Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
  • C12M27/14—Rotation or movement of the cells support, e.g. rotated hollow fibers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
  • C12M25/16—Particles; Beads; Granular material; Encapsulation
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M27/00—Means for mixing, agitating or circulating fluids in the vessel
  • C12M27/02—Stirrer or mobile mixing elements
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
  • C12M29/04—Filters; Permeable or porous membranes or plates, e.g. dialysis
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M45/00—Means for pre-treatment of biological substances
  • C12M45/02—Means for pre-treatment of biological substances by mechanical forces; Stirring; Trituration; Comminuting
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
  • C12M47/10—Separation or concentration of fermentation products

Definitions

• the invention relates to a system for producing extracellular vesicles from producing 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.
• Extracellular vesicles have been identified as effective means for administering drugs, in a personalized or targeted way, in the human body. They first have a native biocompatibility and an immune tolerance. They may also include theranostic nanoparticles, both for imaging certain parts of the body and for delivering active principles having therapeutic functions.
• the extracellular vesicles also have an intercellular communication function: they make it possible, for example, to transport lipids, membrane and cytoplasmic proteins and / or nucleotides of the cellular cytoplasm, such as mRNAs, microRNAs or long non-coding RNAs. , between different cells.
• 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.
• 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 for or in addition to cellular therapies.
• One method is to produce extracellular vesicles from umbilical vein vein endothelial cells (HUVEC) by subjecting these cells to hydrodynamic stresses mimicking stresses exerted under physiological conditions within the blood capillaries or under pathological conditions. when stenosis of blood vessels. These constraints are caused by the passage of the producing cells in microfluidic channels for four hours.
• a microfluidic chip includes two hundred channels in which the cells are transported in a laminar flow to produce vesicles in a parallel fashion.
• Watson et al. (Watson, D. C, Bayik, D., Srivatsan, A., Bergamaschi, C., Valentin, A., Niu, G., ... & Jones, J. C, 2016, Efficient production and enhanced tumor delivery engineered extracellular vesicles, Biomaterials, 105, 195-205) describe a method of producing vesicles to increase the amount of vesicles produced. This method consists in cultivating HEK293 cells in culture flasks, then in hollow fiber membranes (Hollow Fiber Membrane). The central passage of the hollow fibers makes it possible to convey the culture medium to the producer cells.
• Producing cells are first seeded around this passage, where they produce vesicles in an inter-fiber space.
• the liquid medium comprised 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 amounts of seeded cells, for example of the order of 5 ⁇ 10 8 cells, resulting in a yield of approximately 6000 extracellular vesicles per cell and a very low purity ratio (e.g. 1, 09.10 9 particles per microgram of protein).
• this production is not high enough and too slow compared to the applications mentioned above.
• this method is described using production cells corresponding to a cell line that is particularly resistant to culture in serum-free medium: this method may not be transposable to vesicle production by producing cells such as stem cells, for example human, less resistant and particularly suitable for targeted therapeutic applications.
• An object of the invention is to provide a solution for producing extracellular vesicles in large quantities from producing cells, more rapidly than with known methods, under GMP-compliant conditions. Another object of the invention is to propose a solution for increasing the efficiency of the vesicle production system, that is to say the ratio between the number of vesicles produced and the number of production cells introduced into the production system. Another object of the invention is to provide a system adapted to produce extracellular vesicles from a wide range of adherent producer cells, regardless of the resistance of the cell type introduced into the production system and resistant or not to a deficiency in serum. 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 reduce its manufacturing cost.
• an object of the invention is a fluid system for producing extracellular vesicles from producer cells, comprising at least one container, a liquid medium contained by the container and producing 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 form and the dimensions of the container being adapted to the generation of a turbulent flow of the liquid medium in the container.
• the agitator of the liquid medium and the dimensions of the container are adapted to control a flow of the liquid medium, the Kolmogorov length of the flow being less than or equal to 75 ⁇ , and preferably to 50 ⁇ ;
• the fluidic system comprises an output and a connection connected to the output, the connectors being capable of comprising liquid medium and extracellular vesicles;
• the agitator is a rotary stirrer whose rotation speed (s), shape, size are adapted, with the shape and the 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 ⁇ and 300 ⁇ ;
• the fluidic system comprises an extracellular vesicle separator, fluidly connected to the receptacle so as to be able to reintroduce into the receptacle a liquid medium depleted of vesicles.
• Another subject 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
• 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 ⁇ and preferably 50 ⁇ ;
• a separator depletes a portion of the liquid medium collected at the outlet of the container in extracellular vesicles, and the portion of the liquid medium is reintroduced into the container.
• the subject of the invention is also extracellular vesicles that can be obtained by the process for producing extracellular vesicles that are the subject of the invention.
• the subject of the invention is also a pharmaceutical composition comprising extracellular vesicles that can be obtained by the method for producing extracellular vesicles that is the subject of the invention.
