on the solid surface of said chamber, such that it allows vertical observation of the cell culture on the outer face of the protuberance.
  • Said transparent glass piece is positioned on said opening of the solid surface constituting said chamber, and sealed to said upper unit to maintain a closed space between the upper unit and the central unit.
  • said transparent glass piece is sealed to said upper unit with a waterproof glue.
  • the invention relates to a microfluidic cell culture chip in which said upper unit (102) of said upper module (101) and said central unit (105) of said central module (104) are assembled by elements of fixing (201,204) located on each of the bases (103,106) of the upper module (101) and the central module (104), so as to seal said surface of the opening of said upper unit (102) and said upper face.
  • the invention relates to a microfluidic cell culture chip in which said solid and hollow upper unit (102) in which said solid surface comprises a solid rectangular face and four solid faces adjacent to said solid rectangular face and adjacent between them, forming said chamber (203), the free edges of said four solid faces delimiting said surface of the opening of said chamber (206).
  • the invention relates to a microfluidic cell culture chip in which the upper module (101) is composed of bio printed plastic, polycarbonate, tissue culture plastic or SU8.
  • the invention relates to a microfluidic cell culture chip in which the upper module (101) is composed of bio-printed plastic, polycarbonate, tissue culture plastic, glass or SU-8 resin, and said upper module (101) is transparent.
  • the invention relates to a microfluidic cell culture chip in which the base of the upper module (103) is composed of printed organic plastic, polycarbonate, tissue culture plastic or SU8.
  • the invention relates to a microfluidic cell culture chip in which the base of the upper module (103) is composed of bio-printed plastic, polycarbonate, tissue culture plastic, glass or SU-resin. 8, and said base of the upper module (103) is transparent.
  • the invention relates to a microfluidic cell culture chip in which the upper unit (102) is composed of bioimpressed plastic, polycarbonate, tissue culture plastic or SU8.
  • the invention relates to a microfluidic cell culture chip in which the upper unit (102) is composed of bio-printed plastic, polycarbonate, tissue culture plastic, glass or SU-8 resin. , and said upper unit (102) is transparent.
  • the invention relates to a micro-fluidic cell culture in which said surface of the opening of said chamber (203) of said upper unit (102) is positioned above said upper face (6) of the 3D nanostructured porous membrane (5) of said central unit (105) to form a closed space delimited by said solid surface of said upper unit (102) and said upper face (6) of the 3D nanostructured porous membrane (5) .
  • the invention relates to a microfluidic cell culture chip in which said surface of the opening of said chamber (203) of said upper unit (102) is positioned above said upper face (6). ) of the 3D nanostructured porous membrane (5) of said central unit (105), and wherein said at least one solid face of said upper unit (102) has two orifices opening respectively to an inlet / outlet channel (202), to form a closed space delimited by said solid surface of said upper unit (102), and said upper face ( 6) of the 3D nanostructured porous membrane (5) of said central unit (105) able to communicate with the outside of said microfluidic chip only through said two orifices via said input / output channels (202) which open to the outside of the base (109) ( Figures 38 and 43).
  • the upper unit has two orifices opening respectively on an input / output channel.
  • the two input / output channels extend through the base of the upper module, said unit and said base forming an integral part, such that said two input / output channels open out of said upper base.
  • the assembly of the chamber on the nanostructured porous membrane in 3D makes it possible to define a closed space that can communicate with the outside of said micro fluidic chip only through said two orifices via said input / output channels. exit. These channels also allow the introduction of the culture medium into the chamber, and optionally the introduction of cells that are grown on the upper face of said membrane or the outer face of the at least one protuberance.
  • These input / output channels can be coupled to sensors allowing a continuous monitoring of the conditions and culture parameters (CO2, 02 sensors, pressure, temperature, glucose, pH). In an alternative embodiment, these sensors can be directly integrated into the chamber (203).
  • the invention relates to a microfluidic cell culture chip in which said upper module (101) has a height of 50 ⁇ to 2 cm, in particular 1 cm.
  • the invention relates to a micro fluidic cell culture chip wherein said base (103) of said upper module (101) has a height of 50 ⁇ to 2 cm, in particular 1 cm.
  • the invention relates to a micro fluidic cell culture chip wherein said upper unit (102) of said upper module (101) has a height of 50 ⁇ to 2 cm, in particular 1 cm.
  • the invention relates to a microfluidic cell culture chip, comprising an upper module (101), a central module (104) and a lower module (107), and in which the aforementioned modules are assembled such that said surface of the opening of said upper unit (102) of said upper module (101) is positioned above said upper face (2) of the 3D nanostructured porous membrane (5) of said central unit (105) of said module central (104), and the upper orifice (15) of the at least one channel (14), of said upper face of said lower unit (108) of said lower module (107), opens on at least one of said perforations ( 4) of said support (1) of said central unit (105),
  • said surface of the opening of said upper unit (102) and said upper face (2) of the 3D nanostructured porous membrane (5) of said central unit (105) having a shape and a surface identical with each other
  • said upper face said lower unit (108) and said lower face of said support (3) of said central unit (105) having a shape and a surface identical to each other
  • said upper module (101), said central module (104) and said module lower section (107) being connected by fasteners (201, 204) on each of the respective pedestals (103, 106, 109) ( Figures 42, 46 and 47).
  • the fixing elements located on the bases of each of the modules make it possible to lock the modules in pairs, to assemble the upper module with the central module and to assemble the central module with the lower module, or to lock two modules on the third module. , to assemble the central module and the lower module on the upper module.
  • fixing elements may be lugs or male-female elements, preferably male-female elements.
  • the base of the upper module comprises studs (201) as male fastening elements, and the bases of the central and lower modules comprise perforations (204) as female fastening elements, able to lock on said studs of the upper module.
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is entirely opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is wholly opposite said at least one perforation (4) of said support (1):
  • the value of the upper diameter d1 of said at least one perforation (4) is greater than the value of the diameter d3 of said at least one protuberance (8), and that the axis (y), passing through the center of said circular base (13) and perpendicular to said support (1), and the axis (x), passing through the center of the upper section (11) of said perforation (4) and perpendicular to said support (1), are distinct one with respect to the other by a distance of less than or equal to [(value of dl - value of d3) / 2].
