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    • 专利摘要:
    The invention relates to a fluidic system for producing extracellular vesicles from producer cells, comprising at least one container, a liquid medium contained by the container and production cells, characterized in that it also comprises microcarriers suspended in the liquid medium, the majority of the producer cells being adherent to the surface of the microcarriers, and a liquid medium stirrer, the agitator and the dimensions of the container being adapted to control a turbulent flow of the liquid medium in the container.
    公开号:FR3068361A1
    申请号:FR1756183
    申请日:2017-06-30
    公开日:2019-01-04
    发明作者:Florence Gazeau；Amanda Karine Andriola Silva；Otto-Wilhelm Merten；Claire WILHELM；Max Piffoux
    申请人:Centre National de la Recherche Scientifique CNRS；Universite Paris Diderot Paris 7；Genethon；
    IPC主号:
    专利说明:

    FLUIDIC SYSTEM FOR PRODUCING EXTRACELLULAR VESICLES AND ASSOCIATED METHOD
    FIELD OF THE INVENTION
    The invention relates to a system for producing extracellular vesicles from producer cells, a method for producing and recovering such vesicles and vesicles produced by such a system, for example used in cell therapy and in regenerative medicine.
    STATE OF THE ART
    Cells are known to release extracellular vesicles into their environment, for example, in vivo, into the biological fluids of an organism. Extracellular vesicles have been identified as effective means for delivering drugs, either individually or in a targeted manner, to the human body. First, they have native biocompatibility and immune tolerance. They can also comprise theranostic nanoparticles, making it possible both to image certain parts of the body and to deliver active principles having therapeutic functions. Extracellular vesicles also have a function of intercellular communication: they allow, for example, to transport lipids, membrane and cytoplasmic proteins and / or nucleotides of the cell cytoplasm, such as non-coding mRNA, microRNA or long RNA , between different cells.
    In particular, the use of extracellular vesicles may make it possible to solve known problems during the therapeutic use of cells, such as cell replication, differentiation, vascular occlusions, the risks of rejection and the difficulties of storage and freezing. There is therefore an industrial need for the production of cell vesicles in quantities sufficient for therapeutic use, in particular as a replacement or in addition to cellular therapies.
    To this end, Piffoux et al. (Piffoux, M., Silva, AK, Lugagne, JB, Hersen, P., Wilhelm, C., & Gazeau, F., 2017, Extracellular Vesicle Production Loaded with Nanoparticles and Drugs in a Trade-off between Loading, Yield and Purity: Towards a Personalized Drug Delivery System, Advanced Biosystems) describe the comparison of different methods of producing extracellular vesicles.
    A first method consists in producing extracellular vesicles from endothelial cells of the umbilical cord vein (HUVEC), by subjecting these cells to hydrodynamic stresses mimicking the stresses exerted under physiological conditions within the blood capillaries or under pathological conditions during stenosis of blood vessels. These constraints are caused by the passage of producer cells in microfluidic channels, for four hours. A microfluidic chip includes two hundred channels in which cells are transported in a laminar flow, to produce vesicles in a parallelized fashion.
    However, this method presents sizing problems: the quantities of vesicles produced by a microfluidic chip are not adapted to the quantities required for the abovementioned applications. In addition, the yield of extracellular vesicles produced per cell introduced into such a chip (approximately 2.10 4 vesicles per cell) is much lower than the maximum theoretical yield of vesicles produced by a cell, for example of the order of 3.5 × 10 6 vesicles. per cell for an MSC type cell (acronym for mesenchymal stem cell in English). Finally, this method requires compliance with so-called GMP standards (English acronym for Good Manufacturing Practices), necessary for the manufacture of medicines.
    A second method commonly used in the literature and described by Piffoux et al. consists in cultivating HUVECs in a DMEM type culture medium (English acronym for Dulbecco's Modified Eagle's Medium) without serum, for three days (technique called starvation in English, or serum deficiency). The absence of serum leads to cellular stress triggering a release of vesicles by the producer cells. This method has a higher yield and makes it possible to produce a larger quantity of vesicles than the method using a microfluidic chip (approximately 4.10 4 vesicles per producer cell). However, the calculated yield corresponds to a production time much longer than the production time of the previous method. This method does not produce a sufficient quantity of extracellular vesicles for the above-mentioned applications. Finally, this method does not make it possible to produce vesicles continuously since it induces cell death.
    Watson et al. (Watson, DC, Bayik, D., Srivatsan, A., Bergamaschi, C., Valentin, A., Niu, G., ... & Jones, JC, 2016, Efficient production and enhanced tumor delivery of engineered extracellular vesicles , Biomaterials, 105, 195-205) describe a method of producing vesicles which makes it possible to increase the quantity of vesicles produced. This method consists in cultivating HEK293 type cells in culture flasks, then in hollow fiber membranes. The central passage of the hollow fibers makes it possible to convey the culture medium to the producer cells. The producer cells are first sown around this passage, where they produce vesicles in an inter-fiber space. The liquid medium included in the inter-fiber space is collected three times a week, making it possible to produce approximately 3.10 12 vesicles in several weeks, for very large quantities of seeded cells, for example of the order of 5.10 8 cells, causing a yield of around 6000 extracellular vesicles per cell and a very low purity ratio (for example
    1.09.10 9 particles per microgram of protein). However, this production is not high enough and too slow with regard to the aforementioned applications. In addition, this method is described using producer cells corresponding to a cell line particularly resistant to culture in a medium lacking serum: this method may not be transposable to production of vesicles by producer cells such as stem cells, for example human, less resistant and particularly suitable for the targeted therapeutic applications.
    SUMMARY OF THE INVENTION
    An object of the invention is to provide a solution for producing extracellular vesicles in large quantities from producer cells, more quickly than with known methods, under conditions conforming to G.M.P. Another object of the invention is to propose a solution making it possible to increase the yield of the vesicle production system, that is to say the ratio between the number of vesicles produced and the number of producer cells introduced into the system. of production. Another object of the invention is to propose a system adapted to produce extracellular vesicles from a wide range of adherent producer cells, whatever the resistance of the type of cell introduced into the production system and resistant or not to a serum deficiency. Another object of the invention is to provide a solution for producing and recovering extracellular vesicles continuously. Finally, another object of the invention is to simplify the structure of the fluidic system for the production of vesicles and to reduce its manufacturing cost.
    In particular, an object of the invention is a fluidic system for producing extracellular vesicles from producer cells, comprising at least one container, a liquid medium contained by the container and producer cells, characterized in that it also comprises microcarriers suspended in the liquid medium, the majority of the producing cells being adherent to the surface of the microcarriers, and a liquid medium agitator, the agitator and the shape and the dimensions of the container being adapted to the generation of a turbulent flow liquid medium in the container.
    We understand that with such a system, it is possible to produce vesicles in large quantities, and in a system adapted to G.M.P. It is also understood that such a system is simpler and less expensive to manufacture than the systems known for producing extracellular vesicles.