• the pharmaceutical composition comprising extracellular vesicles may be used in regenerative medicine.
• extracellular vesicle generally refers to a vesicle released endogenously by a producer cell, whose diameter is between 30 nm and 5000 nm.
• An extracellular vesicle corresponds in particular to an exosome and / or a microvesicle and / or a cell apoptotic body.
• microcarrier and “microcarrier” designate a spherical matrix allowing the growth of producing cells adhering to its surface or inside and whose maximum size is between 50 ⁇ and 500 ⁇ , and preferably between 100 ⁇ and 300 ⁇ irr.
• the microcarriers are generally beads whose density is chosen 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.
• FIG. 1 schematically illustrates a fluid system for the production of extracellular vesicles
• FIG. 2 illustrates the number of extracellular vesicles produced by HUVEC cells in a fluidic system for different agitations
• FIG. 3 illustrates the number of extracellular vesicles produced by HUVEC cells in a fluid system for different agitations
• FIG. 4 illustrates the number of extracellular vesicles produced by MSC cells in a fluidic system for different agitations
• FIG. 5 illustrates the influence of Kolmogorov length on the number of extracellular vesicles produced by HUVEC and MSC cells
• FIG. 6 illustrates adherent producer cells on the surface of microcarriers
• FIG. 7 illustrates adherent producer cells on the surface of microcarriers
• FIG. 8 illustrates the yield of the production of extracellular vesicles for different stirring times, for different production cells and for different stirring conditions
• FIG. 9 illustrates the yield of the production of extracellular vesicles for different producer cells, and for different stirring conditions, after 240 minutes of agitation compared with the serum starvation method for 72 hours;
• FIG. 10 illustrates the concentration of adherent producer cells on the microcarriers before and after stirring, for different stirring conditions
• FIG. 11 illustrates the metabolism of HUVEC producing cells under agitation conditions for the production of extracellular vesicles
• Figure 12 illustrates the metabolism of HUVEC producing cells under agitation conditions for the production of extracellular vesicles
• Figure 13 illustrates the metabolism of murine MSC-producing cells under agitation conditions for the production of extracellular EV vesicles
• Figure 14 illustrates the metabolism of murine MSC-producing cells under agitation conditions for the production of extracellular vesicles
• FIG. 15 is an electron cryo-microscopy microphotograph of extracellular vesicles produced by a fluidic system
• FIG. 16 illustrates the extracellular vesicle diameter distribution produced by the fluidic system
• FIG. 17 illustrates the extracellular vesicle purity given by the ratio between the number of particles and the mass of proteins produced by the fluidic system in the liquid medium as compared with the serum deprivation method for 72 hours;
• FIG. 18 illustrates the pro-angiogenic properties of a liquid medium comprising extracellular vesicles produced by the fluidic system
• FIG. 19 illustrates the pro-angiogenic properties of a liquid medium comprising extracellular vesicles produced by the fluidic system, the serum deprivation method or the spontaneous vesicle release method;
• FIG. 20 illustrates the metabolic activity of cardiomyocytes (H9C2 line) after a day's incubation in culture media comprising extracellular vesicles produced by the fluidic system;
• FIG. 21 illustrates the metabolic activity of cardiomyocytes (H9C2 line) after two days of incubation in various culture media comprising extracellular vesicles produced by the fluidic system;
• FIG. 22 illustrates the dose effect of an incubation of a liquid culture medium comprising a variable concentration of extracellular vesicles produced by a fluidic system on the proliferation of cardiomyocytes;
• FIG. 23 illustrates the proliferation of cardiomyocytes (H9C2 line) after two days of incubation in the presence of a liquid culture medium comprising extracellular vesicles;
• FIG. 24 illustrates the use of an extracellular vesicle composition produced by the fluidic system as a pharmaceutical composition in a poloxamer gel for the treatment of fistulas between the can and the caecum in rats;
• FIG. 25 illustrates the use of an extracellular vesicle composition produced by the fluidic system as a composition in a poloxamer gel for the treatment of fistulas between the can and the caecum in rats.
• FIG. 26 illustrates the proteomic profile of extracellular vesicles produced by the method according to the invention compared with the profile of extracellular vesicles produced by the conventional production methods according to the prior art.
• the length of Kolmogorov (or Kolmogorov dimension or eddy length) is the length from which the viscosity of a fluid allows to dissipate the kinetic energy of a flow of this fluid. In practice, the length of Kolmogorov corresponds to the size of the smallest vortices in a turbulent flow. This length LK is calculated in Kolmogorov's publication (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):
• N p is the unadjusted power number (or number of Newton) of the stirrer in the liquid medium
• D is the diameter of the stirrer (in meters)
• N is the speed of rotation (in revolutions per minute) second)
• V is the volume of liquid medium (in cubic meters).
• the fluidic system 1 for producing extracellular vesicles EV is intended for the large quantity production of extracellular vesicles EV in a container 4.
• the invention is not limited to this embodiment and may comprise a series of connected containers 4. fluidically in parallel or in series.