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is wholly opposite said at least one perforation (4) of said support (1):
  • the value of the upper diameter d1 of said at least one perforation (4) is greater than the value of the diameter d3 of said at least one protuberance (8), and that the axis (y), passing through the center of said circular base (13) and perpendicular to said support (1), and the axis (x), passing through the center of the upper section (11) of said perforation (4) and perpendicular to said support (1), are distinct one with respect to the other by a distance of less than or equal to [(value of dl - value of d3) / 2]. and when
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is wholly opposite said at least one perforation (4) of said support (1):
  • the value of the upper diameter d1 of said at least one perforation (4) is greater than the value of the diameter d3 of said at least one protuberance (8), and that the axis (y), passing through the center of said circular base (13) and perpendicular to said support (1), and the axis (x), passing through the center of the upper section (11) of said perforation (4) and perpendicular to said support (1), are distinct one with respect to the other by a distance of less than or equal to [(value of dl - value of d3) / 2]. and when
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is wholly opposite said at least one perforation (4) of said support (1):
  • the value of the upper diameter d1 of said at least one perforation (4) is greater than the value of the diameter d3 of said at least one protuberance (8), and that the axis (y), passing through the center of said circular base (13) and perpendicular to said support (1), and the axis (x), passing through the center of the upper section (11) of said perforation (4) and perpendicular to said support (1), are distinct one with respect to the other by a distance of less than or equal to [(value of dl - value of d3) / 2]. and when
  • the axis (y) passing through the center of said circular base (13) of said protuberance (8) and perpendicular to said support (1), and the axis (t) passing through the center of said upper orifice (15); ) of said channel (14) and perpendicular to said support (1) are coincident or distinct from each other by a distance less than or equal to [value of d4 / 2].
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is partially opposite said at least one perforation (4) of said support (1):
  • the value of the upper diameter d1 of said at least one perforation (4) is smaller than the value of the diameter d3 of said at least one protuberance (8), and that the axis (y), passing through the center of said circular base (13) and perpendicular to said support (1), and the axis (x) passing through the center of the upper section (11) of said perforation (4) and perpendicular to said support (1) are merged or distinct from each other by a value less than or equal to [(value of dl 12) + (4/10 of value of d3)] .
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is partially opposite said at least one perforation (4) of said support (1):
  • the value of the upper diameter d1 of said at least one perforation (4) is smaller than the value of the diameter d3 of said at least one protuberance (8), and that the axis (y), passing through the center of said circular base (13) and perpendicular to said support (1), and the axis (x), passing through the center of the upper section (11) of said perforation (4) and perpendicular to said support (1), are merged, or distinct from each other by a value less than or equal to [[(value of d3 + value of d1) / 2] - 10 ⁇ ].
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is partially opposite said at least one perforation (4) of said support (1):
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is partially opposite said at least one perforation (4) of said support (1):
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is partially opposite said at least one perforation (4) of said support (1):
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is partially opposite said at least one perforation (4) of said support (1):
  • the invention relates to a microfluidic cell culture chip, wherein said inner face (10) of said protuberance (8) is partially opposite said upper orifice (15) of said channel (14): when said inner face (10) of said at least one protuberance (8) is partially opposite said at least one perforation (4) of said support (1):
  • the central unit contains:
  • a support consisting of a nonabsorbable membrane (1), comprising an upper face (2) and a lower face (3), perforated by at least one perforation (4)
  • a 3D nanostructured porous membrane (5) comprising an upper face (6) and a lower face (7), and comprising at least one protuberance (8), said at least one protuberance comprising an outer face (9) and a face internal portion (10) and forming a relief structure on the side of the upper face (6) of the 3D nanostructured membrane (5), said protuberance being in particular in the form of a hollow dome having a circular base (13), said upper face ( 6) of the nanostructured membrane in 3D being positioned integrally on said lower face (2) of said support and said at least one protuberance (8) being on the side of the upper face (2) of said support (1).
  • the invention relates to a microfluidic cell culture chip in which said upper unit (102) of said upper module (101) and said central unit (105) of said central module (104) are assembled so that said surface of the opening of said upper unit (102) is positioned above said upper face (2) of the support consisting of a non-resorbable membrane (1) of said central unit (105), to form a delimited space by said solid surface of said upper unit (102), said upper face (2) of the support consisting of a non-absorbable membrane (1) and the outer face (9) of the protuberances,
  • said upper unit (102) of said upper module (101) and said central unit (105) of said central module (104) being assembled by fastening elements (201,204) located on each of the bases (103,106) of the upper module (101) and of the central module (104), so as to sealingly assemble said surface of the opening of said upper unit (102) and said upper face (2) of the support consisting of a non-resorbable membrane (1) of said central unit (105) and advantageously said at least one solid face of said upper unit (102) has two orifices respectively opening on an input / output channel (202), allowing said closed space to communicate with the outside of said microfluidic chip via said input / output channels (202) which open out of the base (109).
  • the invention relates to a microfluidic cell culture chip in which said inner face (10) of said at least one protuberance (8) is wholly or partly facing said at least one perforation (4). ) of said carrier (1).
  • the nanostructured membrane in 3D comprising at least one protuberance and the support perforated by at least one perforation are positioned relative to one another so that the inner face of said protuberance is wholly or partly facing the circular section. said perforation at the upper face of the support.
  • the invention relates to a microfluidic chip of cell culture in which said inner face (10) of said at least one protuberance (8) is totally facing said at least one perforation (4) of said support (1):
  • the invention relates to a microfluidic cell culture chip, comprising an upper module (101), a central module (104) and a lower module (107), and in which the aforesaid modules are assembled such that said surface of the opening of said upper unit (102) of said upper module
  • the invention also relates to a method for manufacturing a microfluidic cell culture chip according to the invention, wherein the manufacture of said central unit (105) of said central module (104) comprises:
  • the first step of manufacturing the central module is a step of extruding a resorbable polymeric solution through one or more perforations of said support consisting of a non-resorbable material, that is to say a material that can not be eliminated neither by a physical process or a chemical process in an aqueous solvent.
  • the resorbable polymeric material used for the solution is initially in the form of a viscous liquid to allow extrusion through the perforations of the support.
  • viscous is used herein to refer to a fluid that has resistance to deformation under shear stress, which is at least the same as that of pure water and preferably substantially larger than that of pure water .
  • a viscous liquid used for extrusion circulates more slowly than pure water, property due to its higher viscosity than pure water.
  • the viscosity property of the liquid used for the extrusion is important, because such a viscous liquid allows a better preservation of the shape of the protrusion, during the extrusion process, than a solution that has the viscosity of pure water .
  • the preferred range of viscosity of the liquid for extrusion is from 1 (pure water) to 100 Pa.s, and preferably the viscosity of the liquid is from 0.1 Pa.s to 1 Pa.s.
  • the term "extrusion” is used herein to define a step in which viscous liquid material is forced through a die to give a predetermined shape by said die to said viscous material.
  • the matrix is constituted by the support comprising at least one perforation and said material in the form of viscous liquid is constituted by the resorbable polymer.
  • the invention relates to a method for manufacturing a microfluidic cell culture chip, in which the step of extruding said resorbable polymer solution (22) through the at least one perforation ( 4) of said support (1) makes it possible to form 3D nanostructures (23) on the side of said upper face (2) of said dome-shaped support (1).
  • the 3D nanostructure obtained after the extrusion through the perforations and the polymerization of said resorbable polymeric solution has the technical effect of serving as a mold for the formation of the protuberances of the nanostructured porous membrane in 3D.
  • Extrusion is stopped when the resorbable polymeric solution has formed a 3D nanostructure of desired length to serve as a mold for the desired protuberance.
  • the resorbable polymeric solution is then left unrestrained to allow its polymerization or gelation, and thus give solid 3D nanostructures which protrude from the side of the upper face of said support through the perforations.
