EP3882385B1 - Fabrication automatique de matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules - Google Patents

Fabrication automatique de matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules Download PDF

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EP3882385B1
EP3882385B1 EP21160776.7A EP21160776A EP3882385B1 EP 3882385 B1 EP3882385 B1 EP 3882385B1 EP 21160776 A EP21160776 A EP 21160776A EP 3882385 B1 EP3882385 B1 EP 3882385B1
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Prior art keywords
nanofibres
deposition table
dimensional
nanofibre
deposition
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German (de)
English (en)
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EP3882385C0 (fr
EP3882385A1 (fr
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António Manuel GODINHO COMPLETO
Paula Alexandrina DE AGUIAR PEREIRA MARQUES
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Universidade de Aveiro
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Universidade de Aveiro
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    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H1/00—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres
    • D04H1/70—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres
    • D04H1/72—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged
    • D04H1/728—Non-woven fabrics formed wholly or mainly of staple fibres or like relatively short fibres characterised by the method of forming fleeces or layers, e.g. reorientation of fibres the fibres being randomly arranged by electro-spinning
    • D—TEXTILES; PAPER
    • D01—NATURAL OR MAN-MADE THREADS OR FIBRES; SPINNING
    • D01D—MECHANICAL METHODS OR APPARATUS IN THE MANUFACTURE OF ARTIFICIAL FILAMENTS, THREADS, FIBRES, BRISTLES OR RIBBONS
    • D01D5/00—Formation of filaments, threads, or the like
    • D01D5/0007—Electro-spinning
    • D01D5/0061—Electro-spinning characterised by the electro-spinning apparatus
    • D01D5/0076—Electro-spinning characterised by the electro-spinning apparatus characterised by the collecting device, e.g. drum, wheel, endless belt, plate or grid
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H3/00—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length
    • D04H3/016—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length characterised by the fineness
    • D—TEXTILES; PAPER
    • D04—BRAIDING; LACE-MAKING; KNITTING; TRIMMINGS; NON-WOVEN FABRICS
    • D04H—MAKING TEXTILE FABRICS, e.g. FROM FIBRES OR FILAMENTARY MATERIAL; FABRICS MADE BY SUCH PROCESSES OR APPARATUS, e.g. FELTS, NON-WOVEN FABRICS; COTTON-WOOL; WADDING ; NON-WOVEN FABRICS FROM STAPLE FIBRES, FILAMENTS OR YARNS, BONDED WITH AT LEAST ONE WEB-LIKE MATERIAL DURING THEIR CONSOLIDATION
    • D04H3/00—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length
    • D04H3/02—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length characterised by the method of forming fleeces or layers, e.g. reorientation of yarns or filaments
    • D04H3/04—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length characterised by the method of forming fleeces or layers, e.g. reorientation of yarns or filaments in rectilinear paths, e.g. crossing at right angles
    • D04H3/045—Non-woven fabrics formed wholly or mainly of yarns or like filamentary material of substantial length characterised by the method of forming fleeces or layers, e.g. reorientation of yarns or filaments in rectilinear paths, e.g. crossing at right angles for net manufacturing

Definitions

  • This invention relates to a system and a process for the automated manufacture of three-dimensional cell matrices by electrospinning from nanofibres of controlled alignment and uniform cell distribution throughout their thickness.
  • the present invention has many applications in various areas, in the manufacture of products or structures, on a nanometric scale, which depend on high surface area, such as in biotechnology, in the pharmaceutical and in tissue engineering areas, in particular in regenerative medicine.
  • nanomaterials associated with the different possibilities of morphologies and functionalities reveal a series of possibilities for new fields of application and drive the progress in the processing of these nanostructures.
  • the electrospinning or electrostatic spinning method is very advantageous, since the fibres obtained with this technique have a high surface area, combined with a low production cost and the possibility of being formed from a wide variety of polymers or composites.
  • This technique is based on the application of high voltage (5-50 KV) and low current (0.5-1 ⁇ A) electric fields for the production of very small diameter fibres. In this process, the electrostatic forces control the formation and deposition of these fibres.
  • the key configuration of a generic electrospinning process consists of a syringe, where the molten polymer or polymeric solution is introduced, which is connected to a capillary tube, a diffuser pump, which controls the flow of the polymeric solution to be supplied, so that a drop of solution is always maintained at the tip of the capillary tube, a metal collector, maintained at zero potential (grounded), where the fibres produced will be collected, a high voltage source, responsible for producing a difference in potential between the tip of the capillary tube and the collector.
  • a high voltage source responsible for producing a difference in potential between the tip of the capillary tube and the collector.
  • the balance of electrostatic charges to which the droplet is subjected namely the surface tension force of the solution and the force exerted by the applied electric field, begins to suffer an imbalance and, from a certain critical value of electric field, a jet of polymeric material from the capillary tube is projected and accelerated towards the collector.
  • the jet with the polymeric solution suffers evaporation of a large part of its solvent, thus ensuring that the fibres formed have enough rigidity to support their own weight.
  • the solvent that remains in the solution such as moisture, allows the adhesion of one fibre to another, as they are deposited in layers, forming a non-woven web.
  • the electrospun fibres form a two-dimensional, randomly oriented blanket or fabric due to the instability of the jet path.
  • Oriented fibre networks have the possibility of developing anisotropic properties in materials. These relationships are quite obvious in the field of tissue engineering.
  • Typical examples include the production of polymeric meshes, containing aligned fibres, used as substrates for culture and regeneration of neural cells, due to the inherently anisotropic nature of nerves and their regenerative mechanisms.
  • the scaffold material has a three-dimensional structure of controlled porosity, in order to allow the development of the three-dimensional cell matrix throughout the depth of the matrix.
  • document US20110018174A1 discloses the production of aligned electrospun fibres, with location and orientation control of the fibres, using for this purpose a device that provides a voltage depending on the selected time, whereby that voltage is applied to a collector with multiple electrodes.
  • said document does not disclose a process capable of forming a three-dimensional matrix of aligned fibres in any desired thickness.
  • the limitations related to the current processes of electrospinning the aligned fibres are mainly related to the fact that, as the aligned and electrically charged fibres are deposited one over the other, the increasing electric charge tends to repel the new fibres from being deposited, preventing their correct alignment and limiting their thickness to a few tenths of a millimetre of the matrix of the formed fibres.