• the container 4 contains a liquid medium 5.
• the container 4 may be a tank, a flange, for example 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 extracellular EV 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 schematically in FIG. 1 is 1 L.
• the container 4 typically comprises a gas inlet and a gas outlet, through which an atmosphere comprising O 2 and CO 2 concentrations adapted to the cell culture, for example comprising 5% of CO2. This atmosphere can come from a suitable gas injector / mixer or a CO2 controlled atmosphere oven.
• a second pump 17 makes it possible to control this gaseous flow in the receptacle 4.
• the receptacle 4 also comprises an outlet 9 capable of comprising liquid medium 5 and extracellular vesicles EV. This output is supplemented by a means of separation and / or filtration of the microcarriers 3 to not recover the microcarriers 3 outside the container 4. This outlet 9 allows to extract from the container 4 EV extracellular vesicles produced.
• the container 4 may also comprise at least one inlet 8 adapted to introduce the liquid medium 5 into the container 4.
• the liquid medium 5 may be generally a saline solution, for example isotonic.
• the liquid medium 5 is a liquid culture medium with or addition of compounds for culturing the cells of interest, or supplemented medium in serum previously purified extracellular vesicles or serum-free medium, so as not to contaminate the extracellular vesicles EV produced by the fluidic system 1 by proteins or other vesicles from a serum.
• a serum-free DMEM-type liquid medium can be used.
• the maximum volume of liquid medium 5 is determined in part by the container 4. This maximum volume may 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 The minimum amount of liquid medium contained by the container 4 is partly determined by the choice of the stirrer 7 for agitating a liquid medium 5.
• the fluidic system 1 also comprises microcarriers 3 suspended in the liquid medium 5.
• the microcarriers 3 may be microbeads 14, for example Dextran, each microbead 14 may be covered with a layer of collagen or other material necessary for culture of cells.
• Other materials can be used for the manufacture of microcarriers 3, such as glass, polystyrene, polyacrylamide, collagen and / or alginate.
• all of the microcarriers adapted for cell culture are suitable for the production of extracellular EV vesicles.
• the density of the microcarriers 3 may be slightly greater than that of the liquid medium 5.
• the density of the microspheres 14 in Dextran is, for example, 1.04.
• the maximum size of the microcarriers 3 can be between 50 ⁇ and 500 ⁇ , preferably between 100 ⁇ and 300 Mm, and preferably between 130 ⁇ and 210 ⁇ .
• the microcarriers 3 may for example be microbeads 1 of Cytodex type 1 (registered trademark). It is possible to rehydrate and sterilize a powder formed by these microbeads 1 before use. 5 g of PBS can be used, then in serum-free culture media (eg, DMEM) at 4 ° C before use.
• the fluidic system 1 also comprises producing cells 6.
• the extracellular vesicles EV are produced by the fluidic system 1 from these producer cells 6.
• the production cells 6 can be cultured, before the production of extracellular vesicles EV by the fluidic system 1 on the surface of microcarriers 3 in a suitable cell culture medium.
• no cell transfer is necessary between the culture of the producer cells 6 and the production of the extracellular vesicles EV, which makes it possible to avoid any contamination and to simplify the process as a whole.
• the majority of the producing cells 6 are adherent to the surface of the microcarriers 3, even if a minor proportion of producing cells 6 can be detached, for example by stirring the liquid medium 5.
• producing cells 6 are then suspended in the In general, any type of producing cells 6 may be used, including nonadherent producer cells, and preferably adhering producer cells.
• Producing cells 6 may be for example multipotent cells, or induced pluripotent stem cells (IPS or IPSCs, Induced Pluripotent Stem Cells). They may also be genetically modified cells and / or tumor lines.
• the container 4 also comprises an agitator 7 for stirring the liquid medium 5.
• the stirrer 7 can be a paddle wheel, whose blades are at least partially immersed in the liquid medium 5, and put in motion by a transmission of magnetic forces.
• the stirrer 7 may also be a liquid medium infusion system 5 at a rate sufficient to agitate the liquid medium contained by the container, or a system with rotating walls (for example arranged on rollers).
• the agitator 7 and the dimensions of the container 4 are adapted to control a turbulent flow of the liquid medium 5 in the vessel 4.
• turbulent flow is meant a flow whose Reynolds number is greater than 2000.
• the Reynolds number can example be calculated by the formula (4).
• the Reynolds number Re of the flow of liquid medium 5 is greater than 7,000, preferably 10,000 and preferably 12,000.
• the agitator 7 used in the exemplary embodiments of the invention comprises a blade 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 causes a flow of the liquid medium 5.
• the agitator is adapted to control a flow, which, given the dimensions of the container 4, is turbulent. In the case of the stirrer 7 illustrated in FIG.
• the agitator 7 is adapted to control a flow in which the length LK is less than or equal to 75 ⁇ and preferably 50 m.