  • the polymerization is carried out directly on the support consisting of a non-resorbable membrane and having at least one perforation.
  • the 3D nanostructures obtained make it possible to form a mold for obtaining the protuberances of the nanostructured porous membrane in 3D.
  • the surface formed by the upper face of the support and the 3D nanostructures formed by extrusion and protruding from the side of the upper face of said support through the perforations, is then covered by a porous membrane consisting of a polyelectrolyte film.
  • Polyelectrolytes have the property of being able to take the form of any type of 3D structure made up of any type of material (even resorbable), and to preserve the 3D shape thus given, during the removal of the 3D structure having served as mold .
  • This polyelectrolyte film is a multilayer film, made using the layer-by-layer technique.
  • the purpose of this technique is to adsorb successive layers of polyelectrolytes on the surface formed by the upper face of the support and 3D nanostructures protruding through the perforations of said support, to create a plurality of thin layers of polyelectrolytes.
  • the polyelectrolytes used are PAH and PSS.
  • the PAH and PSS solutions are prepared at a concentration of 1 mg / ml with 0.5 mol / l of NaCl.
  • the multilayer polyelectrolyte film is constructed by starting indifferently with one of the polyelectrolytes and terminating indifferently with one of the polyelectrolytes, but taking into account the alternation of the layers between oppositely charged polyelectrolytes.
  • This method makes it possible to obtain a final multilayer polyelectrolyte film that constitutes a continuous film over the entire surface formed by the upper face of the support and the 3D nanostructures protruding through the perforations of said support.
  • this final multilayer polyelectrolyte film has protrusions obtained by molding during the application of successive layers of polyelectrolytes on the 3D nanostructures resulting from the extrusion through the perforations of the support.
  • the mold formed by the 3D nanostructures of resorbable polymeric material is dissolved.
  • a support consisting of a non-resorbable material that is to say a material that can not be removed by a physical process or a chemical process in an aqueous solvent, makes it possible to retain said support intact at the end of this dissolution step.
  • the dissolution of the resorbable polymeric material provides a continuous multilayer polyelectrolyte film firmly bonded to the support, and which has protrusions retaining the shape of the 3D nanostructures on which they have been molded.
  • This continuous film thus constitutes the nanostructured porous membrane in 3D.
  • the final structure consisting of the porous nanostructured membrane in 3D fixed on the support, is easily manipulated and forms the central module of said micro fluidic cell culture chip.
  • This central module can be disinfected by washing three times consecutively with 70% ethanol and then washing three to four times consecutively with cell culture medium to remove any remaining trace of ethanol.
  • the invention relates to a method for manufacturing a microfluidic cell culture chip, in which the step of extruding said resorbable polymer solution (22) through the at least one perforation ( 4) of said support (1) is carried out using a pumping system.
  • This extrusion of the resorbable polymeric solution is carried out using a pumping system that can be controlled manually or mechanically (by a motor).
  • the pump system may be constituted for example by a syringe or a plunger or to induce a continuous flow of said resorbable polymer solution (22) through said at least one perforation (4).
  • said resorbable polymer solution (22) may be extruded through said at least one perforation (4) using two parallel flat plates that can be moved toward each other, either manually, either mechanically, using a motor for example.
  • One of the flat plates is positioned on the outer face of the upper module (204) and the other flat plate is positioned on the outer face of the lower module (101), the two flat plates being thus positioned parallel to one another. compared to each other.
  • the extrusion can be carried out in a liquid or in the air.
  • the invention relates to a method for manufacturing a microfluidic cell culture chip, in which the step of extruding said resorbable polymer solution (22) through the at least one perforation ( 4) of said support (1), is made in a liquid.
  • the liquid is chosen so as not to cause the resorption of the polymer used to form the protuberance.
  • the magnitude of the static pressure of fluid that acts against the formation in length of the 3D nanostructure is 9.8 milliPascals. Since the Young's modulus of the polyelectrolyte solution used as a resorbable polymer for extrusion is generally in the range of 100 to 400 MegaPascals, there is then a negligible reduction in the length of the 3D nanostructure formed during extrusion (e.g., in the worst case, for a Young's modulus of 100 megaPascals, reducing the length will be about 10 "5%).
  • the invention relates to a method for manufacturing a microfluidic cell culture chip, in which the step of extruding said resorbable polymer solution (22) through the at least one perforation ( 4) of said support (1) is made in the air.
  • the invention also relates to a method of alternative manufacture of a microfluidic cell culture chip according to the invention, wherein the manufacture of said central unit (105) of said central module (104) comprises:
  • the invention also relates to a method of alternative manufacture of a microfluidic cell culture chip according to the invention, wherein the manufacture of said central unit (105) of said central module (104) comprises:
  • the invention also relates to a method of alternative manufacture of a micro-fluidic cell culture chip according to the invention, in which the manufacture of the central module comprising the central unit, comprises:
  • step (i) of the method described previously corresponds to the assembly of the support part I with the mold H, in particular a mold H1 comprising 100 molded 3D nanostructures (208), in an alignment reproducible and specific which is guided by the flat section of the pieces and the alignment pin (217) of the support piece I which fits into the alignment hole (218) of the mold H1.
  • the solid face (215) of said support part I is placed on the upper face (209) of the mold H, so that the 3D molded nanostructures (208) of said mold H are positioned at the cutting (216) of said solid face of the support part I.
  • the chamber thus formed by the connection of the mold H with the support part I is then filled with the resorbable polymer (22) according to step (ii).
  • the mold H is then removed from the support part I according to step (iv).
  • the base of the support piece I is then formed at the level of the blank (216) by a resorbable polymer matrix (210) comprising at least one negative mold of at least one 3D nanostructure
  • the support piece I is then assembled with the perforated piece G, in particular a perforated piece G1 comprising 100 perforations (4), according to step (v), in a reproducible and specific alignment which is guided by the flat section of the pieces. and the alignment pin (217) of the support piece I which fits into the alignment hole (218) of the perforated piece G (Fig. 70).
  • the perforated piece G consists of a support (1) comprising at least one perforation (4), said support (1) being inserted into a base (106).
  • the choice of the perforated part (Gl, G2, gl, g2) depends on the choice of the mold (H1, H2, h1, h2) in step (i), the shape and the number of 3D nanostructures of the mold (H1, H2, h1, h2) to be identical to the shape and number of perforations of the perforated piece (Gl, G2, gl, g2) ( Figures 67, 68, 72, 73).
  • the upper face (2) of the support (1) of the perforated piece G is positioned in contact with the lower face (212) of said resorbable polymer matrix (210).
  • the perforations (4) of the perforated part G are then aligned with the negative molds of the 3D nanostructures (211) of said matrix (210) thanks to the alignment pin (217) of the support part I which engages in the alignment hole (218) of the perforated piece G.
  • the assembled pieces I and G1 are then turned over as shown in FIG. 70-E, so as to have the piece G1 above the support piece I.