  • cell matrices of nanofibres with a high thickness, i.e. in the order of several millimetres, with control of the alignment of the fibres throughout the thickness, with a uniform distribution of cells throughout the volume of the three-dimensional nanofibre matrix, with control of the distance between aligned nanofibres (inplane porosity) in the deposition plane forming two-dimensional nanofibre meshes and control of the degree of compaction (porosity throughout the thickness) between layers of the deposited two-dimensional nanofibre meshes.
  • the porosity of these nanofibre matrices is of utmost relevance in enhancing cell migration and multiplication, as well as the delivery of nutrients to the cells, throughout the volume of the three-dimensional nanofibre matrix.
  • the present invention proposes to solve the problems of the prior art, described above, through the implementation of a system and a process for the automated manufacture of three-dimensional cell matrices, which can present various patterns of fibre alignment, throughout the thickness of the matrix and with a uniform distribution of cells throughout the thickness of the matrix, this thickness being dependent on the number of layers of fibres deposited, the thickness of the fibres and the degree of compaction between layers.
  • the present invention relates to a system and process for the automated manufacture of three-dimensional cell matrices, by electrospinning, from nanofibres with controlled alignment and uniform cell distribution throughout their thickness.
  • the system of the present invention comprises a module (A) for forming nanofibres by electrospinning, a module (B) for collecting the formed nanofibres, a module (C) for deposition of the collected nanofibres, a module (D) for electropulverisation of cells, a vacuum pump and a computational unit according to claim 1.
  • the module (B), nanofibre collector comprises two collecting cylinders, with coaxial axes and perpendicular to the axis of the electrospinning capillary tube, where each cylinder is provided with continuous rotation movement by an electric motor controlled by a computerised unit, in which the electrospun nanofibres are collected on the surfaces and between the surfaces of the collecting cylinders, also comprising this module (B), brushes for removing the nanofibres, which remain on the cylindrical surfaces, and where the module (C), of fibre deposition, comprises a deposition table, able to be moved linearly, in the direction parallel to its surface and in the direction of the axis of the electrospinning capillary tube, and rotationally, around its longitudinal axis, assures that the electrospinning process occurs in a continuous and automatic way, with formation of three-dimensional matrices with uniform distribution of the cells throughout the thickness of the three-dimensional cell matrix and with alignment and distance between nanofibres controlled according to the
  • the present invention relates to a continuous and automated process for forming three-dimensional cell matrices with nanofibres of controlled alignment and uniform cell distribution throughout their thickness, according to claim 3.
  • the process of the present invention allows to obtain, in a totally automatic way and without manual intervention, three-dimensional cell matrices of aligned polymeric fibres, with a uniform cell distribution throughout the thickness of the matrix, which can present several alignment patterns, of the nanofibres that compose them, throughout their thickness, being the thickness of the matrix dependent on the number of fibre layers deposited, the thickness of the fibres and the degree of compaction between layers.
  • This process has the additional advantage of being versatile, simple, and operating in automatic and continuous mode, so it is not necessary to produce a series of layers of nanofibre meshes with a certain alignment, to manually add other layers with different alignments and to avoid the laborious manual process of cell seeding of the three-dimensional matrices of nanofibres previously manufactured, overcoming the limited capacity of this manual process to ensure the uniformity/control of the cells distribution in the three-dimensional matrices of nanofibres throughout the thickness.
  • This invention relates to a system and a process for the automated manufacture of three-dimensional cell matrices, obtaining nanofibres with controlled alignment and uniform cell distribution throughout their thickness, with the possibility of producing various fibre alignment patterns throughout the matrix thickness with controlled thickness and uniform cell distribution throughout the matrix thickness, this thickness being dependent on the number of layers of deposited nanofibre meshes, the thickness of the nanofibres and the degree of compaction between layers.
  • the system of the present invention comprises a module (A) for forming nanofibres by electrospinning, a module (B) for collecting the produced nanofibres, a module (C) for deposition of the collected nanofibres, and a module (D) for electropulverisation of cells.
  • Module (A) comprises a syringe to contain a polymer solution, connected to an injector pump, connected to an electrospinning capillary tube, which is connected to a voltage source, configured to provide positive polarity.
  • Module (A) is aligned perpendicularly with the axes of the two collector cylinders of module (B), the nanofibre collector.
  • Module (B) comprises two collector cylinders, each equipped with a continuous rotation movement, through the action of an electric motor, which is controlled by a computerised unit.
  • the distance between the top faces of the two cylinders is equal to the diameter of the nanofibre deposition table of module (C), and the cylindrical surfaces of the collector cylinders are made of conductive material, and may have negative or neutral polarity.
  • the nanofibre of polymeric material formed by electrospinning from the capillary tube of the module (A), with positive polarity, moves by the action of an electric field towards a collector module (C), and these are collected on the cylindrical surfaces and between the cylindrical surfaces of the collector cylinders, which are in continuous rotation.
  • the collector module (C) has brushes to remove the nanofibres that remain on the cylindrical surfaces at the end of each fibre production cycle, thus ensuring the continuous electrospinning of the nanofibres on and between the rotating cylindrical surfaces of the collector cylinders.
  • the nanofibres electrospun between the cylindrical surfaces of the collector cylinders, in continuous rotation, are deposited on the surface of the deposition table of the deposition module.
  • the table has a substantially circular shape and is positioned between the generatrices of the two collector cylinders, with its surface perpendicular to the electrospinning capillary tube axis.
  • the deposition table has holes extending from its surface to a chamber inside the table, which is connected by a channel to a vacuum pump.
  • the suction force generated by the vacuum, in the holes of the deposition table surface attaches the deposited nanofibres to the table.
  • the linear movement of the deposition table, parallel to its surface, and the rotation movement, around its longitudinal axis, are performed by electric motors controlled by a computerised unit.
  • the nanofibres, continuously deposited and attached to the surface of the deposition table, are aligned in different directions by the rotation movement of the deposition table, and the distance between the deposited nanofibres is controlled by the linear movement of the deposition table.
  • the continuous deposition of nanofibres, by the cylindrical surfaces of the collector cylinders, allows the formation of a two-dimensional nanofibre mesh of controlled organization and distribution on the surface of the deposition table.
  • the control of the distance between the fibres deposited on the deposition table allows controlling the porosity of the mesh in its plane.
  • the controlled movement of the deposition table in the direction and opposite orientation to the electrospinning capillary tube (direction of the matrix thickness) after the formation of the two-dimensional nanofibre mesh on the surface of the deposition table allows the deposition of a new two-dimensional nanofibre mesh over the previous mesh with a new organisation of the nanofibres, the number of nanofibre mesh layers accumulated on the table is defined by the desired thickness of the three-dimensional cell matrix, the thickness of the deposited nanofibres and the magnitude of the vacuum pressure generated on the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers and therefore the porosity of the three-dimensional matrix throughout the thickness.