    The invention is advantageously supplemented by the following characteristics, taken individually or in any of their technically possible combinations:
    the agitator of the liquid medium and the dimensions of the container are suitable for controlling a flow of the liquid medium, the Kolmogorov length of the flow being less than or equal to 75 μm, and preferably 50 μm;
    the fluidic system comprises an outlet and a connector connected to the outlet, the connector being capable of comprising liquid medium and extracellular vesicles;
    - The agitator is a rotary agitator whose rotation speed (s), shape, size are adapted, with the shape and dimensions of the container, to the generation of a turbulent flow of the liquid medium in the container;
    the microcarriers are microbeads, the diameter of the microbeads being between 100 μm and 300 μm;
    - The fluid system comprises a separator of extracellular vesicles, fluidly connected to the container so as to be capable of reintroducing into the container a liquid medium depleted in vesicles.
    Another object of the invention is a process for the ex vivo production of extracellular vesicles from producer cells, comprising:
    a control of an agitator causing a turbulent flow of a liquid medium in a container, the container comprising an outlet, the liquid medium comprising producer cells adhering to the surface of microcarriers, the microcarriers being in suspension in the liquid medium, and
    - A collection of the liquid medium comprising extracellular vesicles at the outlet of the container.
    The process is advantageously supplemented by the following characteristics, taken individually or in any of their technically possible combinations:
    - the liquid medium is stirred for more than thirty minutes;
    - the agitator is controlled to cause a flow of the liquid medium, the Kolmogorov length of the flow being less than or equal to 75 μm and preferably 50 μm;
    - A separator depletes part of the liquid medium collected at the outlet of the container in extracellular vesicles, and the part of the liquid medium is reintroduced into the container.
    The subject of the invention is also extracellular vesicles capable of being obtained by the process for the production of extracellular vesicles which is the subject of the invention.
    The subject of the invention is also a pharmaceutical composition comprising extracellular vesicles capable of being obtained by the process for the production of extracellular vesicles which is the subject of the invention.
    Advantageously, the pharmaceutical composition comprising extracellular vesicles can be used in regenerative medicine.
    DEFINITIONS
    The term "extracellular vesicle" generally designates a vesicle released endogenously by a producer cell, the diameter of which is between 30 nm and 5000 nm. An extracellular vesicle corresponds in particular to an unexosome and / or a microvesicle and / or a cellular apoptotic body.
    The terms “microcarrier” and “microsupport” designate a spherical matrix allowing the growth of producer cells adherent to its surface or inside and whose maximum size is between 50 pm and 500 pm, and preferably between 100 pm and 300 pm . The microcarriers are generally beads whose density is chosen to be substantially close to that of the liquid culture medium of the producer cells. Thus, a gentle mixture allows the beads to remain in suspension in the liquid culture medium.
    PRESENTATION OF THE FIGURES
    Other characteristics and advantages will also emerge from the description which follows, which is purely illustrative and not limiting, and should be read with reference to the appended figures, among which:
    - Figure 1 schematically illustrates a fluid system for the production of extracellular vesicles;
    - Figure 2 illustrates the number of extracellular vesicles produced by HUVEC cells in a fluid system for different agitations;
    - Figure 3 illustrates the number of extracellular vesicles produced by HUVEC cells in a fluid system for different agitations;
    - Figure 4 illustrates the number of extracellular vesicles produced by MSC cells in a fluid system for different agitations;
    - Figure 5 illustrates the influence of Kolmogorov length on the number of extracellular vesicles produced by HUVEC and MSC cells;
    - Figure 6 illustrates producer cells adhering to the surface of microcarriers;
    - Figure 7 illustrates producer cells adhering to the surface of microcarriers;
    - Figure 8 illustrates the yield of the production of extracellular vesicles for different durations of agitation, for different producer cells and for different conditions of agitation;
    - Figure 9 illustrates the yield of the production of extracellular vesicles for different producer cells, and for different shaking conditions, after 240 minutes of shaking in comparison with the method of deprivation of serum for 72 h;
    - Figure 10 illustrates the concentration of adherent producer cells on the microcarriers before and after shaking, for different shaking conditions;
    - Figure 11 illustrates the metabolism of HUVEC-type producer cells under shaking conditions for the production of extracellular vesicles;
    - Figure 12 illustrates the metabolism of producer cells of the HUVEC type under shaking conditions for the production of extracellular vesicles;
    - Figure 13 illustrates the metabolism of murine MSC-type producer cells under shaking conditions for the production of extracellular EV vesicles;
    - Figure 14 illustrates the metabolism of murine MSC-type producer cells under shaking conditions for the production of extracellular vesicles;
    - Figure 15 is a photomicrograph in cryo-electron microscopy of extracellular vesicles produced by a fluid system;
    - Figure 16 illustrates the distribution of the diameter of extracellular vesicles produced by the fluidic system;
    FIG. 17 illustrates the purity in extracellular vesicles given by the ratio between the number of particles and the mass of proteins produced by the fluidic system in the liquid medium in comparison with the method of deprivation of serum for 72 h;
    - Figure 18 illustrates the pro-angiogenic properties of a liquid medium comprising extracellular vesicles produced by the fluid system;
    - Figure 19 illustrates the pro-angiogenic properties of a liquid medium comprising extracellular vesicles produced by the fluid system, the serum deprivation method or the spontaneous vesicle release method;
    - Figure 20 illustrates the metabolic activity of cardiomyocytes (line H9C2) after a day of incubation in culture media comprising extracellular vesicles produced by the fluid system;
    - Figure 21 illustrates the metabolic activity of cardiomyocytes (line H9C2) after two days of incubation in different culture media comprising extracellular vesicles produced by the fluidic system;
    - Figure 22 illustrates the dose effect of an incubation of a liquid culture medium comprising a variable concentration of extracellular vesicles produced by a fluid system on the proliferation of cardiomyocytes;
    - Figure 23 illustrates the proliferation of cardiomyocytes (line H9C2) after two days of incubation in the presence of a liquid culture medium comprising extracellular vesicles;
    FIG. 24 illustrates the use of a composition of extracellular vesicles produced by the fluid system as a pharmaceutical composition in a poloxamer gel for the treatment of fistulas between the can and the cecum in rats;
    FIG. 25 illustrates the use of a composition of extracellular vesicles produced by the fluidic system as a pharmaceutical composition in a poloxamer gel for the treatment of fistulas between the can and the cecum in rats.
    DETAILED DESCRITPION
    Theoretical elements
    The Kolmogorov length (or Kolmogorov dimension or Adeddy length) is the length from which the viscosity of a fluid dissipates the kinetic energy of a flow of this fluid. In practice, the Kolmogorov length corresponds to the size of the smallest vortices in a turbulent flow. This length Lk is calculated in the publication by Kolmogorov (Kolmogorov, AN, 1941, January, The local structure of turbulence in incompressible viscous fluid for very large Reynolds numbers, In Dokl. Akad. Nauk, SSSR, Vol. 30, No. 4 , pp. 301-305) and described by the following formula (1):
    V = 3/4. ε -1 / 4 (1) in which v is the kinematic viscosity of the flowing liquid medium and ε is the energy dissipated in the fluid per unit mass (or energy injection rate in the fluid).