  • the walls of the perforations of the part G 1 allow the formation of a chamber, so as to facilitate the formation of the protuberances (8) by the application of at least one continuous layer of at least one polyelectrolyte according to step (vi ), on the continuous surface formed by the lower face (3) of the support (1) and the lower face (212) of said resorbable polymer matrix (210) comprising at least one negative mold of said at least one 3D nanostructure (211). ), at the perforations (4) of said support (1).
  • step (vii) the resorbable polymer forming said matrix (210) of negative molds (211) is dissolved according to step (vii).
  • the support piece I is then removed from the perforated piece G1.
  • the perforated piece G1 then comprises, on the side of the lower face (3) of the support (1), a nanostructured porous membrane in 3D (5), and on the side of the upper face (2) of the support (1), said at least one protuberance in the extension of said at least one perforation of the support of the perforated piece Gl, ( Figure 66).
  • the upper face (6) of the 3D nanostructured porous membrane (5) is integral (in contact) with the lower face (3) of the support consisting of a non-resorbable membrane (1).
  • This perforated piece G1 thus obtained comprising a support (1) with perforations (4) and the nanostructured membrane in 3D (5) comprising protuberances (8), constitutes the central module of the microfluidic chip and is used for cell culture. on each side of said at least one protuberance.
  • said support piece (I), said mold (H1, H2) and said perforated piece (G1, G2) are circular in shape, in particular making it possible to adapt to cell culture dishes. a diameter of 35 mm.
  • FIG. 67 shows a particular embodiment of the method in which said mold (H1) and said perforated piece (G1) respectively comprise 100 molded 3D nanostructures and 100 perforations.
  • FIG. 68 shows a particular embodiment of the method in which said mold (H2) and said perforated piece (G2) respectively comprise 9 molded 3D nanostructures and 9 perforations.
  • Such a method makes it possible to obtain a central unit with 9 protuberances.
  • the choice of the mold and the perforated part depends on the number of protuberances desired for the nanostructured membrane in 3D of the central unit.
  • the central module constituted by the perforated piece G with the protuberances obtained at the end of the process detailed above can be placed on a cell culture chamber, such as a cell culture dish. diameter 35 mm containing culture medium, through a part F, as shown in Figure 71.
  • the central module obtained by the process described above is placed on a lower module as described in the present invention, comprising at least one channel for collecting the secretions of the at least one protuberance.
  • the lower module is of identical shape and dimensions to said central module.
  • the lower module is assembled to the central module in a reproducible and specific alignment which is guided by the flat section of the workpieces and the alignment pin of the workpiece of the lower module which fits into the alignment hole of the central module.
  • the lower module comprises a number of channels identical to the number of perforations and therefore protuberances of the central module, so that the assembly of said central module with said lower module allows the alignment of the channels with the perforations and therefore the protuberances, to collect cell secretions via a microfluidic system.
  • the lower module is replaced by the part F.
  • This part F used as support of the central module on the culture box as shown in FIGS. 67, 68 and 71 has square openings allowing the circulation of the culture medium to provide nutrients to growing cells on the inner face of said at least one protuberance.
  • said support piece (i), said mold (h1, h2) and said perforated piece (g1, g2) are square in shape.
  • FIG. 72 shows a particular embodiment of the method in which said mold (h1) and said perforated piece (gl) respectively comprise 100 molded 3D nanostructures and 100 perforations.
  • Such a method makes it possible to obtain a central unit with 100 protuberances.
  • FIG. 73 shows a particular embodiment of the method in which said mold (h2) and said perforated piece (g2) respectively comprise 9 molded 3D nanostructures and 9 perforations.
  • Such a method makes it possible to obtain a central unit with 9 protuberances.
  • the choice of the mold and the perforated part depends on the number of protuberances desired for the nanostructured membrane in 3D of the central unit.
  • the resorbable polymeric solution is preferably carried out with chitosan, agarose or alginate.
  • the invention relates to a process for manufacturing a microfluidic cell culture chip, wherein said resorbable polymer solution (22) is chitosan.
  • the resorbable mold can be prepared by dissolving 2% chitosan in 2% acetic acid overnight, then diluting to 1.5% chitosan with ethanol. The chitosan solution is then polymerized in a hot bath at 5M NaOH: ethanol at a 1: 1 ratio.
  • the invention relates to a process for manufacturing a microfluidic cell culture chip, in which the step of polymerizing said resorbable polymer solution (22), said resorbable polymer solution (22) being chitosan, is performed by incubation with a 2% solution of acetic acid.
  • the dissolution is carried out by incubation overnight with a 2% solution of acetic acid, according to a protocol well known to those skilled in the art.
  • the invention relates to a method for manufacturing a microfluidic cell culture chip, wherein said resorbable polymer solution (22) is agarose.
  • the resorbable mold can be prepared by heating and dissolving 40 ⁇ g / ml of agarose in PBS (phosphate buffered saline). The agarose is polymerized by placing the resulting solution at a temperature below its gel point.
  • PBS phosphate buffered saline
  • the invention relates to a process for manufacturing a microfluidic cell culture chip, in which the step of polymerizing said resorbable polymer solution (22), said resorbable polymer solution (22) being agarose, is performed by incubation at a temperature above the gelling temperature of the agarose.
  • the dissolution is carried out by slow heating from room temperature to a temperature of 70 ° C for 120 minutes and then allowing the temperature of the agarose to return to room temperature on one night.
  • This heating can be carried out in a water bath. It is important that the temperature increases slowly to minimize thermal convection currents that could damage the 3D nanostructured porous membrane.
  • Variants of this heating protocol include the addition of DMSO in water bath water to modify the gelation properties of the agarose.
  • the invention relates to a method for manufacturing a microfluidic cell culture chip, wherein said resorbable polymer solution (22) is alginate.
  • the invention relates to a process for manufacturing a microfluidic cell culture chip, in which the step of polymerizing said resorbable polymer solution, said resorbable polymer solution (22) being alginate, is performed by overnight incubation with a Ca 2+ free solution and supplemented with a Ca 2+ ion chelating agent, such as EDTA or EGTA.
  • a Ca 2+ ion chelating agent such as EDTA or EGTA.
  • the multilayer film of polyelectrolytes has as variable parameters:
  • the film is composed of 15 layers of 2 nm thick, polyelectrolytes.
  • the hydrophobicity of the final multilayer film can also be modified.
  • the extrusion of the 3D nanostructure may be subject to the following defects due to the pumping system used for extrusion:
  • a protuberance thus formed from 3D nanostructures with a translation defect or an extrusion defect may continue to perform its technical function initially provided within the device, however, as the protuberance thus formed has a less optimal shape, the performance of it within the device is also less optimal. However, the device can continue to perform its intended function, but with reduced performance.
  • the protuberance can have different changes such as:
  • the culture medium used for all the experiments is a serum-free Keratinocyte medium (KSFM, Keratinocyte Serum Free Medium) (Life Technologies, Carlsbad, CA, Ref 17005-075) supplemented with 5 ng / ml of epidermal growth factor. (EGF) and 50 ⁇ g / mL of bovine pituitary extract.