  • the electropulverisation module of the cells can basically consist of a syringe to contain a solution with cells in suspension, connected to an injector pump, connected to an electropulverisation capillary tube, which is connected to a voltage source.
  • the voltage source is configured to provide positive polarity, being aligned with the ring-shaped collector, with internal diameter equal to the diameter of the deposition table, with negative or neutral polarity.
  • the electropulverisation of the cells starts from the capillary tube with positive polarity, in which the solution with cells moves by the action of an electric field towards the collector ring.
  • the solution with cells is seeded onto the nanofibre meshes deposited on the deposition table, which is in concentric position with the collector ring. In this position, the deposition table starts a rotation movement for a certain period of time, so that the surface of the nanofibre mesh is uniformly distributed with cells.
  • the number of nanofibre mesh layers deposited on the deposition table and the number of times the electropulverisation of the cells occurs is defined by the desired thickness of the three-dimensional cell matrix, as well as by the thickness of the deposited nanofibres, the magnitude of the vacuum pressure generated on the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers and thus the porosity of the three-dimensional matrix throughout the thickness and the desired cell density of the matrix.
  • the successive layers of two-dimensional nanofibre meshes, deposited on the surface of the deposition table, are kept in position on the deposition table by the action of the vacuum generated in the holes of the upper surface of the deposition table which communicate with a chamber in its interior connected to the vacuum pump.
  • Pressure control in the vacuum pump is also intended to control the degree of compaction between the two-dimensional nanofibre mesh layers formed and thus the porosity in the direction perpendicular to the plane of the deposited nanofibre mesh layer.
  • the control of the distance between the electrospinning capillary tube with positive polarity and the collector cylinders, the control of the speed of rotation of the collector cylinders, the control of the linear and rotation movements of the deposition table, the control of the pressure of the vacuum pump, the control of the distance between the electrospinning capillary tube of the cells with positive polarity and the collector ring, the control of the voltage of the capillary tubes, of the cylindrical surfaces of the collector cylinders and the collector ring are performed by a computerised control unit that, depending on the alignment and distance between nanofibres desired for each deposited two-dimensional layer and on the thickness of the matrix desired and the cell density desired, programs the sequence of all the movements, vacuum pressure and polarity of the electrodes required, based on a computer program developed for this purpose.
  • the system of the present invention has the capacity to continuously and automatically produce three-dimensional cell matrices with nanofibres of controlled alignment and uniform cell distribution throughout their thickness.
  • matrices with various alignment patterns and with different distances between nanofibres (porosity), throughout the matrix thickness, and with a uniform distribution of tissue component cells, throughout the matrix thickness, in a controlled manner.
  • the surface of the collection table provided with holes, which are subjected to vacuum pressure, allows the attachment of the fibres to this table, also allowing the control and definition of the degree of compaction (porosity) to be presented between the different layers of deposited two-dimensional nanofibre meshes, as well as the support and deposit of the nanofibres on the deposition table.
  • the separation of the deposited fibres which occurs by the effect of nanofibres stretching on the surface of the collector cylinders, during the continuous movement of these cylinders, and the linear movement of the deposition table in the direction and opposite orientation to the electrospinning capillary tube, contributes advantageously to the deposition of successive layers of two-dimensional fibre meshes, and the linear movement of the deposition table, to the concentric position, with the collector ring of the electropulverisation module of the cells.
  • the solution with cells which is electropulverised over the nanofibre meshes deposited on the deposition table, the rotation movement of the deposition table, in this position, for a certain period of time, ensures the uniform distribution of the cells on the surface of the nanofibre mesh, while the linear movement, returning the deposition table to the fibre collector module, allows a new deposition of another layer or layers of two-dimensional nanofibre meshes.
  • the alternating and controlled movement of the deposition table between the nanofibre deposition position and the cell electropulverisation position allows to automatically manufacture a three-dimensional cell matrix with nanofibres of controlled alignment and uniform cell distribution throughout the thickness, where the number of nanofibre mesh layers deposited on the deposition table and the number of times that the electropulverisation of the cells occurs are defined by the desired thickness of the three-dimensional cell matrix, by the thickness of the deposited nanofibres, by the magnitude of the vacuum pressure generated on the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers and therefore the porosity of the matrix throughout the thickness and by the cell density desired for the matrix.
  • the present invention relates to a system and a process for the automated manufacture of three-dimensional cell matrices.
  • the system of the present invention also comprises a computerised control unit and the electronics necessary for its proper functioning, actuators, namely the actuators of the linear and rotation movement of the deposition table, as well as all the electric wiring for the distribution of energy to the various components of the system, as well as all the devices and accessories necessary to guarantee the sterilisation, humidity and temperature conditions required to ensure the survival of the cells deposited on the nanofibre meshes during the manufacturing time of the three-dimensional cell matrix.
  • system (1) of the present invention comprises:
  • the nanofibre formation module (A) comprises:
  • the nanofibre collector module (B) comprises:
  • the nanofibre deposition module comprises:
  • the cell electropulverisation module (D) comprises:
  • the cylindrical surfaces of the collector cylinders (34, 36)) are made of conductive material, and these can have negative or neutral polarity (33).
  • the electrospun nanofibres (5) are collected on the cylindrical surfaces (34, 36) and between the cylindrical surfaces (34, 36).
  • Module (B) has brushes (32, 35) to remove the nanofibres that remain on the cylindrical surfaces (34,36) maintaining the electrical continuity of these surfaces (34,36) thus ensuring the continued electrospinning of the nanofibres on and between the cylindrical surfaces in rotation (31).
  • the nanofibres deposition module (C) comprises a deposition table (10), of circular shape, positioned between the generatrices of the collector cylinders (6, 30), with a flat surface perpendicular to the axis of the electrospinning capillary tube (4), where the nanofibres, electrified between the collector cylinders (6, 30), are deposited by rotation action (31) of them.
  • the deposition table (10) has a surface with holes (9), extending to a chamber (42), which is located inside the table (10), this chamber (42) being connected by a channel (11) to a vacuum pump (21).
  • the deposited nanofibres (8) are attached to the deposition table (10) by action of the suction force (43), generated by the vacuum in the holes of the surface of the deposition table (10).