• the rotational speed of the stirrer 7 can be controlled at 100 rpm (rotations per minute), the diameter of a blade wheel is 10.8 cm and the the volume of liquid medium contained by the container 4 is 400 ml.
• the measured NP power number of the blade wheel in the liquid medium 5, by the formula (3), is substantially equal to 3.2.
• the energy dissipated per unit mass ⁇ , calculated by the formula (2), is equal to 5.44 ⁇ 10 1 J. kg 1 .
• the length of Kolmogorov LK calculated by the formula (1) is thus equal to 41.8 ⁇ .
• the container 4 may be disposable or sterilized before 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 producer cells 6, comprising serum, in the container 4. This incubation allows the culture medium to be oxygenated and to cover the surface of the microbeads 14 with at least a partial layer. 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 producing cells 6 per microbead 14.
• the production cells 6 and the microbeads 14 are then shaken and then sedimented, so as to bring into contact the microbeads 14 and the producer cells 6, and promote the adhesion of the production cells 6 on the surface of the microbeads 14.
• the stirring can be periodically repeated, so as to promote the homogeneity of the adhesion of the producing 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 slight stirring 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).
• Extracellular vesicles EV are produced in a container 4 containing a liquid medium 5, for example without serum, microcarriers 3 and producer cells 6 adherent to the surface of the microcarriers 3.
• the medium used before production for the cultivation of the production cells 6 on the microcarriers 3 comprising serum the container 4 is washed three to four times with serum-free DMEM liquid medium, each washing corresponding for example to a volume of approximately 400 ml.
• the agitation of the liquid medium 5 is then controlled by the stirrer 7 so as to cause a turbulent flow in the vessel 4.
• the stirring 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 ⁇ and preferably 50 ⁇ .
• the agitation of the liquid medium is controlled at least for half an hour, preferably for more than one hour, and preferably for more than two hours.
• Extracellular vesicle production EV can be measured during production.
• the agitation can be momentarily interrupted.
• the microbeads 14 are allowed to settle at the bottom of the container 4, and then a sample of liquid medium 5 comprising extracellular vesicles EV is taken.
• the sample is centrifuged at 2000 G for 10 minutes to remove cell debris.
• the supernatant is analyzed by a monitoring method particle count (or NTA, acronym for Nanoparticie Tracking Analysis) in order to count the number of extracellular vesicles EV and to deduce the extracellular vesicle EV concentration of the samples. It can be verified that the concentration of extracellular vesicles EV at the beginning of the agitation is close to zero or negligible.
• NTA acronym for Nanoparticie Tracking Analysis
• Extracellular EV vesicles produced can also be observed and / or counted by transmission electron cryo-microscopy.
• a drop of 2.7 ⁇ of solution comprising extracellular vesicles EV is deposited on a grid adapted to cryomicroscopy, then dipped in liquid ethane, resulting in an almost instantaneous freezing of said drop, avoiding the formation of ice crystals.
• the grid supporting the extracellular vesicles EV is introduced into the microscope and the extracellular vesicles EV are observed at a temperature of the order of -170 ° C.
• the extracellular vesicles EV 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 adhered to the microcarriers 3 during the extraction of extracellular vesicles EV 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 may comprise an extracellular vesicle separator EV.
• the separator 15 comprises an inlet of the separator 10, wherein the liquid medium comprising extracellular vesicles EV from the container 4 can be conveyed directly or indirectly.
• the separator 15 may also comprise a first outlet 1 1 of the separator, by which the liquid medium 5 is likely to leave the separator 15 with a concentration of EV extracellular vesicles smaller than the inlet 10 of the separator 15, or substantially zero.
• the separator 15 may also comprise a second outlet 12 of the separator 15, through which the liquid medium 5 is able to exit the separator 15 with an extracellular vesicle concentration EV higher than at the inlet 10 of the separator 15.
• the extracellular vesicle separator EV may be fluidly connected to the container 4 so as to be able to reintroduce an EV vesicle-depleted liquid medium into the container 4, for example through the inlet 8 of the container 4.
• the production and / or extraction of extracellular vesicles EV can be carried out continuously, with a substantially constant volume of liquid medium 5 in the container 4.
• 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 allows the liquid medium 5 contained in the manifold 19 to be fed to the inlet 10 of the separator 15 via another connector.
• the first output 1 1 of the separator 15 is connected to the container 4 via a connector, so as to reintroduce liquid medium 5 depleted extracellular vesicles EV in the container 4.
• the second output 12 of the separator 15 is connected to the manifold 19 via a connector , so as to enrich the liquid medium contained in the collector 19 in extracellular vesicles EV.
• the inlet 10 of the separator 15 may be directly connected to the outlet 9 of the container 4 (or via a first pump 16).
• the first outlet 1 1 of the separator 15 is connected to the container 4 and the second outlet 12 of the separator 15 is connected to the manifold 19.
• Several separators can also be arranged in series to vary the degree of separation in 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 rate of a first pump 16.