  • KSFM Keratinocyte Serum Free Medium
  • EGF epidermal growth factor
  • the epithelial cell lines of prostate and stromal cells are maintained in this medium and are cultured in an atmosphere at 37 ° C and 5% C0 2 .
  • the subculturing of the cells in a fresh medium is carried out every three days for the epithelial cells and every two days for the stromal cells.
  • the cells are washed with a solution of Dulbecco's calcium phosphate phosphate buffer (D-PBS) without calcium and without magnesium (Life Technologies, Ref.11490), and then incubated with 1 ml of 0.25 mg Trypsin-EDTA. mL, at 37 ° C, (Lonza, Basel, CH, Ref CC-5012) for about 7 minutes.
  • D-PBS Dulbecco's calcium phosphate phosphate buffer
  • Trypsin-EDTA 0.25 mg Trypsin-EDTA.
  • mL at 37 ° C, (Lonza, Basel, CH, Ref CC-5012) for about 7 minutes.
  • the cell culture medium was supplemented daily with fresh culture medium.
  • a chemical dissociation of the cells is carried out by incubation for 5 minutes at 37 ° C. with 1 ml of 0.25 mg / ml trypsin-EDTA (Life Technologies, Ref 25300-054) in PBS medium without calcium and without magnesium.
  • a microfluidic chip according to the invention is sterilized by circulating a 70% ethanol solution (volume / volume) through the channels, then drying the entire microfluidic system in an oven at a temperature between 35 ° C. C and 45 ° C for at least 30 minutes, then exposing it to UV radiation and ozone for 40 min. 3.
  • a 70% ethanol solution volume / volume
  • the nanostructured 3D porous membrane consists of successive layers of polyelectrolytes alternating a positively charged polyelectrolyte layer and a negatively charged polyelectrolyte layer. According to the manufacturing method, this same membrane constitutes the protuberances.
  • ECM extracellular matrix preparation
  • the Matrigel® matrix used here is a commercial product manufactured by Corning®.
  • EHS Engelbreth-Holm-Swarm
  • This material is composed of approximately 60% laminin, 30% collagen IV, and 8% entactin.
  • Entactin is a bridging molecule that interacts with laminin and collagen IV, and contributes to the structural organization of these extracellular matrix molecules.
  • Corning® Matrigel® matrix also contains heparan sulfate (perlecan) proteoglycans, transforming growth factor ⁇ (TGF- ⁇ ), epidermal growth factor, insulin-like growth factor, fibroblast growth factor, a tissue plasminogen activator and other growth factors that are naturally present in the EHS tumor. It also contains residual matrix metalloproteinases derived from tumor cells.
  • Matrigel® can be used alone to functionalize the porous membrane, at a concentration of 6 mg / ml, or in admixture with type I collagen at a concentration of between 0.75 and 2.5 mg / ml. 4. Introduction of both cell types and cellular co-culture in the central unit
  • the epithelial cells are introduced to form a contiguous layer of cells, that is to say a cell culture at the confluence stage.
  • the epithelial cells are introduced on the inner faces of the protuberances of the central unit.
  • the inner surface of the protuberances of the central unit is thus covered with a dense monolayer of epithelial cells, which serves as a physio logically support for the growth and differentiation of human cells, isolated from the patient's urine.
  • the introduction of the cells on the inner face of the protuberances can be done according to 3 modes:
  • the cells are introduced via the channels of the lower module.
  • This mode of introduction of the cells after a pre-assembly of the three modules is preferred to the other two modes, because it prevents any bacterial contamination because the previously sterilized system is kept closed.
  • the epithelial cells are introduced at a concentration of 3 ⁇ 10 6 cells / ml in the central unit, either directly via the perforations (1st and 2nd mode) with a syringe or via the channels of the lower unit. (3rd mode) using an automated fluidic system and controlled in pressure and flow (Fluigent) or a syringe pump.
  • a syringe pump with adjustable flow rate is preferred to provide a gentle and controlled introduction of the cells.
  • Stable and continuous flow is delivered by the use of pressure pumps (Fluigent, France). Pressurized containers containing culture medium are maintained in a controlled temperature chamber and C0 2 . The flow rate is adjusted to about 5-10 mL / h (10 mbar) and cell adhesion and proliferation is observed over time. All samples are kept in a humidified incubator at 37 ° C and 5% C0 2 .
  • the central unit comprises protuberances of a height of 350 ⁇ with a circular base of 150 ⁇ in diameter.
  • the area of the inner surface of the protuberance is then 329 700 ⁇ 2 , on which about 50 epithelial cells are counted at the confluence stage (layer of contiguous cells), or about one cell for 66 ⁇ 2 .
  • the stromal cells are dispensed on the porous membrane at the external and internal faces of the protuberances.
  • the introduction of the cells on the inner face of the protuberances can be done according to two modes:
  • the stromal cells are introduced via the inlet / outlet channels of the upper module at a concentration of 3 ⁇ 10 6 cells / ml in the central unit, either directly using a syringe (1st mode), either via the channels of the upper module (2nd mode) using an automated fluidic system and controlled in pressure and flow (Fluigent) or a syringe pump.
  • the stromal cells form a layer of confluent (or contiguous) cells, their simple adhesion on the outer face in this example is sufficient.
  • the ratio between epithelial cells and stromal cells is 1: 2.
  • the porous membrane at the outer and inner faces of the protuberances is functionalized with 90 ⁇ l of a solution of Matrigel® diluted to 6 mg / ml, then seeded to finally obtain 7000 epithelial cells / cm 2 and 14 000 stromal cells (fibroblasts) / cm 2 .
  • the culture medium introduced via the inlet / outlet channels of the upper module and via the channels of the lower module to feed the cell cultures, is identical on both sides of the protuberances and is constituted by KSFM culture medium supplemented with 5 ng / mL of epidermal growth factor (EGF) and 50 ⁇ g / mL of bovine pituitary extract.
  • EGF epidermal growth factor
  • epithelial cells may be non-tumorigenic commercial cell lines (prostate or bladder or kidney) or commercial primary cultures.
  • These stromal cells can be: either fibroblasts (commercial primary cultures or lineages),
  • mesenchymal cells commercial cultures or lines
  • the two types of cell used to form these cell monolayers are called “neutral” or “healthy”, they are non-tumorigenic and play only a role of basal layer.
  • These "neutral” cells form at the confluence stage a very contiguous layer of cells on the inner and outer surface of the protuberances, establishing tight junctions that can be characterized by immunofluorescence and imaging (see labeling of E-cadherin part 5).
  • 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.
  • the co-culture can be established interchangeably, that is to say that 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.
  • the polyelectrolyte layer between the two cell types makes it possible to form a porous barrier by virtue of its mesh of positively and negatively charged polyelectrolytes.
  • immunolabeling is performed.
  • This immunolabeling is therefore performed on dead cells (fixed by PFA) and this visualization has the sole purpose of controlling that the co-culture is well in place and the methodology of introduction of the cells is correct.