  • the deposition table (10) moves linearly parallel to its surface (22) and has a rotation movement (24) around its longitudinal axis, the linear and rotation movements of the table are performed by electric motors (39, 40) controlled by a computerised unit (20), the control of these movements allow defining the two-dimensional organisation of the nanofibre mesh deposited on the deposition table (81, 82, 83, 84).
  • the continuously deposited nanofibres (8) attached to the surface (9) of the deposition table (10) are aligned in different directions by the rotation movement of the deposition table (24).
  • the distance between the deposited nanofibres is controlled by the linear movement of the deposition table (22).
  • the continuous deposition of nanofibres, by the cylindrical collector surfaces (34, 36), allows the formation of a two-dimensional nanofibre mesh (81, 82, 83, 84), of controlled organisation and distribution, over the surface of the deposition table (10).
  • the control of the distance (45, 47, 49, 52) between the nanofibres deposited on the deposition table (10) allows controlling the porosity of the two-dimensional mesh on the plane thereof.
  • the linear movement of the deposition table in the direction and opposite orientation (60) to the electrospinning capillary tube (4) determines the end of an electrospinning cycle.
  • the number of layers (64, 65, 66) of nanofibre meshes deposited onto the table is defined by the desired thickness (67) for each three-dimensional cell matrix (68), the thickness of the deposited nanofibres and the magnitude of the vacuum pressure generated on the surface of the deposition table (10) which controls the level of compaction of the nanofibre mesh layers and hence the porosity of the three-dimensional matrix (68) throughout the thickness (67).
  • the cell electropulverisation module (D) comprises a container (14), for containing and delivering a solution with cells in suspension, typically a syringe, and an injection pump, connected to an electropulverisation capillary tube (13), connected to a voltage source (17), configured to provide positive polarity (16), an adjustable length support (15), a ring-shaped collector (12) with an internal diameter identical to the diameter of the deposition table (10), this ring having negative or neutral polarity (18).
  • the deposition table (10) After deposition of one or more mesh layers (81,82,83,84) of nanofibres over the deposition table (10), it moves linearly (55) towards the cell electropulverisation module (D), until it is centred with the ring-shaped collector (12), which presents negative or neutral polarity (18). In this position, the deposition table (10) starts a rotation movement (57) on its axis and the vacuum system is turned off, initiating the seeding of the cells (56) from the electropulverisation capillary tube (13), with positive polarity (16), on the nanofibre meshes (81,82,83,84), during a certain period of time, thus leaving the surface of the nanofibre mesh with cells (58) uniformly distributed.
  • the deposition table (10) moves (59) again to the fibre collector module, in order to proceed to a new deposition cycle of another layer or layers of two-dimensional nanofibre meshes with controlled orientation and distance between nanofibres.
  • the deposition table (10) is alternately moved (55, 59) between the position of the nanofibre collector module, where the nanofibres are deposited, and the electropulverisation module, where the cells (56) are seeded in a controlled manner by the computerised unit (20), thus obtaining a three-dimensional cell matrix (68) with nanofibres of controlled alignment and uniform cell distribution throughout the thickness (67).
  • the number of nanofibre mesh layers deposited (64,65,66) over the deposition table (10) and the number of times the electropulverisation of the cells (56) occurs over these is defined by the thickness (67) desired for the three-dimensional cell matrix (68), by the thickness of the deposited nanofibres, the magnitude of the vacuum pressure generated at the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers and thus the porosity of the three-dimensional matrix throughout the thickness and the cell density desired for the three-dimensional cell matrix (68).
  • the successive two-dimensional layers of nanofibre meshes, deposited (81,82,83,84) onto the deposition table (10), are held in position by the action of a suction force (43) generated by the vacuum pressure in the holes of the surface (9) of the deposition table (10), which communicate with a chamber (42), in its interior, connected by a channel (11) to the vacuum pump (21).
  • control of the pressure in the vacuum pump (21) is also intended to control the suction forces (43) on the fibres and the degree of compaction between the formed two-dimensional nanofibre mesh layers (81,82,83,84) and, consequently, the porosity in the direction perpendicular to the plane of the deposited fibre layer.
  • the control of the distance between the electropulverisation capillary tube (4) with positive polarity and the generatrix of the collector cylinders (6, 30), the control of the rotation speed (31) of the collector cylinders (6, 30), the control of the linear (22) and rotation movements (24) of the deposition table, control of the pressure of the vacuum pump (21), control of the distance between the electropulverisation capillary tube (13) of the cells with positive polarity and the collector ring (12), control of the voltage on the capillary tubes (38) and the cylindrical surfaces (34, 36) of the collector cylinders (6, 30) and the collector ring (12) is performed by a computerised control unit (20), which, depending on the desired alignment and distance of the nanofibres for each deposited two-dimensional nanofibre mesh layer (81, 82, 83, 84), the desired thickness (67) of the matrix (68) and the desired cell density, programmes the sequence of all the necessary movements, vacuum pressure and polarity of the electrodes on the basis of a computer programme specifically developed for this
  • the process of the present invention is carried out in several steps using the manufacturing system (1), as described in the previous section.
  • the production of three-dimensional cell matrices with nanofibres of controlled alignment and uniform cell distribution throughout the thickness occurs from the alternating linear movement (55, 59) of the deposition table (10) between the position of the nanofibre collector module, where the aligned nanofibres (8) are deposited, and the electropulverisation module, where the cells (56) are seeded onto the two-dimensional nanofibre mesh layers (81, 82, 83, 84).
  • the continuously deposited nanofibres (8) attached to the surface of the deposition table (10) are aligned in different directions by the rotation movement of the deposition table (24), the distance between the deposited nanofibres is controlled by the linear movement (22) of the deposition table (10), the continuous deposition of nanofibres by the cylindrical surfaces (34, 36) of the collector cylinders (6, 30) allows the formation of two-dimensional nanofibre meshes (81,82,83,84) of controlled organization and distribution on the surface (9) of the deposition table (10), the control of the distance (45, 47, 49, 52) between the fibres deposited on the deposition table (10) allows controlling the porosity of the two-dimensional mesh in the plane thereof.
  • the controlled linear movement of the deposition table in the direction and opposite orientation (60) to the electrospinning capillary tube (4) allows the deposition of a new two-dimensional nanofibre mesh over the previous mesh with a new organisation of the nanofibres, being the number of layers (64,65, 66) of nanofibre meshes deposited on the table (10) defined by the desired thickness (67) of the three-dimensional cell matrix (68), the thickness of the deposited nanofibres and the magnitude of the vacuum pressure generated on the surface of the deposition table (9) which controls the level of compaction of the nanofibre mesh layers and thus the porosity of the three-dimensional matrix throughout the thickness (67).