    Zhou et al. (Zhou, G., Kresta, SM, 1996, Impact of tank geometry on the maximum turbulence energy dissipation rate for impellers, AlChE journal, 42 (9), 2476-2490) describe the relationship between ε and the geometry of a container in which a liquid medium is agitated by impeller of the impeller type. This relation is given by the following formula (2):
    N „.D S .N 3 (2) in which N p is the dimensionless power number (or Newton number) of the agitator in the liquid medium, D is the diameter of the agitator (in meters), N is the speed of rotation (in number of revolutions per second) and V is the volume of liquid medium (in cubic meters). This relation is used for the calculation of ε corresponding to the geometry of a container and an agitator used for the implementation of the invention. The number of powers N p is given in a known manner by formula (3):
    where P is the power supplied by the agitator, and p is the density of the liquid medium. Formula (3) can be adjusted as described in Nienow et al. (Nienow, A. W., & Miles, D., 1971, Impeller power numbers in closed vessels, Industrial & Engineering Chemistry Process Design and Development, 10 (1), 41-43) or Zhou et al. (Zhou, G., Kresta, SM, 1996, Impact of tank geometry on the maximum turbulence energy dissipation rate for impellers, AlChE journal, 42 (9), 2476-2490) as a function of the Reynolds number of the medium flow liquid. It is also possible to calculate the Reynolds number of the system by the following formula (4):
    General architecture of the fluidic system
    Figure 1 schematically illustrates a fluidic system 1 for the production of EV extracellular vesicles. The fluidic system 1 for producing EV extracellular vesicles aims at producing a large quantity of extracellular EV vesicles in a container 4. However, the invention is not limited to this embodiment and may comprise a series of connected containers 4 fluidly in parallel or in series.
    The container 4 contains a liquid medium 5. The container 4 can be a tank, a flange, for example made of glass or plastic, or any other container adapted to contain a liquid medium 5. The volume of the container 4 is one factors making it possible to produce EV extracellular vesicles in large quantities: this volume can be between 50 mL and 500 L, preferably between 100 mL and 100 L, and preferably between 500 mL and 10 L. The volume of the container 4 illustrated diagrammatically in Figure 1 is 1 L. The container 4 typically includes a gas inlet and a gas outlet, through which an atmosphere can flow comprising O2 and CO2 concentrations suitable for cell culture, for example comprising 5% CO2 . This atmosphere can come from a suitable gas injector / mixer or from an oven with controlled CO2 atmosphere. A second pump 17 makes it possible to control this gas flow in the container 4. The container 4 also includes an outlet 9 capable of comprising liquid medium 5 and extracellular vesicles EV. This outlet is completed by a means of separation and / or filtration of the microcarriers 3 making it possible not to recover microcarriers 3 outside the container 4. This outlet 9 makes it possible to extract from the container 4 the EV extracellular vesicles produced. The container 4 can also include at least one inlet 8 adapted to introduce the liquid medium 5 into the container
    4.
    The liquid medium 5 can generally be a saline, for example isotonic solution. Preferably, the liquid medium 5 is a liquid culture medium with or addition of compounds allowing the culture of the cells of interest, or a medium supplemented with serum previously purified from the extracellular vesicles or a medium without serum, making it possible not to contaminate the vesicles extracellular EV produced by the fluidic system 1 by proteins or other vesicles from a serum. A liquid medium 5 of DMEM type without serum can be used. The maximum volume of liquid medium 5 is determined in part by the container 4. This maximum volume can also be between 50 mL and 500 L, preferably between 100 mL and 100 L, and more preferably between 500 mL and 10 L. The volume minimum of liquid medium 5 contained in container 4 is partly determined by the choice of agitator 7 making it possible to agitate liquid medium 5.
    The fluidic system 1 also comprises microcarriers 3 suspended in the liquid medium 5. The microcarriers 3 can be microbeads 14, for example made of Dextran, each microbead 14 can be covered with a layer of collagen or other material necessary for the culture cells. Other materials can be used for the manufacture of microcarriers 3, such as glass, polystyrene, polyacrylamide, collagen and / or alginate. In general, all of the microcarriers adapted for cell culture are suitable for the production of extracellular EV vesicles. The density of the microcarriers 3 may for example be slightly greater than that of the liquid medium 5. The density of the microbeads 14 made of Dextran is for example 1.04. This density allows the microbeads 14 to be suspended in the liquid medium 5 by slightly agitating the liquid medium 5, the drag of each microcarrier 3 in the liquid medium 5 being dependent on the density of the microcarrier 3. The maximum size of the microcarriers 3 can be between 50 pm and 500 pm, preferably between 100 pm and 300 pm, and preferably between 130 pm and 210 pm.
    The microcarriers 3 can for example be microbeads 14 of the Cytodex 1 type (registered trademark). A powder formed by these microbeads 14 can be rehydrated and sterilized before use. 5 g of PBS can be used, then in a culture medium (for example DMEM) without serum, at 4 ° C. before use.
    The fluid system 1 also includes producer cells 6. The extracellular EV vesicles are produced by the fluid system 1 from these producer cells 6. The producer cells 6 can be cultured, before the production of extracellular EV vesicles by the fluid system 1 , on the surface of the microcarriers 3 in a suitable cell culture medium. Thus, no transfer of cells is necessary between the culture of the producer cells 6 and the production of the extracellular EV vesicles, which makes it possible to avoid any contamination and to simplify the whole process. The majority of the producer cells 6 are adherent to the surface of the microcarriers 3, even if a minority proportion of producer cells 6 can be detached, for example by agitation of the liquid medium 5. The other producer cells are then and suspended in the liquid medium 5 or sedimented at the bottom of the container 4. In general, any type of producer cells 6 can be used, including non-adherent producer cells, and preferably adherent producer cells 6. The producer cells 6 can be, for example, multipotent cells or induced pluripotent stem cells (IPS or IPSCs, Induced Pluripotent Stem Cells in English). They can also be genetically modified cells and / or tumor lines.
    The container 4 also comprises an agitator 7 making it possible to agitate the liquid medium 5. The agitator 7 can be an impeller, the blades of which are at least partly immersed in the liquid medium 5, and set in motion by a transmission of magnetic forces. The agitator 7 can also be a liquid medium infusion system 5 at a rate sufficient to agitate the liquid medium 5 contained by the container, or a system with rotating walls (for example arranged on rollers). The agitator and the dimensions of the container 4 are suitable for controlling a turbulent flow of the liquid medium 5 in the container 4. By turbulent flow is meant a flow whose Reynolds number is greater than 2000. The Reynolds number can for example be calculated by formula (4). Preferably, the Reynolds Re number of the flow of liquid medium 5 is greater than 7,000, preferentially 10,000 and preferentially 12,000.