• FIG. 2 illustrates the number of extracellular vesicles EV produced in a fluidic system 1 for different agitations controlled by the stirrer 7.
• the left ordinate corresponds to the numbers of extracellular vesicles EV 4.
• Each column corresponds to an extracellular vesicle production EV for different rotational speeds of the agitator 7 in the container 4.
• the ordinate on the right corresponds to the length LK driven by the stirrer 7 during the production.
• extracellular vesicle EV calculated by the formulas (1), (2) and (3).
• the extracellular vesicles EV are produced from HUVEC-type producer cells 6 in the vessel 4 using a concentration of 3 ⁇ l -1 of microcarriers 3 in 50 ml of liquid medium in a spinner flask. 100 ml.,
• a significantly high production of extracellular vesicles EV is observable by controlling a flow of liquid medium in which the length LK is equal to 35 ⁇ (production corresponding to column 300 RPM) compared to extracellular vesicle production EV in lower stirring conditions in which the length LK is equal to 75 ⁇ and preferably 50 ⁇ (production corresponding to the column 150 RPM)
• Figure 3 illustrates the number of extracellular vesicles EV produced in a fluidic system 1 for different agitators controlled by the agitator 7.
• Twenty million production cells 6 of the HUVEC type are using a concentration of 3 gL 1 of microcarriers 3 in 350 mL of liquid medium in a 1000 ml spinner flask.
• the left ordinate corresponds to the numbers of extracellular vesicles EV produced in the vessel 4.
• Each column corresponds to production of extracellular vesicles EV for different rotational speeds of the agitator 7 in the container 4.
• the ordinate on the right corresponds to the length entrained during the production of extracellular vesicles EV, calculated by the formulas (1), (2 ) and (3).
• a significantly high production of extracellular vesicles EV is observable by controlling a flow of liquid medium in which the length U is less than 40 ⁇ compared to the extracellular vesicle production EV under lower agitation conditions (production corresponding to the columns 125 RPM, 150 RPM and 175 RPM).
• FIG. 4 illustrates the number of extracellular vesicles EV produced in a fluidic system 1 for different stirrings controlled by the stirrer 7.
• MSC mesenchymal stem cell acronym
• the microcarriers 3 are introduced at a concentration of 3 ⁇ l 1 in 200 ml of liquid medium in a 500 ml spinner flask.
• the left ordinate corresponds to the numbers of extracellular vesicles EV produced in the receptacle 4.
• Each column corresponds to an extracellular vesicle production EV for different rotational speeds of the agitator 7 in the receptacle 4.
• the ordinate on the right corresponds to the length LK entrained during the production of extracellular vesicles EV, calculated by formulas (1), (2) and (3).
• a significantly high production of extracellular vesicles EV is observable by controlling a flow of liquid medium in which the length LK is equal to 35 ⁇ (production corresponding to the 175 RPM column), compared to extracellular vesicle production EV under conditions lower agitation in which the length LK is equal to 50 m.
• Figure 5 illustrates the influence of Kolmogorov LK length on the number of EV extracellular vesicles produced.
• Length LK is a scale parameter for the production of extracellular vesicles EV.
• the squares (a) correspond to the extracellular vesicle production EV shown in Figure 2 for different lengths
• the diamonds (b) correspond to the extracellular vesicle production EV shown in Figure 3 for different lengths U
• the triangles (c) correspond to Extracellular vesicle production EV illustrated in FIG. 4 for different lengths U.
• U 50 ⁇
• FIG. 6 illustrates producing cells 6 adhered to the surface of microcarriers 3, in this case microbeads 14, suspended in the liquid medium 5, before the agitation corresponding to an extracellular vesicle production EV. Producing cells 6 adherent to the surface of microcarriers 3 are visible and quantifiable.
• FIG. 7 illustrates producing cells 6 adhered to the surface of microcarriers 3, in this case microbeads 14, suspended in the liquid medium 5, after the agitation corresponding to an extracellular vesicle production EV.
• Producing cells 6 adherent to the surface of 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 stirring for the production of extracellular vesicles EV makes it possible to verify that the stirring conditions described above, for example an agitation causing a flow in which the LK length is less than 75 ⁇ and preferentially to 50 ⁇ irr, do not cause the detachment of the producer cells 6 microporteurs 3.
• FIG. 1 illustrates producing cells 6 adhered to the surface of microcarriers 3, in this case microbeads 14, suspended in the liquid medium 5, after the agitation corresponding to an extracellular vesicle production EV.
• curve (a) illustrates the evolution, during agitation, of the ratio between the number of particles produced (including extracellular vesicles EV) and between the number of producing cells 6 introduced into the container 4, the producing cells 6 being of the murine MSC type, and the flow of the liquid medium 5 being characterized by a length LK substantially equal to 35 ⁇ .
• Curve (b) illustrates the same evolution, the producer 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 m.