  • the process for manufacturing three-dimensional cell matrices with nanofibres of controlled alignment and uniform cell distribution throughout the thickness of the present invention comprises the following steps:
  • the controlled movement (27) of the deposition table (10) in the direction and opposite orientation (60) to the electrospinning capillary tube (4) after a set of layers of deposited fibre meshes followed by electropulverisation of the cells (56) allows the accumulation of successive layers of two-dimensional nanofibre meshes (81,82,83, 84) with cells (58) and the formation of a three-dimensional cell matrix (68) with nanofibres of controlled alignment and uniform cell distribution with thickness (67) dependent on the number of layers (64,65,66) of deposited fibres, the thickness of the nanofibres and the degree of compaction between layers which is controlled by vacuum pressure and the vacuum pressure attaches the fibres to the table.
  • the obtained matrix thickness varies according to the number of deposited layers of nanofibre meshes, the thickness of these nanofibres and the degree of compaction between layers, the latter controlled by the vacuum pressure exerted on the layers of fibres deposited on the surface of the deposition table.
  • the alignment and distance, between the nanofibres in each layer is controlled by the different rotation and linear movements of the deposition table in each fibre deposition step.
  • the cell density of the three-dimensional nanofibre matrix is defined by the number of deposited nanofibre layers, the thickness of the deposited nanofibres, the magnitude of the vacuum pressure generated at the surface of the deposition table that controls the level of compaction of the nanofibre mesh layers, the number of times the electropulverisation of the cells (56) over the two-dimensional nanofibre mesh occurs and the duration of the cell electropulverisation period on the meshes.
  • Example Production of a three-dimensional cell matrix with nanofibres of controlled alignment and uniform cell distribution throughout the thickness.
  • This example concerns the production of a cell matrix composed of 48 layers of aligned polymeric nanofibre meshes and cells from a chondrocyte cell line for cartilage engineering, with a total thickness of 3 mm.
  • PCL polycaprolactone
  • DCM dichloromethane
  • DMF dimethylformamide
  • the molten polymer was then electrospun using a capillary tube (4) with a flow of 2.5 mL/h, a voltage of 25 kV and a working distance of 15 cm from the cylindrical surfaces (34,36) of the collector cylinders (6,30).
  • the collector cylinders (6,30) have a diameter of 80 mm and are moved with a continuous rotational speed of 10 rpm and the cylindrical surfaces (34,36) were subjected to a negative voltage of -3 kV.
  • the deposition table has a diameter of 6 mm, and the holes on its surface (9) are subjected to a vacuum pressure of 3300 Pa.
  • a vacuum pressure 3300 Pa.
  • different combinations of speeds and duty strokes were programmed in linear movement (22) and rotation movement (24) of the deposition table (10).
  • the 48 layers of the deposited two-dimensional nanofibre meshes resulted from performing 6 consecutive cycles (6 times) of 8 layers of two-dimensional meshes with different alignments (81), (82), (83), (84), (85), (86), (87) and (88) in the following order, with the speed and stroke characteristics of the deposition table (10) for each mesh as follows:
  • chondrocyte cell line C28 / I2 was used and maintained at 37°C in a humidified atmosphere of 5% CO2 in air, in DMEM / F-12 (Sigma - Aldrich) supplemented with 10% (v / v) of FBS (Sigma -Aldrich), 1% (v / v) P / S (Sigma - Aldrich) and 0.25 ⁇ g / mL of amphotericin B.
  • Cells were harvested pre-confluence using a trypsin / EDTA (0.25%; Sigma-Aldrich) solution. 1.00 ⁇ 10 ⁇ 6 chondrocytes were suspended in 154 ⁇ L of culture medium and poured into a 5 mL plastic syringe.
  • the chondrocyte suspension was subjected to electropulverisation with a flow rate of 2 mL / h at +12.5 kV through 27G capillary tube/blind needle (13) (0.4 mm diameter and 1.5 mm length) with a distance between the needle and the ring-shaped collector (12) of 70 mm, the deposition table (10) was positioned concentrically with the collector ring (12). The collector ring was subjected to a voltage of -1 kV. In this position the deposition table (10) started the rotation movement (57) at a speed of 5 rpm for 3s.
  • each four layers of two-dimensional nanofibres mesh deposited on the deposition table (10) it moved 0.0625 mm in the direction and opposite orientation (60) to the electrospinning capillary tube (4), which corresponds to the average thickness of the four layers of deposited two-dimensional nanofibres mesh.
  • the deposition table moved in the opposite orientation of the capillary tube about 3 mm, corresponding to the thickness of the matrix (67) obtained at the end of the 48 deposited layers.
  • the deposition table moves (55) to the position of the cell electropulverisation module, the vacuum on the deposition table is turned off and the cells (58) are seeded on the meshes, then the deposition table (10) moves (59) to the nanofibre collector module for new deposition of four layers of nanofibre meshes (85, 86, 87, 88), the vacuum system is turned on again.
  • This automated and alternated process between the deposition of the nanofibre meshes and the seeding of cells (58) on them was repeated until the final thickness (67) of the aligned nanofibre cell matrix was reached (65).
  • the three-dimensional matrix of aligned fibres obtained in this example exhibits, like native cartilage, a preferential alignment of the fibres in its surface area parallel to the surface, in the intermediate area it does not show any preferential alignment, and in the deeper area the fibres are aligned in a vertical manner relative to the surface.