    The agitator 7 used in the exemplary embodiments of the invention comprises a paddle wheel arranged in a container 4 and set in motion by a magnetic force transmission system. The speed of the impeller in the liquid medium 5 causes the liquid medium 5 to flow. The agitator is adapted to control a flow which, taking into account the dimensions of the container 4, is turbulent. In the case of the agitator 7 illustrated in FIG. 1, several parameters make it possible to calculate a value representative of the turbulence of the liquid medium 5, in particular the kinematic viscosity v of the liquid medium 5, the dimensions of the container 4 and in particular the volume V of liquid medium 5 contained in the container 4, the power number N p corresponding to the submerged part of the paddle wheel, the diameter D of the agitator and in particular of the wheel, the speed N of rotation of the wheel. The user can thus calculate, as a function of these parameters, values representative of the turbulence of the flow, and in particular the length of Kolmogorov Lk, as given by equations (1), (2) and (3) . In particular, the agitator 7 is suitable for controlling a flow in which the length Lk is less than or equal to 75 μm and preferably to 50 μm.
    In an exemplary embodiment of the fluidic system 1, the speed of rotation of the agitator 7 can be controlled at 100 rpm (rotations per minute), the diameter of a paddle wheel is 10.8 cm and the volume of liquid medium contained in container 4 is 400 mL. The number of power Np measured from the paddle wheel in the liquid medium 5, by the formula (3), is substantially equal to 3.2. The energy dissipated per unit of mass ε, calculated, by formula (2), is equal to 5.44.10 -1 J.kg -1 . The length of Kolmogorov Lk calculated by formula (1) is thus equal to 41, pm.
    Preparation of microcarriers and producer cells
    The container 4 can be for single use or sterilized before any introduction of liquid medium 5, microcarriers 3 and producer cells 6. The microcarriers 3, in this case microbeads 14, are also sterilized. The microbeads 14 are incubated in the culture medium of the producing cells 6, comprising serum, in the container 4. This incubation makes it possible to oxygenate the culture medium and to cover the surface of the microbeads 14 with a layer, at least partial , of proteins, promoting the adhesion of the producing cells 6 to the surface of the microbeads 14.
    The producer cells 6, before being introduced into the fluidic system 1, are suspended using a medium comprising trypsin. They can then be centrifuged at 300 G for five minutes to be concentrated in the pellet of a tube, so as to replace the medium comprising trypsin with a DMEM medium. The producer cells 6 are then introduced into the container 5, comprising culture medium and the microbeads 14, in an amount corresponding substantially to 5 to 20 producer cells 6 per microbead 14. The producer cells 6 and the microbeads 14 are then agitated and then sedimented, so as to bring the microbeads 14 into contact with the producer cells 6, and promote the adhesion of the producer cells 6 to the surface of the microbeads 14. Agitation can resume periodically, so as to promote the homogeneity of the adhesion of the producer cells 6 to the surface of the microbeads 14, for example every 45 minutes for 5 to 24 hours. The culture of the producer cells is then carried out with gentle agitation of the culture medium (for example the rotation of a paddle wheel at a speed of 20 rpm), as well as a regular replacement of the culture medium (for example a replacement from 5% to 40% of the culture medium each day).
    Example of production of EV extracellular vesicles
    The extracellular EV vesicles are produced in a container 4 containing a liquid medium 5, for example without serum, microcarriers 3 and producer cells 6 adhering to the surface of the microcarriers
    3. The medium used before production for the culture of the producer cells 6 on the microcarriers 3 comprising serum, the container 4 is washed three to four times with liquid medium 5 DMEM without serum, each washing corresponding for example to a volume d '' about 400 mL. The agitation of the liquid medium 5 is then controlled by the agitator 7 so as to cause a turbulent flow in the container 4. The agitation is preferably adjusted so as to control a flow of the liquid medium 5 in which the length of Kolmogorov Lk is less than or equal to 75 pm and preferably 50 pm. The stirring of the liquid medium 5 is controlled at least for half an hour, preferably for more than an hour, and preferably for more than two hours. EV extracellular vesicle production can be measured during production. For this purpose, the agitation can be temporarily interrupted. The microbeads 14 are allowed to sediment at the bottom of the container 4, then a sample of liquid medium 5 is taken comprising extracellular EV vesicles. The sample is centrifuged at 2000 G for 10 minutes, in order to remove cellular debris. The supernatant is analyzed by an individual particle tracking method (or NTA, acronym for Nanoparticle Tracking Analysis) so as to count the number of extracellular EV vesicles and to deduce the concentration of extracellular EV vesicles from the samples. We can verify that the concentration of extracellular EV vesicles at the start of agitation is close to zero or negligible.
    The EV extracellular vesicles produced can also be observed and / or counted by transmission electron microscopy. For this purpose, a drop of 2.7 μL of solution comprising EV extracellular vesicles is placed on a grid suitable for cryomicroscopy, then immersed in liquid ethane, causing said drop to be almost instantaneous, preventing the formation of ice crystals. The grid supporting the EV extracellular vesicles is introduced into the microscope and the extracellular EV vesicles are observed at a temperature of the order of -170 ° C.
    Separation of extracellular vesicles
    The extracellular EV vesicles produced in the container 4 can be extracted from the container 4 by the outlet 9 of the container 4, suspended in liquid medium 5. A filter 18 can be arranged at the outlet 9 so as to filter the microcarriers 3 and the producer cells 6 adhering to the microcarriers 3 during the extraction of extracellular EV vesicles from the container 4. A connector 13 is fluidly connected to the outlet 9, allowing the transport of the liquid medium 5 comprising the extracellular EV vesicles produced.
    The fluidic system 1 can comprise a separator 15 of extracellular vesicles EV. The separator 15 comprises an inlet to the separator 10, into which the liquid medium 5 comprising extracellular vesicles EV from the container 4 can be conveyed directly or indirectly. The separator 15 can also include a first outlet 11 from the separator, by which the liquid medium 5 is capable of leaving the separator 15 with a concentration of extracellular vesicles EV smaller than at the inlet 10 of the separator 15, or even substantially zero. The separator 15 may also include a second outlet 12 from the separator 15, through which the liquid medium 5 is capable of leaving the separator 15 with a higher concentration of extracellular vesicles EV than at the inlet 10 of the separator 15.
    In general, the separator 15 of extracellular vesicles EV can be fluidly connected to the container 4 so as to be capable of reintroducing a liquid medium 5 depleted in vesicles EV in the container 4, for example by the inlet 8 of the container 4. Thus , the production and / or the extraction of extracellular EV vesicles can be carried out continuously, with a volume of liquid medium 5 substantially constant in the container 4.
    In the embodiment of a fluid system 1 illustrated in FIG. 1, the liquid medium 5 can be extracted from the container 4 by a first pump 16, via a connector 13, so as to transport the liquid medium 5 in a collector 19. Another first pump 16 makes it possible to route the liquid medium 5 contained in the collector 19 to the inlet 10 of the separator 15, via another connector. The first outlet 11 of the separator 15 is connected to the container 4 via a connector, so as to reintroduce liquid medium 5 depleted in extracellular vesicles EV in the container 4. The second outlet 12 of the separator 15 is connected to the collector 19 via a connector, so as to enrich the liquid medium 5 contained in the collector 19 with extracellular vesicles EV. Alternatively, the inlet 10 of the separator 15 can be directly connected to the outlet 9 of the container 4 (or via a first pump 16). The first outlet 11 of the separator 15 is connected to the container 4 and the second outlet 12 of the separator 15 is connected to the collector 19. Several separators can also be arranged in series to vary the degree of separation into extracellular vesicles EV in the liquid medium 5 , and / or in parallel to adapt the flow of liquid medium 5 in each separator 15 to the flow of a first pump 16.