• Curve (c) illustrates the same evolution, the producer cells 6 being of HUVEC type, and the flow of the liquid medium 5 being characterized by a length LK substantially equal to 35 m.
• Curve (d) illustrates the same evolution, producing cells 6 being of human MSC type, and the flow of liquid medium 5 being characterized by a length LK substantially equal to 35 m.
• Curve (e) illustrates the same evolution, the production cells being of HUVEC type, and the flow of the liquid medium being characterized by a length LK substantially equal to 50 Mm.
• Curve (f) illustrates the same evolution, cells producing cells being of the murine MSC type, and the flow of the liquid medium being characterized by a length LK substantially equal to 50 ⁇ m.
• the curve (g) illustrates the same evolution, the producer 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 ⁇ .
• Curve (h) illustrates the same evolution, the producer cells being of murine MSC type, and the flow of liquid medium 5 being characterized by a length LK substantially equal to 53 ⁇ .
• Figure 9 illustrates the yield of extracellular vesicle production EV for different producer cells 6, and for different agitation conditions after 240 minutes of agitation. The four columns on the left illustrate the yield of EV extracellular vesicle production using murine MSC-like producer cells.
• the production yield for three stirring conditions corresponding to a stirring resulting in a length LK of 50 ⁇ (first column), 47 ⁇ (second column) and 35 ⁇ (third column) is compared to the production yield according to the method of serum deficiency (or starvation method or serum deprivation in English).
• Three columns illustrate the yield of EV extracellular vesicle production using HUVEC type 6 producer cells.
• the production yield for two stirring conditions corresponding to a stirring resulting in a length LK of 50 ⁇ (fourth column) and 47 ⁇ (fifth column) is compared with the production yield according to the serum deficiency method.
• the four rightmost columns of the figure illustrate the yield of EV extracellular vesicle production using human MSC-like producer cells.
• the production yield for three stirring conditions corresponding to a stirring resulting in a length LK of 50 ⁇ (eighth column), 35 ⁇ (ninth column) and 33 ⁇ (tenth column) is compared to the production yield according to the method of deficiency in serum.
• Figure 10 illustrates the concentration of producer cells 6 adherent on the microcarriers 3 before and after stirring, for different stirring conditions.
• Figure 11 illustrates the metabolic activity of HUVEC type 6 producing cells under agitation conditions for the production of EV extracellular vesicles.
• the stirring of the liquid medium is controlled by an agitator 7 rotating at 75 RPM, in a spinner flask of 1 L, resulting in a flow characterized by a length LK substantially equal to 50 ⁇ , for 240 minutes.
• the metabolism is measured by observing the variation of the wavelength emitted by the reagent Alamar blue in the liquid medium 5. No significant decrease in the metabolism of the producing cells 6 is observable under these stirring conditions.
• Figure 12 illustrates the metabolism of HUVEC producing cells 6 under agitation conditions for the production of EV extracellular vesicles.
• Stirring of the liquid medium is controlled by an agitator 7 rotating at 125 RPM in a 1L spinner flask, causing a flow characterized by a length substantially equal to 35 ⁇ for 240 minutes.
• the metabolism is measured by observing the variation of the wavelength emitted by the reagent Alamar blue in the liquid medium 5.
• a decrease, or even a disappearance of the metabolism of the producer cells 6 is observable after 250 minutes of agitation. This decrease in cellular metabolism does not, however, prevent the production of extracellular EV vesicles during agitation.
• Figure 13 illustrates the metabolism of MSC-like producing cells 6 under agitation conditions for the production of EV extracellular vesicles.
• the stirring of the liquid medium 5 is controlled by an agitator 7 rotated at 75 RPM in a 1L spinner flask, resulting in a flow characterized by a length LK substantially equal to 50 ⁇ , for 240 minutes.
• the metabolism is measured by observing the variation in wavelength emitted by the Alamar blue reagent in the liquid medium 5. No significant decrease in the metabolism of the producing cells 6 is observable under these stirring conditions.
• Figure 14 illustrates the metabolism of MSC type 6 producing cells under agitation conditions for the production of extracellular EV vesicles.
• the stirring of the liquid medium is controlled by an agitator 7 rotating at 125 RPM in a 1L spinner flask, resulting in a flow characterized by a length LK substantially equal to 35 ⁇ for 240 minutes.
• the metabolism is measured by observing the variation in wavelength emitted by the Alamar blue reagent in the liquid medium 5. No significant decrease in the metabolism of the producing cells 6 is observable under these stirring conditions.
• the conditions of low agitation corresponding to a stirring resulting in a flow characterized by a length LK substantially equal to 50 ⁇ m, make it possible to reuse the cells 6 for subsequent extracellular EV vesicle production.
• 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.
• Photomicrography is performed using the transmission electron cryomicroscopy technique (cryo-TEM).
• Figure 16 illustrates the extracellular vesicle diameter distribution EV produced by fluid system 1 measured by cryo TEM.