  • the cells are visualized in the central module by immunostaining.
  • Phalloidin is used to identify cortical actin filaments, which follow the contours of the plasma membrane and therefore provide a means to delineate the extent of the cell and its membrane.
  • E-cadherin is used to detect cell-cell junctions.
  • Immunostaining is accomplished by introducing ⁇ -cadherin with a syringe pump via the channels at room temperature.
  • the cells are then washed with solution A and permeabilized for 3 minutes with a solution A supplemented with 0.1% Triton TX-100. Washing with a TBS solution is performed for 10 minutes, followed by a second wash with PBS solution for 30 minutes. The auto-fluorescence of the PFA is inactivated by the NH 4 C1 contained in the TBS solution. Non-specific sites are blocked by incubation with a solution of PBS 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 abl416) diluted 1:50 in a solution of PBS at 0.1% Tween-20 and 1% BSA.
  • the cultures are then washed for 30 minutes with a solution of PBS, and then incubated with a cytochrome Cy3-coupled anti-mouse secondary antibody (Jackson, Ref 115-162-062) diluted 1/1000 and Phalloidin FITC ( Sigma, P5282) diluted 1: 1000 in 0.1% Tween-20 PBS and 1% BSA for 20 minutes.
  • a cytochrome Cy3-coupled anti-mouse secondary antibody Jackson, Ref 115-162-062
  • Phalloidin FITC Sigma, P5282
  • the nuclei are counter-stained with Hoechst Stain (Life Technologies, Ref H-1399), diluted 1: 7000, for 5 minutes. The cells are then washed for 10 minutes and the Dako fluorescent medium is manually introduced.
  • Focal points of adhesion were detected by labeling using Vinculin.
  • the cells are pre- permeabilized for 40 seconds with Triton X-100 and fixed with 4% PBS (v / v) PBS for 20 minutes, then washed once with PBS solution.
  • the cells are incubated with a solution of 0.1% BSA and 10% goat serum for one hour.
  • the cells are then incubated for one hour with a primary antibody against Vinculin (Sigma, Ref V9131) diluted 1: 700 in a solution of PBS 0.05% Tween 20 and 5% goat serum, and washed. 4 consecutive times for 45 minutes with a PBS solution.
  • a primary antibody against Vinculin Sigma, Ref V9131
  • the cells are then incubated with cytochrome Cy5-coupled anti-mouse antibody diluted 1/500 in 0.05% PBS Tween 20 and 5% goat serum (Jackson).
  • the central module is then washed 4 times for 15 minutes with a solution of PBS.
  • the nuclei and actin are stained as described above.
  • the co-culture is observed by fluorescence microscopy or can be observed by other modes of microscopy such as phase contrast microscopy, lensless imaging, confocal microscopy, light microscopy.
  • cell images are recorded using a sensor without a lens. SEM analyzes are also performed.
  • the fluorescence images of the central module containing the co-culture of cells are obtained using a Zeiss Axiolmager ZI microscope with a 20x objective equipped with the right Apotome module for the deep acquisitions of z-stack field, with the image taken every 3 mm in the z-axis, for a 150 mm diameter tube.
  • the images are recorded using an AxioCam MRm digital monochrome camera mounted on the microscope. 6.
  • the cell cultures in the central unit can be monitored in real time by a phase-contrast microscopic observation which makes it possible to visualize the unmarked and living cells, due to the transparency of the materials constituting the modules.
  • 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.
  • the microfluidic chip thus provided with cells, can be used for the diagnosis of a patient.
  • cells are isolated from a patient urine sample of at least 50 ml, in particular from 50 to 100 ml.
  • the isolation is carried out by centrifugation of the urine sample at a low speed, in particular 800 g for 5 min, allowing sedimentation of the cells contained in the urine sample. This centrifugation step is well known to those skilled in the art.
  • the pellet of sedimented cells is then resuspended in culture medium and the cell suspension is directly introduced into the microfluidic chip according to the invention, which means that the cells do not require pre-culture before their introduction into the chip. .
  • the concentration of cells obtained from the urine sample is or ranges from a few hundred cells to several thousand.
  • the cells isolated from the patient's urine can be introduced on the side of the protuberance which supports the culture of the epithelial cells, or on the side of the protuberance side that supports the culture of the stromal cells. In other words, these cultures being interchangeable on both sides of the protuberance, the cells isolated from the patient's urine can be introduced both on the inner face and on the outer face of the protuberances.
  • the cells isolated from the patient's urine are introduced on the side of the protuberance side that supports the culture of the epithelial cells.
  • they are introduced either via the channels of the lower unit, when the monolayer of epithelial cells is formed on the side of the inner face of the protuberances, or via the inlet / outlet channels of the upper module when the monolayer of epithelial cells is formed on the side of the outer face of the protuberances.
  • Cells isolated from the patient's urine are exfoliated uroepithelial (or urothelial) cells, including both bladder, prostate and kidney epithelial cells.
  • the inner surface of the protuberances is covered with a pre-formed layer of epithelial cells previously cultured, the outer face of the protuberances is covered with a pre-formed layer of fibroblasts (stromal cells), and the Isolated cells are delivered via the channels of the lower module.
  • stromal cells fibroblasts
  • the proliferation of the isolated cells is then followed, in order to observe the progression of the proliferation of isolated cells in the device and to examine whether this proliferation results in the replacement of healthy basal cells and affects the overall secretory profile of the tissue.
  • the epithelial cells of patients are stimulated by addition of 0.1 ng / mL of DHT (Dihydrotestosterone) on the external or internal surface of the protuberance This stimulation of the cells by DHT lasts between 24h and 48h.
  • DHT Dihydrotestosterone
  • this stimulation can be performed indifferently on one side or the other of the protuberances.
  • Epithelial cells can also be stimulated by the addition of mibolerone (non-metabolized hormone).
  • mibolerone non-metabolized hormone
  • the stimulation of the epithelial cells is thus carried out after the adhesion of the two cell types on either side of the protuberances, and after their growth to the confluence stage.
  • the secretions can be recovered when the isolated cells of the patient adhere and insert into this pre-formed layer of healthy epithelial cells on the side of the inner surface of the protuberances, and which is supported by a layer of healthy fibroblasts on the side of the face. external protuberances.
  • the adhesion of the isolated cells of the patient lasts about 3 hours and their integration lasts about 6 hours.
  • the final recovery of secretions for the secretome analysis is done after allowing at least 12 hours.
  • the secretions are recovered after 24 to 48 hours of stimulation with DHT.
  • the secretome is analyzed by mass spectrometry.
  • the secretions can be analyzed online by sensors incorporated in said chip.
  • the search for specific markers by immunological methods can also be carried out in the recovered secretions.
  • PSA Prostate Specific Antigen
  • a prostate cancer reference biomarker For example, the detection of PSA (Prostate Specific Antigen), a prostate cancer reference biomarker, can be performed.
  • the volume of secretions recovered after 24 hours is about 2 nL.
  • the reaction is stopped by the addition of 100 ⁇ l of sulfuric acid and the absorbance is detected using an ELISA plate reader at 450 nm.