    Influence of agitation on the production of EV extracellular vesicles
    FIG. 2 illustrates the number of extracellular EV vesicles produced in a fluidic system 1 for different agitations controlled by the agitator 7. The ordinate on the left corresponds to the numbers of extracellular EV vesicles produced in the container 4. Each column corresponds to a production of EV extracellular vesicles for different speeds of rotation of the agitator 7 in the container 4. The ordinate on the right corresponds to the length Lk entrained by the agitator 7 during the production of extracellular EV vesicles, calculated by the formulas (1) , (2) and (3). The extracellular EV vesicles are produced from producer cells 6 of the HUVEC type in the container 4 using a concentration of 3 gL -1 of microcarriers 3 in 50 ml of liquid medium 5 in a flask with stirring (spinner flask in English). 100 ml. Significantly high production of EV extracellular vesicles is observable by controlling a flow of liquid medium 5 in which the length Lk is equal to 35 μm (production corresponding to the column 300 RPM) relative to the production of EV extracellular vesicles under conditions of weaker stirring in which the length Lk is equal to 75 μm and preferably to 50 μm (production corresponding to the column 150 RPM).
    FIG. 3 illustrates the number of extracellular EV vesicles produced in a fluidic system 1 for different agitations controlled by the agitator 7. Twenty million producer cells 6 of the HUVEC type are used, using a concentration of 3 g. L ' 1 of microcarriers 3 in 350 ml of liquid medium 5 in a 1000 ml flask spinner. The ordinate on the left corresponds to the numbers of extracellular EV vesicles produced in the container 4. Each column corresponds to a production of extracellular EV vesicles for different rotational speeds of the agitator 7 in the container 4. The ordinate on the right corresponds to the length Lk entrained during the production of extracellular EV vesicles, calculated by the formulas (1), (2) and (3). Significantly high production of EV extracellular vesicles is observable by controlling a flow of liquid medium 5 in which the length Lk is less than 40 μm compared to the production of extracellular EV vesicles under lower stirring conditions (production corresponding to the columns 125 RPM, 150 RPM and 175 RPM).
    FIG. 4 illustrates the number of extracellular EV vesicles produced in a fluidic system 1 for different agitations controlled by the agitator 7. Producing cells 6 of MSC type (acronym of mesenchymal stem cell in English) are used, and microcarriers 3 are introduced at a concentration of 3 gL ' 1 in 200 ml of liquid medium 5 in a 500 ml flask spinner. The ordinate on the left corresponds to the numbers of extracellular EV vesicles produced in the container 4. Each column corresponds to a production of extracellular EV vesicles for different rotational speeds of the agitator 7 in the container 4. The ordinate on the right corresponds to the length Lk entrained during the production of extracellular EV vesicles, calculated by the formulas (1), (2) and (3). Significantly high production of EV extracellular vesicles is observable by controlling a flow of liquid medium in which the length Lk is equal to 35 μm (production corresponding to column 175 RPM), compared to the production of extracellular EV vesicles under conditions lower agitation in which the length Lk is equal to 50 μm.
    Figure 5 illustrates the influence of Kolmogorov Lk length on the number of EV extracellular vesicles produced. The length Lk is a scale parameter concerning the production of the extracellular EV vesicles. The squares (a) correspond to the productions of extracellular EV vesicles illustrated in FIG. 2 for different lengths Lk, the diamonds (b) correspond to the productions of extracellular EV vesicles illustrated in FIG. 3 for different lengths Lk and the triangles (c) correspond to the production of extracellular EV vesicles illustrated in FIG. 4 for different lengths Lk. A characteristic value of Lk characterizing the change in slope of the production of EV extracellular vesicles as a function of Lk can be extracted from the diagram of FIG. 5, substantially equal to Lk = 50 μm. Thus, for values of Lk less than or equal to 50 μm, the production of extracellular EV vesicles increases significantly.
    FIG. 6 illustrates producer cells 6 adhering to the surface of microcarriers 3, in this case microbeads 14, suspended in the liquid medium 5, before the agitation corresponding to the production of extracellular EV vesicles. Producing cells 6 adhering to the surface of the microcarriers 3 are visible and quantifiable.
    FIG. 7 illustrates producer cells 6 adhering to the surface of microcarriers 3, in this case microbeads 14, suspended in the liquid medium 5, after the agitation corresponding to a production of EV extracellular vesicles. Producing cells 6 adhering to the surface of the microcarriers 3 are visible and quantifiable. The comparison between the number of producer cells adhering to the surface of the microcarriers 3 before and after the agitation for the production of extracellular EV vesicles makes it possible to verify that the agitation conditions described above, for example an agitation causing a flow in which the length Lk is less than 75 μm and preferably 50 μm, do not cause the detachment of the producer cells 6 from the microcarriers 3.
    FIG. 8 illustrates the yield of the production of EV extracellular vesicles for different durations of agitation, for different producer cells, and for different agitation conditions, corresponding to different lengths Lk of the flow controlled by the agitator 7. The curve (a) illustrates the evolution, during agitation, of the ratio between the number of particles produced (including extracellular EV vesicles) and between the number of producer cells 6 introduced into the container 4, the producer cells 6 being murine MSC type, and the flow of the liquid medium 5 being characterized by a length Lk substantially equal to 35 pm. Curve (b) illustrates the same evolution, the producing cells 6 being of human MSC type, and the flow of the liquid medium 5 being characterized by a length Lk substantially equal to 33 μm. Curve (c) illustrates the same evolution, the producing cells 6 being of the HUVEC type, and the flow of the liquid medium 5 being characterized by a length Lk substantially equal to 35 μm. Curve (d) illustrates the same evolution, the producing cells 6 being of the human MSC type, and the flow of the liquid medium 5 being characterized by a length Lk substantially equal to 35 μm. Curve (e) illustrates the same evolution, the producer cells being of the HUVEC type, and the flow of the liquid medium 5 being characterized by a length Lk substantially equal to 50 μm. Curve (f) illustrates the same evolution, the producer cells being of the murine MSC type, and the flow of the liquid medium 5 being characterized by a length Lk substantially equal to 50 μm. Curve (g) illustrates the same evolution, the producing cells 6 being of the human MSC type, and the flow of the liquid medium 5 being characterized by a length Lk substantially equal to 50 μm. Curve (h) illustrates the same evolution, the producing cells being of the murine MSC type, and the flow of the liquid medium 5 being characterized by a length Lk substantially equal to 53 μm. Thus, EV extracellular vesicles are produced at higher yields than those known, for a duration of agitation greater than half an hour.