• the distribution (a) corresponds to extracellular EV vesicles produced by murine MSC cells with agitation causing a flow characterized by a length LK substantially equal to 35 ⁇ (condition of strong agitation).
• the distribution (b) corresponds to extracellular EV vesicles produced with agitation causing a flow characterized by a length LK substantially equal to 50 ⁇ (low agitation condition).
• the median diameter of the extracellular EV vesicles produced under low agitation conditions is greater than the median diameter of the extracellular EV vesicles produced under conditions of strong agitation.
• the size of the extracellular vesicles EV may be substantially between 30 and 500 nm.
• Figure 17 illustrates the purity of the extracellular EV vesicles produced by the fluidic system 1 in the liquid medium indicated by the ratio of the number of particles to the microgram protein mass.
• different entities may be produced by the producing cells 6, in this case extracellular vesicles EV but also protein aggregates. Quantification of particles by individual particle monitoring (or NTA for Nanoparticle Tracking Analysis) does not allow To differentiate these different entities, it is also advantageous to quantify the ratio between the number of particles measured by NTA and the mass of proteins produced, defining the purity of the extracellular vesicles EV. Columns (a) illustrated in Fig.
• FIG. 17 correspond to extracellular vesicle production EV from murine MSC-like producer cells 6, and columns (b) correspond to extracellular vesicle production EV from cell-producing cells 6.
• human MSC type The two left-hand columns correspond to an extracellular vesicle production EV under conditions of strong agitation, and the two right-hand columns correspond to an extracellular EV vesicle production according to the serum deficiency method.
• the extracellular vesicle purity EV of the medium obtained after the production is comparable in both methods.
• Figure 18 illustrates the pro-angiogenic properties of a serum-free liquid medium comprising extracellular EV vesicles produced by murine MSC cells by fluidic system 1.
• Panel B of FIG. 18 is a photograph of the same surface, after 4 hours of incubation in the liquid medium comprising the extracellular vesicles EV.
• Panel C of FIG. 18 is a photograph of the same surface after 9 hours of incubation in the liquid medium comprising extracellular vesicles EV. During the experiment, HUVEC cells cover the part of the surface on which no cells are present at the beginning of the experiment. So, the liquid medium comprising the extracellular vesicles EV has pro-angiogenic and / or pro-proliferative properties.
• Figure 19 illustrates the pro-angiogenic properties of a liquid medium comprising extracellular EV 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 h (corresponding to panel A of FIG. 18) and 9 h (corresponding to panel C of FIG. 18) for each incubation condition.
• the first column (“complete medium”) corresponds to an incubation in a culture medium of HUVEC cells (corresponding to a positive control).
• the second column (“ctrl neg”) corresponds to an incubation in a liquid medium (culture medium) without extracellular EV vesicles, serum-free where a given volume of PBS was added.
• the third column (“strong agitation 10/1”) corresponds to an incubation in a liquid culture medium where the same given volume of PBS was added comprising extracellular EV vesicles produced by a fluidic system 1 under conditions of strong agitation. in which the amount of producer cells 6 introduced corresponds to 10 murine MSC producer cells for a recipient HUVEC cell.
• the fourth column (“low agitation 10/1”) corresponds to an incubation in a culture medium where the same given volume of PBS was added comprising extracellular EV vesicles produced by a fluidic system 1 under conditions of low agitation, in which the amount of producer cells 6 introduced corresponds to 10 murine MSC producer cells for a recipient HUVEC cell.
• the fifth column corresponds to an incubation in a culture medium of HUVEC cells, depleted in exosomes, the amount of introduced producer cells 6 corresponding to 10 producer cells 6 for a recipient HUVEC cell.
• the sixth column corresponds to an incubation in a liquid medium of culture where the same given volume of PBS was added comprising serum-deficient EV extracellular vesicles, the amount of introduced producer cells 6 corresponding to 10 murine MSC producer cells for a recipient HUVEC cell. Incubation in a liquid medium comprising extracellular EV vesicles produced under high agitation conditions ("strong 10/1 agitation”) significantly overcomes the initially cell-free portion.
• Figure 20 illustrates the proliferation of cardiomyocytes after one day of incubation in different liquid media, as measured by alamar blue included in the incubation medium.
• the first column corresponds to an incubation in a medium adapted to the culture of cardiomyocytes H9C2 ("complete medium", positive control).
• the second column corresponds to an incubation in PBS.
• the third column corresponds to an incubation in a liquid medium comprising extracellular EV vesicles produced by a fluid system 1 under conditions of strong agitation ("strong stirring 10/1"), the quantity of producing cells 6 introduced corresponding to 10 cells. producing 6 for a recipient cell.
• the fourth column corresponds to an incubation in a liquid medium comprising extracellular EV vesicles produced by a fluid system 1 under conditions of low agitation ("low agitation 10/1"), the amount of producer cells 6 introduced corresponding to 10 cells. producing 6 for a recipient cell.