  • the microfluidic cell culture chip according to the invention is used for the screening of molecules.
  • the microfluidic cell culture chip according to the invention is used to determine the effect of a treatment for urological cancer on a patient suffering from urological cancer.
  • the secretome analysis of the cells isolated from the patient's urine, inserted into the epithelial cell culture on the protuberance, is performed before and after the patient's treatment, and / or during the treatment.
  • 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 affected.
  • Figure 1 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 the diameter d3 of the circular base of said protuberance, and wherein the protuberance is entirely opposite the perforation.
  • Figure 2 Schematic cross-sectional view of a central unit having a protrusion in the nanostructured membrane in 3D, 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. 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 entirely opposite the perforation.
  • Figure 3 Schematic cross-sectional view of a central unit having a protrusion in the nanostructured membrane in 3D, 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 the diameter d2 of the lower section of said perforation, and the value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is partly opposite the perforation.
  • Figure 4 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 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 partly opposite the perforation.
  • Figure 5 Schematic cross-sectional view of a central unit having a protrusion in the nanostructured membrane in 3D, 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. 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 partly opposite the perforation.
  • Figure 6 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 diameter d2 of the lower section of said perforation, and is less than value of the diameter d3 of the circular base of said protuberance, and wherein the protuberance is partly opposite the perforation.
  • Figure 7 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 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 entirely opposite the perforation.
  • Figure 8 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 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 entirely opposite the perforation.
  • Figure 9 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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. 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 entirely opposite the perforation.
  • Figure 10 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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. 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 partly opposite the perforation.
  • Figure 11 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 entirely opposite the perforation.
  • Figure 12 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, and a perforation in the support, and wherein the protuberance is inclined along an axis (z) with respect to the vertical axis (y).
  • Figure 13 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, and a perforation in the support, and wherein the inner face of the protuberance is covered with a set of a first type at the confluence stage, and the outer surface of the protuberance is covered with a set of a second cell type at the confluence stage.
  • FIG. 14 is a schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, and a perforation in the support, and in which 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 channel, 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 protrusion is in full facing the channel.
  • Figure 15 is a schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 channel, 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 protrusion is in full facing the channel.
  • Figure 16 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 channel whose diameter value 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 in which the protuberance is entirely facing the channel.
  • Figure 17 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 channel, 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 protrusion is in full facing the channel.
  • Figure 18 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 channel, 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 protrusion is in full facing the channel.
  • Figure 19 Schematic cross-sectional view of a central unit having a protrusion in the nanostructured membrane in 3D, 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 channel, 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 in which the protuberance is partly opposite the channel.
  • Figure 20 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 channel, 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 partly opposite the channel.
  • Figure 21 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 smaller 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 channel, 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 partly opposite the channel.
  • Figure 22 is a schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 smaller 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 channel, 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 partly opposite the channel.
  • Figure 23 is a schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 smaller 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 channel whose diameter value 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 in which the protuberance is partly opposite the channel.
  • Figure 24 Schematic cross-sectional view of a central unit having a protrusion in the nanostructured membrane in 3D, 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 channel, 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 partly opposite the channel.
  • FIG. 25 Diagrammatic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, and a perforation in the support, and in which 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 channel, 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 partly opposite the channel.
  • Figure 26 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 channel, 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 in which the protuberance is partly opposite the channel.
  • Figure 27 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 d3 of the diameter of the circular base of said protuberance, said central unit being positioned on a lower unit comprising a channel, 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 partly opposite the channel.
  • Figure 28 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 channel, 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 partly opposite the channel.
  • Figure 29 Schematic cross-sectional view of a central unit having a protrusion in the nanostructured membrane in 3D, 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 channel, 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 partly opposite the channel.
  • Figure 30 Schematic cross-sectional view of a central unit having a protuberance in the nanostructured membrane in 3D, 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 channel whose diameter value 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 in which the protuberance is partly opposite the channel.
  • Figure 31 Schematic cross-sectional view of a central unit having two protuberances in the nanostructured membrane in 3D, and two perforations in the support, and wherein the value of the diameter dl 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 channels, whose diameter value d4 the upper orifice of each of the channels 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 openings of the two channels opening respectively on a tank, opening on the outside of the lower module via an outlet channel (protuberance entirely in look at the canal).
  • FIG. 32 is a schematic cross-sectional view of a central unit comprising two protuberances in the nanostructured membrane in 3D, and two perforations in the support, and in which the value of the diameter d1 of the upper section of each of the perforations is equal to 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 channels, whose diameter value d4 the upper orifice of each of the channels 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 openings of the two channels opening at the outside of the lower module at two distinct locations (protuberance in full facing the channel).
  • Figure 33 Schematic cross-sectional view of a central unit having two protuberances in the nanostructured membrane in 3D, 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 channels, whose value of the diameter d4 of the upper orifice of each of the channels 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 channels are connected together so that the two lower openings of the two channels open out of the lower module at the same location (protuberance in full opposite the channel).
  • Figure 34 Schematic cross-sectional view of a central unit having two protuberances in the nanostructured membrane in 3D, and two perforations in the support, and wherein the value of the diameter dl 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 channels, whose diameter value d4 the upper orifice of each of the channels 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 openings of the two channels opening respectively on a tank, each of the tanks opening out of the lower module at the same location, via output channels connected to each other (protuberance in full facing the channel).
  • Figure 35 Schematic cross-sectional view of a central unit having two protuberances in the nanostructured membrane in 3D, 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 channels, whose diameter value d4 of the upper orifice of each of the channels 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 channels are connected to each other by so that the two lower openings of the two channels open at the same location on a reservoir, which opens out outside the lower module via an output channel (protuberance in full facing the channel).
  • Figure 36 Diagrammatic cross-sectional view of a central unit having two protuberances in the nanostructured membrane in 3D, and two perforations in the support, and in which 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 channels, whose diameter value d4 the upper orifice of each of the channels 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 openings of the two channels open respectively at two distinct locations on the same tank, which opens out of the lower module via a output channel (protuberance in full facing the channel).
  • Figure 37 Schematic cross-sectional view of a central unit having four protuberances in the nanostructured membrane in 3D, 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 channels, whose diameter value d4 the upper orifice of each of the channels is equal to the value of the diameter d2 of the lower section of each of the perforations, and the two lower openings of a first set of two channels open respectively to two different locations on a first reservoir, and the two lower ports of a second set of two channels open respectively to ux distinct locations on a second reservoir, the first and the second reservoir opening respectively outside the lower module in separate locations (protuberance in all facing the channel).
  • Figure 38 Schematic perspective view of the upper module.
  • Figure 39 Schematic perspective view of the central module.
  • Figure 40 Schematic perspective view taken above the upper face of the porous membrane nanostructured in 3D, a central unit having a set of protuberances.
  • Figure 41 Schematic perspective view of the lower module.
  • Figure 42 Schematic perspective view of the micro fluidic chip comprising the assembly of the upper module, the central module and the lower module.