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Claims (14)

  1. Un système de fabrication automatique de matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur comprenant (a) un module de formation de nanofibres par électrofilage, (b) un module collecteur de nanofibres, (c) un module de dépôt des nanofibres collectées, qui forme des mailles de nanofibres bidimensionnelles (81, 82, 83, 84) à alignement et distance entre nanofibres contrôlés par une combinaison de mouvements linéaire (22) et rotatif (24) de la table de dépôt (10), ces mailles s'accumulent en couches successives formant l'épaisseur de la matrice de cellules (67), la surface avec des trous (9) de la table de dépôt (10) est raccordée par un canal à une pompe à vide (21), (d) un module d'électropulvérisation de cellules, dans lequel le mouvement linéaire (55) de la table de dépôt (10) de la position dans le module collecteur de nanofibres à la position d'électropulvérisation de cellules (56) ensemence les cellules des mailles de nanofibres précédemment déposées sur la table de dépôt (10) en répétant l'électropulvérisation des cellules de manière alternée avec le dépôt de mailles de nanofibres bidimensionnelles successives obtenant ainsi une distribution uniforme de cellules sur toute l'épaisseur de la matrice de cellules tridimensionnelle, caractérisé en ce que :
    a. Le module de formation de nanofibres comprend un tube capillaire d'électrofilage (4) à polarité positive et un support à longueur ajustable (3) ;
    b. Le module collecteur de nanofibres comprend deux cylindres collecteurs (6, 30) avec des axes coaxiaux et perpendiculaires à l'axe du tube capillaire d'électrofilage (4), chaque cylindre est pourvu d'un mouvement de rotation continu (31) à travers un moteur électrique (7, 29) contrôlé par une unité informatisée (20), la distance entre les faces supérieures des deux cylindres collecteurs (6,30) est égale au diamètre de la table de dépôt des nanofibres (10), les surfaces cylindriques (34, 36) sont faites d'un matériau conducteur à polarité négative ou neutre (33), les nanofibres électrofilées (5) sont collectées sur les surfaces cylindriques (34, 36) et entre les surfaces cylindriques (34, 36), ce module est pourvu des brosses (32, 35) pour éliminer les nanofibres restantes sur les surfaces cylindriques (34, 36) maintenant la continuité électrique des surfaces cylindriques (34, 36) assurant ainsi l'électrofilage continu des nanofibres sur et entre les surfaces cylindriques (34, 36) rotatives (31) ;
    c. Le module de dépôt de nanofibres comprend une table de dépôt (10) au format circulaire placée entre les génératrices des cylindres collecteurs (6, 30) ayant une surface avec des trous (9) perpendiculaire à l'axe du tube capillaire d'électrofilage (4) où les nanofibres électrofilées entre les cylindres collecteurs (6, 30) sont déposées par l'action de rotation (31) de ceux-ci, la table de dépôt (10) a des trous s'étendant de sa surface (9) jusqu'à une chambre (42) qui est à l'intérieur de la table de dépôt (10), cette chambre étant raccordée, par un canal (11), à une pompe à vide (21), la table de dépôt (10) se déplace linéairement (22) parallèlement à sa surface (9) et vers (27) l'axe du tube capillaire d'électrofilage (4), la table de dépôt (10) a un mouvement rotatif (24) autour de son axe longitudinal, les mouvements linéaire (22, 27) et de rotation (24) de la table de dépôt (10) sont exécutés par des moteurs électriques (39, 40, 41) contrôlés par une unité informatisée (20), le contrôle de ces mouvements permet l'organisation bidimensionnelle de la maille de nanofibres (81, 82, 83, 84) permettant le contrôle de son alignement sur la surface de la table de dépôt (10) ainsi que de la distance (44, 45, 47, 49, 52) entre les nanofibres déposées ;
    d. Le module d'électropulvérisation de cellules comprend un conteneur (14) pour contenir et fournir une solution avec des cellules en suspension, typiquement une seringue, et une pompe d'injection, raccordée à un tube capillaire d'électropulvérisation (13), raccordée à une source de tension (17), configurée pour fournir une polarité positive (16), un support à longueur réglable (15), un collecteur en forme d'anneau (12) avec un diamètre interne identique au diamètre de la table de dépôt (10), cet anneau ayant une polarité négative ou neutre (18), la table de dépôt (10) se déplace alternativement (55, 59) entre la position du module collecteur de nanofibres où les nanofibres sont déposées et le module d'électropulvérisation où les cellules (56) sont ensemencées sur les couches de maille de nanofibres bidimensionnelles (81, 82, 83, 84) ;
    dans lequel :
    - les surfaces cylindriques (34, 36) à polarité négative ou neutre (33), collectent les nanofibres (5) à partir du tube capillaire d'électrofilage (4) à polarité positive (38) de manière continue par l'action du mouvement rotatif (31) des cylindres collecteurs (6, 30) ;
    - à travers le mouvement rotatif (31) des cylindres collecteurs (6, 30) contrôlé par l'unité informatisée (20), les nanofibres électrofilées (5) entre les surfaces cylindriques (34, 36) sont déposées (8) sur la surface (9) de la table de dépôt (10) les nanofibres étant fixées à la table de dépôt (10) par l'action de la force d'aspiration (43) générée par le vide dans les trous de la surface (9) de la table de dépôt (10), ces nanofibres (8) fixées à la table de dépôt (10) se séparent de leur partie fixe sur les surfaces cylindriques (34, 36) par un effet d'étirement de leur section transversale, dû au mouvement rotatif continu (31) des cylindres collecteurs (6, 30) ;
    - les nanofibres continuellement déposées qui sont fixées à la surface (9) de la table de dépôt (10) sont alignées dans différentes directions (81, 82, 83, 84) par le mouvement rotatif (24) de la table de dépôt (10), la distance (44, 45, 47, 49, 52) entre les nanofibres déposées est contrôlée pour le mouvement linéaire (22) de la table de dépôt (10), tous les mouvements sont contrôlés par une unité informatisée (20), le dépôt continu de nanofibres (8) sur les surfaces du collecteur cylindrique (34, 36) permet la formation de mailles de nanofibres bidimensionnelles (81, 82, 83, 84) à l'organisation et distribution contrôlées sur la surface (9) de la table de dépôt (10), le contrôle de la distance (44, 45, 47, 49, 52) entre les nanofibres déposées sur la table de dépôt (10) permet de contrôler la porosité de la maille bidimensionnelle sur le plan de celle-ci ;
    - après la formation d'une ou de plusieurs mailles bidimensionnelles (81, 82, 83, 84) de nanofibres sur la surface (9) de la table de dépôt (10), le mouvement linéaire (27) de la table de dépôt (10) dans la direction et orientation opposée (60) au tube capillaire d'électrofilage (4) permet le dépôt de nouvelles mailles de nanofibres bidimensionnelles sur les mailles précédentes, le nombre de couches (64, 65, 66) de mailles de nanofibres déposées sur la table étant défini par l'épaisseur souhaitée (67) de la matrice de cellules tridimensionnelle (68), l'épaisseur des nanofibres déposées et la magnitude de la pression sous vide générée sur la surface (9) de la table de dépôt (10) qui contrôle le niveau de compactage des couches de mailles de nanofibres et ainsi la porosité de la matrice tridimensionnelle sur toute l'épaisseur (67) ;
    - après le dépôt d'une ou de plusieurs couches de mailles de nanofibres sur la table de dépôt (10) elle se déplace linéairement (55) vers le module d'électropulvérisation de cellules jusqu'à ce qu'elle soit concentrique au collecteur en forme d'anneau (12), qui a une polarité négative ou neutre (18), dans cette position la table de dépôt (10) commence le mouvement rotatif (57) sur son axe et le système de vide est éteint, commençant l'électropulvérisation de cellules (56) à partir du tube capillaire (13) à polarité positive (16) sur les mailles de nanofibres (81, 82, 83, 84) pour une période de temps définie en fonction de la densité cellulaire souhaitée, faisant ainsi en sorte que les mailles de nanofibres soient ensemencées avec des cellules (58) distribuées uniformément, après avoir ensemencé les cellules, la table de dépôt (10) se déplace (59) vers le module collecteur de fibres afin de procéder à un nouveau dépôt d'une ou de plusieurs couches de mailles de nanofibres bidimensionnelles à orientation et distance entre les nanofibres contrôlées, intercalant successivement de manière contrôlée par l'unité informatique (20) le dépôt de mailles de nanofibres (81, 82, 83, 84) et l'ensemencement de cellules (56), fabriquant ainsi une matrice de cellules tridimensionnelle (68) avec des nanofibres à alignement contrôlé et à distribution uniforme de cellules sur toute l'épaisseur (67) ;
    - le nombre de couches (64, 65, 66) de mailles de nanofibres déposées sur la table de dépôt (10) et le nombre de fois que l'électropulvérisation de cellules (56) a lieu sur elles est défini par l'épaisseur souhaitée (67) de la matrice de cellules tridimensionnelle (68), par l'épaisseur des nanofibres déposées, la magnitude de la pression sous vide générée à la surface de la table de dépôt (10) qui contrôle le niveau de compactage des couches de maille de nanofibres et, par conséquent, la porosité de la matrice tridimensionnelle sur toute l'épaisseur (67) et la densité de cellules souhaitée de la matrice de cellules tridimensionnelle.