    FIG. 9 illustrates the yield of the production of EV extracellular vesicles for different producer cells 6, and for different shaking conditions, after 240 minutes of shaking. The four columns on the left illustrate the yield from the production of EV extracellular vesicles using murine MSC-type producing cells 6. The production yield for three stirring conditions, corresponding to stirring resulting in a length Lk of 50 μm (first column), 47 μm (second column) and 35 μm (third column) is compared to the production yield according to the method of serum deficiency (or starvation method or deprivation serum in English). The production yield of extracellular EV vesicles under conditions Lk = 47 pm and Lk = 35 pm is significantly higher than under conditions Lk = 50 pm and serum deficiency. Three columns illustrate the yield of the production of EV extracellular vesicles using producer cells 6 of the HUVEC type. The production yield for two stirring conditions, corresponding to stirring resulting in a length Lk of 50 pm (fourth column) and 47 pm (fifth column) is compared to the production yield according to the serum deficiency method. The production yield of EV extracellular vesicles under the Lk = 35 µm condition is higher than under the Lk = 50 µm and serum deficiency conditions. The four rightmost columns of the figure illustrate the yield of the production of EV extracellular vesicles using human MSC-type producing cells 6. The production yield for three stirring conditions, corresponding to stirring resulting in a length Lk of 50 μm (eighth column), 35 μm (ninth column) and 33 μm (tenth column) is compared to the production yield according to the method of serum deficiency. The production yield of EV extracellular vesicles under conditions Lk = 35 pm and Lk = 33 pm is significantly higher than under conditions Lk = 50 pm and serum deficiency.
    FIG. 10 illustrates the concentration of producer cells 6 adhering to the microcarriers 3 before and after shaking, for different shaking conditions. Columns (a) correspond to the concentration of producer cells 6 before agitation (JO) for the production of extracellular EV vesicles and one day after agitation (J1), under so-called “strong” agitation conditions, ie that is to say when the agitator 7 is controlled to cause a flow characterized by a length Lk = 35 μm. Columns (b) correspond to the concentration of producer cells 6 before agitation (JO) for the production of extracellular EV vesicles and one day after agitation (J1), under so-called “weak” agitation conditions, c that is to say when the agitator 7 is controlled to cause a flow characterized by a length Lk = 50 μm. No significant decrease in the concentration of producer cells 6 is observable after the agitation of the liquid medium 5 for the production of EV extracellular vesicles.
    FIG. 11 illustrates the metabolic activity of producer cells 6 of the HUVEC type under agitation conditions for the production of extracellular EV vesicles. The stirring of the liquid medium 5 is controlled by an agitator 7 rotating at 75 RPM, in a 1 L spinner flask, causing a flow characterized by a length Lk substantially equal to 50 pm, for 240 minutes. The metabolism is measured by observing the variation in the wavelength emitted by the reagent Alamar blue in the liquid medium 5. No significant drop in the metabolism of the producing cells 6 is observable under these conditions of agitation.
    FIG. 12 illustrates the metabolism of producer cells 6 of the HUVEC type under agitation conditions for the production of extracellular EV vesicles. The stirring of the liquid medium 5 is controlled by an agitator 7 rotating at 125 RPM in a 1 L spinner flask, causing a flow characterized by a length Lk substantially equal to 35 pm for 240 minutes. The metabolism is measured by observing the variation in the wavelength emitted by the reagent Alamar blue in the liquid medium
    5. A drop or even disappearance of the metabolism of the producing cells is observable after 250 minutes of agitation. This decrease in cell metabolism does not, however, prevent the production of EV extracellular vesicles during agitation.
    FIG. 13 illustrates the metabolism of producer cells 6 of the MSC type under agitation conditions for the production of extracellular EV vesicles. The stirring of the liquid medium 5 is controlled by a stirrer 7 rotating at 75 RPM in a 1 L spinner flask, causing a flow characterized by a length Lk substantially equal to 50 pm, for 240 minutes. The metabolism is measured by observing the variation in wavelength emitted by the reagent Alamar blue in the liquid medium 5. No significant drop in the metabolism of the producing cells 6 is observable under these conditions of agitation.
    FIG. 14 illustrates the metabolism of producer cells 6 of the MSC type under agitation conditions for the production of EV extracellular vesicles. The stirring of the liquid medium 5 is controlled by an agitator 7 rotating at 125 RPM in a 1 L spinner flask, causing a flow characterized by a length Lk substantially equal to 35 pm for 240 minutes. The metabolism is measured by observing the variation in wavelength emitted by the reagent Alamar blue in the liquid medium 5. No significant drop in the metabolism of the producing cells 6 is observable under these conditions of agitation.
    Thus, the conditions of weak agitation, corresponding to agitation resulting in a flow characterized by a length Lk substantially equal to 50 μm, make it possible to reuse the producer cells 6 for production of subsequent EV extracellular vesicles.
    Figure 15 is a photomicrograph of EV extracellular vesicles produced by murine MSC cells by a fluidic system 1. The scale bar corresponds to a length of 200 nm. Microphotography is performed using the transmission electron cryomicroscopy (cryo-TEM) technique.
    FIG. 16 illustrates the distribution of the diameter of the extracellular EV vesicles produced by the fluidic system 1 measured by cryo TEM. Distribution (a) corresponds to EV extracellular vesicles produced by murine MSC cells with agitation resulting in a flow characterized by a length Lk substantially equal to 35 μm (condition of strong agitation). Distribution (b) corresponds to EV extracellular vesicles produced with agitation causing a flow characterized by a length Lk substantially equal to 50 μm (condition of weak agitation). The median diameter of the EV extracellular vesicles produced under conditions of weak agitation is greater than the median diameter of the extracellular EV vesicles produced under conditions of strong agitation. The size of the EV extracellular vesicles can be substantially between 30 and 500 nm.
    FIG. 17 illustrates the purity of the EV extracellular vesicles produced by the fluidic system 1 in the liquid medium 5 indicated by the ratio between the number of particles and the mass of proteins in micrograms. During the production of EV extracellular vesicles, different entities can be produced by producer cells 6, in this case EV extracellular vesicles but also protein aggregates. The quantification of particles by analysis of individual particle monitoring (or NTA for Nanoparticle Tracking Analysis) does not allow to differentiate these different entities: also, it is advantageous to quantify the relationship between the number of particles measured by NTA and the mass of proteins produced, defining purity in EV extracellular vesicles. Columns (a) illustrated in FIG. 17 correspond to a production of EV extracellular vesicles from producer cells 6 of the type
    Murine MSC, and columns (b) correspond to a production of EV extracellular vesicles from producer cells 6 of human MSC type. The two columns on the left correspond to a production of EV extracellular vesicles under conditions of strong agitation, and the two columns on the right correspond to a production of extracellular EV vesicles according to the serum deficiency method. The purity in EV extracellular vesicles of the medium obtained after production is comparable in the two methods.