• the fifth column corresponds to an incubation in a culture medium of cardiomyocytes, depleted in exosomes ("lib 10/1"), the quantity of producing cells 6 introduced corresponding to 10 producer cells 6 for a recipient cell.
• the sixth column corresponds to an incubation in a liquid medium comprising EV extracellular vesicles produced by a method of production in a serum-free "stress" medium, the amount of introduced producer cells 6 corresponding to 10 producer cells 6 for one cell. receptor.
• Incubation conditions in a liquid medium comprising extracellular EV vesicles produced in a fluidic system 1 under conditions of high agitation and / or low agitation result in a significantly higher proliferation of cardiomyocytes compared to incubation conditions in patients.
• PBS and under the conditions "exo" and "stress".
• FIG. 21 illustrates the proliferation of H9C2 cardiomyocytes after two days of incubation.
• Cardiomyocytes incubated in a liquid medium comprising extracellular EV vesicles produced from murine MSC-like producer cells 6 by a fluidic system 1 under conditions of low agitation, under conditions of strong agitation, as well as by spontaneous release in a exosome-depleted complete medium proliferate significantly more than cardiomyocytes incubated in serum deficient medium.
• Cardiomyocytes incubated in a liquid medium comprising extracellular EV vesicles produced under conditions of low agitation and under high agitation conditions proliferated significantly more than cardiomyocytes incubated in serum deficient medium.
• Figure 22 illustrates the dose effect on H9C2 cardiomyocyte proliferation of an incubation of a liquid medium comprising a variable concentration of extracellular EV vesicles produced by a fluidic system 1.
• the metabolic activity of the cardiomyocytes is measured after two days of incubation in a liquid medium by alamar blue.
• the cardiomyocyte metabolism is measured for three incubation conditions: in a liquid medium comprising extracellular EV vesicles produced under low agitation conditions in (a), under low agitation conditions in (b) and in a liquid medium with serum deficiency in (c).
• curves (a) and (b) The measurement of cardiomyocyte metabolism is performed in curves (a) and (b) for different ratio between the concentration of extracellular vesicles EV 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 ratio of extracellular EV vesicle concentrations to cardiomyocytes increases.
• Figure 23 illustrates the proliferation of cardiomyocytes after two days of incubation in the presence of a liquid medium comprising extracellular EV vesicles of murine MSC cells at a concentration of 100,000 extracellular EV vesicles by cardiomyocyte.
• the proliferation of cardiomyocytes after two days is significantly greater when the liquid incubation medium comprises extracellular EV vesicles produced by a fluidic system 1 under conditions of high agitation or low agitation, compared to a liquid medium comprising vesicles.
• extracellular EV obtained by serum deficiency and / or culture medium of cardiomyocytes containing extracellular EV vesicles obtained spontaneous release in complete medium depleted exosomes.
• Fig. 24 illustrates the use of an extracellular vesicle composition EV produced by the fluidic system 1 as a pharmaceutical composition.
• a caecostomy is performed on each rat of a group of rats. The presence of excrement is observed at the opening of a fistula formed by the caecostomy, under three conditions: a control condition, a condition corresponding to a 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 method which is the subject of the invention, for example under conditions of strong agitation ("gel + vesicles").
• Figure 25 illustrates the use of an EV extracellular vesicle composition produced by the fluidic system 1 as a pharmaceutical composition.
• a score is calculated from the observations presented in Figure 24. A score of 1 is assigned when the hole of a fistula shows faeces and a score equal to zero is assigned when the orifice of a fistula does not present no excrement.
• Figure 25 illustrates the mean score, for all the caecostomies and for each of the conditions: control, "gel” and "gel + vesicles”.
• the application of a gel comprising extracellular vesicles EV results in a decrease in the average score of fistula productivity compared to control groups and gel without vesicles and significantly reduces the presence of feces at the fistula orifice in these fistulas. conditions.
• the extracellular vesicle composition EV produced by the fluidic system 1 can be used in regenerative medicine.
• FIG. 26 illustrates the proteomic profile of extracellular vesicles produced by the method according to the invention compared with the extracellular vesicles produced by the conventional production methods according to the prior art, more particularly the proteomic profile of markers conventionally used to characterize the extracellular vesicles.
• the proteomic profile of extracellular vesicles obtained by four different production methods were compared.
• a method of production in flask by deprivation of serum for a period of 72 hours (2D)
• a method of production in bioreactors by deprivation of serum for a duration of 72 hours (3D)
• a production method in medium-speed bioreactor characterized by a Kolmogorov length equal to 50 ⁇ for a duration of 4 hours MS
• a high-speed bioreactor production method characterized by a Kolmogorov length equal to 35 ⁇ for a duration of 4 hours (HS).
• the extracellular vesicles produced by the method according to the invention have a similar proteomic profile to the extracellular vesicles produced by the methods of the prior art.
• a similar proteomic profile is a proteomic profile characterized by the presence and the amount of markers conventionally used to characterize extracellular vesicles.

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