  2. Système de fabrication (1) selon la revendication précédente comprenant une unité de contrôle informatisée (20) et un logiciel informatique.
  3. Un processus automatique pour produire des matrices de cellules tridimensionnelles à alignement contrôlé et nanofibres à distribution uniforme de cellules sur toute l'épaisseur qui a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce qu'il comprend les étapes suivantes:
    a. exposition des deux surfaces cylindriques (34, 36) des cylindres collecteurs (6, 30) au tube capillaire d'électrofilage (4) contenant une solution d'un polymère donné adapté à la fonction de la matrice devant être produite, telle exposition étant exécutée en appliquant une tension négative ou neutre aux deux surfaces cylindriques (34, 36) les cylindres collecteurs (6, 30) étant animés par un mouvement rotatif (31) pour l'électrofilage continu de nanofibres (5) sur et entre ces surfaces (34, 36) dans la zone de la génératrice la plus proche du tube capillaire d'électrofilage (4) ;
    b. dépôt continu des nanofibres électrofilées entre les surfaces cylindriques (34, 36) sur la surface avec des trous (9) de la table de dépôt (10), positionnées entre les génératrices des cylindres collecteurs (6, 30), par l'action du mouvement rotatif continu (31) des cylindres collecteurs (6, 30) ;
    c. application de pression sous vide aux nanofibres déposées de la table de dépôt (10) obtenue à travers des trous sur sa surface (9) pour fixer et compacter les nanofibres à la table de dépôt (10) ;
    d. mouvements linéaire (22) et rotatif (24) de la table de dépôt (10) pour aligner et espacer les nanofibres déposées par les surfaces cylindriques (34, 36), formant une maille de nanofibres bidimensionnelles (81, 82, 83, 84) à organisation et distribution sur la surface de la table de dépôt (10) contrôlées ;
    e. rupture des nanofibres (8) fixées à la table de dépôt (10) par l'effet d'étirement de sa section transversale, par l'action du mouvement rotatif (31) des cylindres collecteurs (6, 30) ;
    f. mouvement linéaire (27) de la table de dépôt (10) dans la direction et orientation opposée (60) au tube capillaire d'électrofilage (4) ;
    g. répétition de cycles tels que décrits dans l'étape (d), (c) et (f) autant de fois que nécessaire afin de déposer des couches successives de mailles de nanofibres bidimensionnelles sur les mailles déposées lors du cycle précédant ;
    h. interruption du processus d'électrofilage de fibres ;
    i. mouvement linéaire (55) de la table de dépôt (10) vers la position concentrique au collecteur en anneau (12) du module d'électropulvérisation de cellules ;
    j. interruption de l'application de pression sous vide sur la surface de la table de dépôt (10) ;
    k. exposition des mailles de nanofibres bidimensionnelles (81, 82, 83, 84) sur la table de dépôt (10) au tube capillaire d'électropulvérisation (13) contenant une solution d'un moyen donné avec des cellules en suspension, cette exposition étant exécutée en appliquant une tension négative ou neutre (18) à l'anneau collecteur (12) autour de la table de dépôt (10) pour une période de temps ;
    l. mouvement rotatif (57) de la table de dépôt (10) pour une période de temps pour l'ensemencement uniforme des cellules et le contrôle de la densité de cellules sur le plan des mailles de nanofibre déposées (81, 82, 83, 84) sur la table de dépôt (10) ;
    m. arrêter l'électropulvérisation des cellules (56) ;
    n. application de pression sous vide à la surface de la table de dépôt (10) ;
    o. mouvement linéaire (59) de la table de dépôt (10) vers la position entre les génératrices des cylindres collecteurs (6, 30) ;
    p. répétition de cycles, tels que décrits dans les étapes (a) à (o), autant de fois que nécessaire, les paramètres de rotation (24) et de mouvement linéaire (22) de la table de dépôt (10) dans l'étape (d) peuvent être modifiés par rapport au cycle précédent, pour former des mailles de nanofibres bidimensionnelles à alignement et distance entre les nanofibres (81, 82, 83, 84) qui sont différents de ceux obtenus lors du cycle précédent ;
    dans lequel :
    le mouvement contrôlé (27) de la table de dépôt (10) dans la direction et orientation opposée (60) au tube capillaire d'électrofilage (4) après chaque couche de fibres déposée suivie d'un électrofilage des cellules permet l'accumulation de couches successives de mailles de nanofibres bidimensionnelles (81, 82, 83, 84) avec des cellules (58) et la formation d'une matrice de cellules tridimensionnelle (68) avec des nanofibres à alignement contrôlé et distribution de cellules contrôlée à épaisseur (67) dépendant du nombre de couches (64, 65, 66) de fibres déposées, de l'épaisseur des nanofibres et du degré de compactage entre les couches qui est contrôlé par pression sous vide et la même pression sous vide fixe les fibres à la table.