  • Figure 44 Photo of the central module. Top view of the upper face of the 3D nanostructured porous membrane comprising a set of protuberances.
  • Figure 45 Photo of the lower module. Top view of the upper face of the lower unit comprising all the upper openings of the channels.
  • Figure 46 Photo of the upper module and the lower module.
  • Figure 47 Photo of the upper module, the central module and the lower module disassembled.
  • Figure 48 Diagrammatic cross-sectional view of the support consisting of a non-absorbable membrane, the central unit, having a perforation.
  • Figure 49 Schematic cross-sectional view of the support consisting of a non-absorbable membrane, 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.
  • Figure 50 Schematic cross-sectional view of the support consisting of a non-absorbable membrane, 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 protuberance molded on said nanostructure in 3D.
  • Figure 51 Diagrammatic cross-sectional view of the support consisting of a non-absorbable membrane and having a perforation, the central unit, on which is positioned integrally the nanostructured membrane in 3D having a hollow protuberance.
  • Figure 52 Confocal microscopic image of the internal surface of a protuberance supporting a culture of epithelial cells at the confluence stage.
  • Figure 53 Schematic cross-sectional view of a mold comprising at least one 3D nanostructure molded on the side of its upper face
  • Figure 54 Schematic cross-sectional view of a mold coated with resorbable polymer on the side of the upper face of said mold
  • Figure 55 Schematic cross-sectional view of a resorbable polymer matrix comprising at least one negative mold of at least one molded 3D nanostructure
  • Figure 56 Schematic cross-sectional view of a resorbable polymer matrix comprising at least one negative mold of at least one molded 3D nanostructure, the lower face of said matrix being covered with a polyelectrolyte layer.
  • Figure 57 Schematic cross-sectional view of a resorbable polymer matrix comprising at least one negative mold of at least one molded 3D nanostructure assembled with a perforated piece comprising a support consisting of a non-absorbable membrane perforated by at least one perforation , said matrix being assembled on the side of its lower face with the upper face of said support, so that the negative mold of the 3D nanostructure is in alignment with said perforation of the support.
  • Figure 58 Schematic cross-sectional view of a resorbable polymer matrix comprising at least one negative mold of at least one molded 3D nanostructure assembled with a perforated piece comprising a support consisting of a non-absorbable membrane perforated by at least one perforation , said matrix being assembled on the side of its lower face with the upper face of said support, so that the negative mold of the 3D nanostructure is in alignment with said perforation of the support, in which the continuous surface constituted by the face lower part of said support and the lower face of said resorbable polymer matrix comprising at least one negative mold at said at least one perforation of said support, is covered with a polyelectrolyte layer to form a nanostructured membrane in 3D comprising at least one protuberance .
  • FIG. 59 Diagrammatic cross-sectional view of the central module corresponding to the perforated piece comprising, on the side of the lower face of the support, a porous membrane nanostructured in 3D 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 piece.
  • Figure 60 Schematic cross-sectional view of a mold comprising at least one 3D nanostructure molded on the side of its upper face, assembled on the side of its upper face with a support member having a cutout in its lower face.
  • FIG. 61 Diagrammatic cross-sectional view of a mold comprising at least one 3D nanostructure molded on the side of its upper face, assembled on the side of its upper face with a support piece having a cut-out in its lower face, where said mold is covered with resorbable polymer on the side of its upper face.
  • FIG. 62 Schematic cross-sectional view of a resorbable polymer matrix comprising at least one negative mold of at least one molded 3D nanostructure, said matrix being formed at the level of the cutout of the support piece
  • FIG. 63 Diagrammatic cross-sectional view of a support piece containing at the level of the cut-out of its solid lower face, a resorbable polymer matrix comprising at least one negative mold of at least one molded 3D nanostructure, said support piece being assembled to a perforated piece comprising a support with at least one perforation, so that said negative mold of at least one molded 3D nanostructure is in alignment with said perforation.
  • Figure 64 Diagrammatic cross-sectional view of a support piece containing at the level of the cutout of its full lower face, a resorbable polymer matrix comprising at least one negative mold of at least one molded 3D nanostructure, said support piece being assembled to a perforated part comprising a support with at least one perforation, so that said negative mold of at least one molded 3D nanostructure is in alignment with said perforation, the continuous surface constituted by the underside of said support and the underside of said resorbable polymer matrix comprising at least one negative mold at said at least one perforation of said support, being covered with a polyelectrolyte layer to form a 3D nanostructured membrane comprising at least one protuberance.
  • FIG. 65 Diagrammatic cross-sectional view of the support of the perforated part comprising, on the side of its lower face, a porous nanostructured membrane in 3D and on the side of its upper face, 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.
  • FIG. 66 Diagrammatic cross-sectional view of the central module corresponding to the perforated part comprising, on the side of the lower face of the support, a porous membrane nanostructured in 3D 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 piece.
  • FIG. 67 Schematic perspective view of a support piece I of circular shape, of a mold H of circular shape and comprising 100 molded 3D nanostructures (H1), of a perforated piece G of circular shape and comprising 100 perforations ( Gl) and a part F, support of the central module.
  • FIG. 68 Schematic perspective view of a support piece I of circular shape, of a mold H of circular shape and comprising 9 molded 3D nanostructures (H2), of a perforated piece G of circular shape and comprising 9 perforations ( G2) and a part F, support of the central module.
  • Figure 69 Schematic perspective views of the assembly of a support piece I of circular shape on a mold H of circular shape and comprising 100 molded 3D nanostructures (H1), via the alignment pin of I and the hole d Hl alignment.
  • A View from above when the two elements are assembled.
  • B View from above when the elements are disassembled.
  • C Side view when both elements are assembled.
  • D Side view when both elements are disassembled.
  • Figure 70 Schematic perspective views of the assembly of a support piece I of circular shape on a perforated piece G of circular shape and comprising 100 perforations (Gl) via the alignment pin of I and the hole d Gl alignment.
  • A View from above when the two elements are assembled.
  • B View from above when the elements are disassembled.
  • C Bottom view when both elements are assembled.
  • D Bottom view when both elements are disassembled.
  • E Side view when the two assembled elements are turned up so that Gl is oriented upwards and I is facing downwards.
  • Figure 71 Schematic perspective views of the assembly of a perforated piece G of circular shape and comprising 100 perforations (Gl) on a part F, support of the central module.
  • Figure 73 Schematic perspective view of a support piece i of square shape, of a mold H of square shape and comprising 9 molded 3D nanostructures (h1), of a perforated piece G of square shape and comprising 9 perforations ( g2).

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US16/300,504 US11857958B2 (en) 2016-05-11 2017-05-11 Cell co-culture chip and process for the production thereof
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JP6983180B2 (ja) 2021-12-17
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FR3051195B1 (fr) 2018-05-18
EP3455342B1 (fr) 2023-10-25
US11857958B2 (en) 2024-01-02
EP3455342A1 (fr) 2019-03-20
US20190168216A1 (en) 2019-06-06
FR3051195A1 (fr) 2017-11-17

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