    FIG. 18 illustrates the pro-angiogenic properties of a serum-free liquid medium 5 comprising EV extracellular vesicles produced by murine MSC cells by the fluidic system 1. Panel A in FIG. 18 is a photograph of a surface on which HUVEC type cells are adherent. Cells have been removed from part of the surface (cell-free area in the middle of the photograph). This photograph is taken at the start of an experiment, at time t = 0 h, during which the cells are covered with a liquid medium 5 comprising extracellular EV vesicles produced by the fluidic system 1. Panel B of FIG. 18 is a photograph of the same surface, after 4 hours of incubation in the liquid medium 5 comprising the extracellular EV vesicles. Panel C of FIG. 18 is a photograph of the same surface, after 9 hours of incubation in the liquid medium 5 comprising the extracellular EV vesicles. During the experiment, cells of the HUVEC type cover the part of the surface on which no cell is present at the start of the experiment. Thus, the liquid medium 5 comprising the extracellular EV vesicles has pro-angiogenic and / or pro-proliferative properties.
    FIG. 19 illustrates the pro-angiogenic properties of a liquid medium 5 comprising EV extracellular vesicles produced by murine MSC cells by the fluidic system 1 under different conditions.
    Each column illustrates the normalized percentage of bank closure between 0 a.m. (corresponding to panel A in Figure 18) and 9 a.m. (corresponding to panel C in Figure 18) for each incubation condition. The first column (“complete medium”) corresponds to an incubation in a culture medium for HUVEC cells (corresponding to a positive control). The second column (“ctrl neg”) corresponds to an incubation in a liquid medium 5 (culture medium) without EV extracellular vesicles, without serum where a given volume of PBS has been added. The third column (“strong agitation 10/1”) corresponds to an incubation in a liquid culture medium 5 to which the same given volume of PBS has been added comprising EV extracellular vesicles produced by a fluidic system 1 under conditions of strong agitation , in which the quantity of producer cells 6 introduced corresponds to 10 producer cells 6 murine MSCs for a receptor HUVEC cell. The fourth column (“weak agitation 10/1”) corresponds to an incubation in a culture medium where the same given volume of PBS was added comprising EV extracellular vesicles produced by a fluidic system 1 under conditions of weak agitation. in which the quantity of producer cells 6 introduced corresponds to 10 producer cells 6 murine MSCs for a receptor HUVEC cell The fifth column (“lib 10/1”) corresponds to an incubation in a culture medium for HUVEC cells, which is downgraded to exosomes, the quantity of producer cells 6 introduced corresponding to 10 producer cells 6 for a receptor HUVEC cell. The sixth column (“stress 10/1”) corresponds to an incubation in a liquid culture medium 5 where the same given volume of PBS has been added comprising extracellular EV vesicles produced by serum deficiency, the quantity of producer cells 6 introduced corresponding to 10 producer cells 6 murine MSCs for a receptor HUVEC cell. Incubation in a liquid medium comprising extracellular EV vesicles produced under conditions of strong agitation (“strong agitation 10/1”) makes it possible to significantly cover the part initially devoid of cells.
    FIG. 20 illustrates the proliferation of cardiomyocytes after a day of incubation in different liquid media, measured by alamar blue included in the incubation medium. The first column corresponds to an incubation in a medium suitable for the culture of H9C2 cardiomyocytes (“complete medium”, positive control). The second column corresponds to an incubation in PBS. The third column corresponds to an incubation in a liquid medium 5 comprising EV extracellular vesicles produced by a fluidic system 1 under conditions of strong agitation (“strong agitation 10/1”), the quantity of producer cells 6 introduced corresponding to 10 producer cells 6 for a receptor cell. The fourth column corresponds to an incubation in a liquid medium 5 comprising EV extracellular vesicles produced by a fluidic system 1 under conditions of weak agitation (“weak agitation 10/1”), the quantity of producer cells 6 introduced corresponding to 10 producer cells 6 for a receptor cell. The fifth column corresponds to an incubation in a cardiomyocyte culture medium, depleted in exosomes (“lib 10/1”), the quantity of producer cells 6 introduced corresponding to 10 producer cells 6 for a receptor cell. The sixth column corresponds to an incubation in a liquid medium 5 comprising EV extracellular vesicles produced by a production method in a medium without “stress” serum, the quantity of producer cells 6 introduced corresponding to 10 producer cells 6 for a receptor cell . The conditions of incubation in a liquid medium 5 comprising EV extracellular vesicles produced in a fluidic system 1 under conditions of strong agitation and / or of weak agitation result in a significantly higher proliferation of the cardiomyocytes compared to the conditions of incubation in PBS, and under "exo" and "stress" conditions.
    Figure 21 illustrates the proliferation of H9C2 cardiomyocytes after two days of incubation. Cardiomyocytes incubated in a liquid medium 5 comprising EV extracellular vesicles produced from producer cells 6 of murine MSC type by a fluidic system 1 under conditions of weak agitation, under conditions of strong agitation, as well as by spontaneous release in a complete medium which is screened in exosomes, proliferate significantly more than cardiomyocytes incubated in a medium with a serum deficiency. Cardiomyocytes incubated in a liquid medium comprising EV extracellular vesicles produced under conditions of weak agitation and under conditions of strong agitation proliferate significantly more than cardiomyocytes incubated in a medium having a serum deficiency.
    FIG. 22 illustrates the dose effect on the proliferation of H9C2 cardiomyocytes of an incubation of a liquid medium 5 comprising a variable concentration of extracellular EV vesicles produced by a fluidic system 1. The metabolic activity of cardiomyocytes is measured after two days incubation in a liquid medium by alamar blue. Cardiomyocyte metabolism is measured for three incubation conditions: in a liquid medium comprising extracellular EV vesicles produced under conditions of weak agitation in (a), under conditions of weak agitation in (b) and in a liquid medium with a serum deficiency in (c). The measurement of the cardiomyocyte metabolism is carried out, in curves (a) and (b), for different relationships between the concentration of extracellular EV vesicles and the concentration of cardiomyocytes, displayed on the abscissa. Curve (a) illustrates the dose effect of proliferation in the presence of extracellular EV vesicles: the metabolism of cardiomyocytes increases when the concentration ratio between extracellular EV vesicles and cardiomyocytes increases.
    FIG. 23 illustrates the proliferation of cardiomyocytes after two days of incubation in the presence of a liquid medium 5 comprising extracellular EV vesicles of murine MSC cells at a concentration of 100,000 extracellular EV vesicles per cardiomyocyte. The proliferation of cardiomyocytes after two days is significantly higher when the liquid incubation medium 5 comprises extracellular EV vesicles produced by a fluidic system 1 under conditions of strong agitation or of weak agitation, compared to a liquid medium comprising vesicles extracellular EV obtained by deficiency in serum and / or in a culture medium of cardiomyocytes containing extracellular EV vesicles obtained spontaneous release in a complete medium which is screened with exosomes.