  4. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon la revendication précédente, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que les cylindres collecteurs (6, 30) ont des surfaces conductrices cylindriques (34, 36) avec un mouvement rotatif continu (31) ces surfaces étant continuellement exposées au tube capillaire d'électrofilage (4), la continuité de la conductivité électrique des surfaces cylindriques (34, 36) étant assurée par un retrait ininterrompu des nanofibres déposées sur ces surfaces par des brosses de nettoyage (32, 35) en contact permanent avec ces surfaces dans la zone génératrice des cylindres collecteurs (6, 30).
  5. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 ou 4, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que la table de dépôt (10) accumule continuellement les nanofibres électrofilées entre les surfaces cylindriques (34, 36) des cylindres collecteurs (6, 30), ladite table de dépôt (10) intégrant des trous allant de sa surface (9) à une cavité en son intérieur (42) cette cavité étant raccordée (11) à une pompe à vide (21) avec contrôle de pression, dans lequel la table de dépôt (10) a un mouvement linéaire (27) contrôlé vers l'axe du tube capillaire d'électrofilage (4).
  6. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 à 5, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que l'alignement et la distance entre les nanofibres déposées sur la surface de la table de dépôt (9) est exécuté par la combinaison de mouvements rotatifs (24) et linéaires (22) de la table de dépôt (10) formant une maille de nanofibres bidimensionnelles (81, 82, 83, 84) à organisation et distribution contrôlées, la porosité sur le plan de la maille bidimensionnelle de nanofibres déposées étant contrôlée par la distance (44, 45, 47, 49, 52) entre les nanofibres déposées.
  7. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 à 6, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que la force d'aspiration (43) générée par le vide dans les trous de la surface (9) de la table de dépôt (10) fixe les nanofibres à la table de dépôt (10) séparant ces nanofibres de la partie encore attachée aux surfaces cylindriques (34, 36) par l'effet d'étirement de leur section transversale, par l'action du mouvement rotatif continu (31) des cylindres collecteurs (6, 30).
  8. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 à 7, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que le mouvement linéaire de la table de dépôt dans la direction et orientation opposée (60) au tube capillaire d'électrofilage (4) permet des couches successives de mailles de nanofibres bidimensionnelles (81, 82, 83, 84) sur la table de dépôt (10) pour former une structure matrice de cellules tridimensionnelle (68) de fibres dans laquelle son épaisseur (67) dépend du nombre de couches (64, 65, 66) de fibres bidimensionnelles déposées, de l'épaisseur des fibres et du degré de compactage entre les couches souhaité par l'action du système et de la pression sous vide.
  9. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 à 8, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que la pompe à vide (21) contrôle le degré de compactage entre les couches de nanofibres bidimensionnelles (81, 82, 83, 84) formées sur la table de dépôt (10) et la porosité dans la direction perpendiculaire au plan de la couche de nanofibres déposée.
  10. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 à 9, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que les divers paramètres de vitesse, position, tension et vide sont contrôlés par une unité de contrôle informatisée (20) et par un logiciel informatique.
  11. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 à 10, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que le mouvement linéaire de la table de dépôt (55) de la position génératrice des cylindres collecteurs (6, 30) à la position du tube capillaire d'électropulvérisation de cellules centrée sur l'anneau collecteur (12) permet l'ensemencement des cellules sur les mailles de nanofibres bidimensionnelles (81, 82, 83, 84) précédemment déposées sur la surface de la table de dépôt (10).
  12. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 à 11, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que à la fin du mouvement de la table de dépôt (55) vers la position du tube capillaire d'électropulvérisation de cellules, l'application de la pression sous vide à la surface de la table de dépôt (9) est interrompue évitant l'aspiration du moyen de suspension de cellules, la pression sous vide étant à nouveau appliquée à la surface de la table de dépôt (9) au début du mouvement linéaire (59) vers la position de dépôt des nanofibres par les cylindres collecteurs (6, 30).
  13. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 à 12, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que le mouvement rotatif (57) de la table de dépôt (10) pour une période de temps, lorsque la dernière est dans la position centrale avec l'anneau collecteur (12) et le tube capillaire d'électropulvérisation de cellules (13), permet un ensemencement des cellules uniforme et un contrôle de densité des cellules sur le plan des mailles de nanofibres déposées (81, 82, 83, 84) sur la table de dépôt (10).
  14. Le processus automatique pour produire des matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur qui, selon l'une quelconque des revendications 3 à 13, a lieu dans le système de l'une quelconque des revendications 1 ou 2, caractérisé en ce que les mouvements linéaires alternatifs (55, 59) de la table de dépôt (10) entre la position génératrice des cylindres collecteurs (6, 39) pour le dépôt des mailles de nanofibres (81, 82, 83, 84) et la position du tube capillaire d'électropulvérisation de cellules (13) permet le dépôt alterné des mailles de nanofibres et l'ensemencement de cellules (56) fabriquant ainsi une matrice de cellules tridimensionnelle (68) avec des nanofibres à alignement contrôlé et distribution uniforme de cellules sur toute l'épaisseur (67).
EP21160776.7A 2020-03-04 2021-03-04 Fabrication automatique de matrices de cellules tridimensionnelles avec des nanofibres à alignement contrôlé et distribution uniforme de cellules Active EP3882385B1 (fr)

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US7134857B2 (en) * 2004-04-08 2006-11-14 Research Triangle Institute Electrospinning of fibers using a rotatable spray head
US7981353B2 (en) 2005-12-12 2011-07-19 University Of Washington Method for controlled electrospinning
CN101410508B (zh) * 2006-01-27 2013-07-03 加利福尼亚大学董事会 仿生支架
US7828539B1 (en) 2007-03-26 2010-11-09 Clemson University Fabrication of three dimensional aligned nanofiber array
US8211352B2 (en) 2009-07-22 2012-07-03 Corning Incorporated Electrospinning process for aligned fiber production
US9005604B2 (en) 2009-12-15 2015-04-14 The United States Of America As Represented By The Administrator Of The National Aeronautics And Space Administration Aligned and electrospun piezoelectric polymer fiber assembly and scaffold
JP2017516601A (ja) * 2014-03-14 2017-06-22 スクリップス ヘルス 軟骨および半月板のマトリックスポリマーのエレクトロスピニング
US20160004706A1 (en) 2014-07-01 2016-01-07 Microsoft Corporation Security trimming of search suggestions
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