    FIG. 24 illustrates the use of a composition of EV extracellular vesicles produced by the fluidic system 1 as a pharmaceutical composition. A caecostomy is performed on each rat in a group of rats. The presence of excrement is observed at the orifice of a fistula formed by the cecostomy, under three conditions: a control condition, a condition corresponding to treatment by the application of a gel comprising poloxamer 407 on the orifice of the fistula of each mouse (“gel”) and a condition corresponding to the application of this gel comprising extracellular EV vesicles produced by a fluidic system 1 according to a process which is the subject of the invention, for example under conditions of high agitation ("gel + vesicles"). The light gray columns correspond to the fistula openings with excrement and the dark gray columns correspond to the fistula openings without excrement. The application of a gel comprising extracellular EV vesicles makes it possible to significantly reduce the presence of excrement at the orifice of the fistula under these conditions and leads to a reduction in the cases of productive fistulas (which release intestinal secretions) compared to control groups and gel without vesicles. Thus, the composition of EV extracellular vesicles produced by the fluidic system 1 can be used in regenerative medicine.
    FIG. 25 illustrates the use of a composition of EV extracellular vesicles produced by the fluidic system 1 as a pharmaceutical composition. A score is calculated from the observations presented in Figure 24. A score equal to 1 is assigned when the opening of a fistula has feces and a score equal to zero is assigned when the opening of a fistula does not no droppings. FIG. 25 illustrates the average score, for all the caecostomies and for each of the conditions: control, "gel" and "gel + vesicles". The application of a gel comprising extracellular EV vesicles leads to a reduction in the average score of productivity of the fistulas compared to the control and gel groups without vesicles and makes it possible to significantly reduce the presence of excrement at the orifice of the fistula in these conditions. Thus, the composition of EV extracellular vesicles produced by the fluidic system 1 can be used in regenerative medicine.
    权利要求:
    Claims (13)
    [1" id="c-fr-0001]
    1. Fluidic system (1) for producing extracellular vesicles (EV) from producer cells (6), comprising at least one container (4), a liquid medium (5) contained by the container (4) and producer cells (6), characterized in that it also comprises microcarriers (3) suspended in the liquid medium (5), the majority of the producing cells (6) being adherent to the surface of the microcarriers (3), and an agitator ( 7) of liquid medium (5), the agitator (7) and the shape and dimensions of the container (4) being adapted to generate a turbulent flow of the liquid medium (5) in the container (4).
    [2" id="c-fr-0002]
    2. Fluidic system (1) according to claim 1, in which the agitator (7) of the liquid medium (5) and the dimensions of the container (4) are adapted to control a flow of the liquid medium (4), the length of Kolmogorov of the flow being less than or equal to 75 pm.
    [3" id="c-fr-0003]
    3. Fluidic system (1) according to claim 1 or 2, comprising an outlet (9) and a connector (13) connected to the outlet (9), the connector (13) being capable of comprising liquid medium (5) and extracellular vesicles (EV).
    [4" id="c-fr-0004]
    4. Fluidic system (1) according to one of claims 1 to 3, wherein an agitator (7) of liquid medium is a rotary agitator whose rotation speed (s), shape and size are adapted, with the shape and the dimensions of the container (4), generating a turbulent flow of the liquid medium (5) in the container.
    [5" id="c-fr-0005]
    5. Fluidic system (1) according to one of claims 1 to 4 wherein the microcarriers (3) are microbeads (14), the diameter of the microbeads (14) being between 100 µm and 300 µm.
    [6" id="c-fr-0006]
    6. Fluidic system (1) according to one of claims 1 to 5 comprising a separator (15) of extracellular vesicles (EV), fluidly connected to the container (4) so as to be capable of reintroducing into the container (4) a liquid medium (5) depleted in extracellular vesicles (EV).
    [7" id="c-fr-0007]
    7. Method for the ex vivo production of extracellular vesicles (EV) from producer cells (6), comprising:
    • a control of an agitator (7) causing a turbulent flow of a liquid medium (5) in a container (4), the container comprising an outlet (9), the liquid medium (5) comprising producer cells (6 ) adhering to the surface of microcarriers (3), the microcarriers (3) being suspended in the liquid medium (5), and • a collection of the liquid medium (5) comprising extracellular vesicles (EV) at the outlet (9) of the container (4).
    [8" id="c-fr-0008]
    8. The method of claim 7 wherein the liquid medium (5) is stirred for more than thirty minutes.
    [9" id="c-fr-0009]
    9. Method according to claim 7 or 8, in which the agitator (7) is controlled to cause a flow of the liquid medium (5), the Kolmogorov length of the flow being less than or equal to 75 μm, preferably less than or equal to 75 pm.
    [10" id="c-fr-0010]
    10. Method according to one of claims 7 to 9 wherein a separator depletes part of the liquid medium (5) collected at the outlet of the container (4) in extracellular vesicle (EV), and in which the part of the liquid medium is reintroduced (5) in the container (4).
    [11" id="c-fr-0011]
    11. Extracellular vesicles (EV) capable of being obtained by the method according to one of claims 7 to 10.
    5
    [12" id="c-fr-0012]
    12. Pharmaceutical composition comprising extracellular vesicles (EV) according to claim 11.
    [13" id="c-fr-0013]
    13. Pharmaceutical composition according to claim 12, for its use in regenerative medicine.
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    同族专利:
    公开号 | 公开日
    US20200385665A1|2020-12-10|
    KR20200034727A|2020-03-31|
    WO2019002608A1|2019-01-03|
    CA3068614A1|2019-01-03|
    EP3645700A1|2020-05-06|
    CN111108186A|2020-05-05|
    AU2018294559A1|2020-02-13|
    FR3068361B1|2021-10-15|
    JP2020528763A|2020-10-01|
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    FR1756183A|FR3068361B1|2017-06-30|2017-06-30|FLUIDIC SYSTEM FOR THE PRODUCTION OF EXTRACELLULAR VESICLES AND ASSOCIATED PROCESS|
    FR1756183|2017-06-30|FR1756183A| FR3068361B1|2017-06-30|2017-06-30|FLUIDIC SYSTEM FOR THE PRODUCTION OF EXTRACELLULAR VESICLES AND ASSOCIATED PROCESS|
    PCT/EP2018/067704| WO2019002608A1|2017-06-30|2018-06-29|Fluid system for producing extracellular vesicles and associated method|
    US16/627,013| US20200385665A1|2017-06-30|2018-06-29|Fluid system for producing extracellular vesicles and associated method|
    CA3068614A| CA3068614A1|2017-06-30|2018-06-29|Fluid system for producing extracellular vesicles and associated method|
    CN201880053335.6A| CN111108186A|2017-06-30|2018-06-29|Fluidic systems for preparing extracellular vesicles and related methods|
    JP2020522409A| JP2020528763A|2017-06-30|2018-06-29|Fluid system for producing extracellular vesicles and related methods|
    EP18737565.4A| EP3645700A1|2017-06-30|2018-06-29|Fluid system for producing extracellular vesicles and associated method|
    AU2018294559A| AU2018294559A1|2017-06-30|2018-06-29|Fluid system for producing extracellular vesicles and associated method|
    KR1020207002967A| KR20200034727A|2017-06-30|2018-06-29|Fluid systems and related methods for producing extracellular vesicles|
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