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
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
  • C12M41/44—Means for regulation, monitoring, measurement or control, e.g. flow regulation of volume or liquid level
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
  • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
  • C12M23/00—Constructional details, e.g. recesses, hinges
  • C12M23/02—Form or structure of the vessel
• • 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
  • C12M29/10—Perfusion
• • 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
  • C12M29/12—Pulsatile flow
• • 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
  • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
  • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
• • 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
  • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation

Definitions

• the invention relates to the field of bioreactors for cell culture.
• the invention relates to a bioreactor (1) for cell culture on a three-dimensional substrate, comprising:
• a culture chamber (2) whose internal walls form a vertical duct, preferably of frustoconical shape, the diameter of which widens regularly from the entrance of the duct to the outlet of the duct,
• the invention also relates to the advantageous use of these bioreactors in tissue engineering, for the production of tissue grafts, in particular bone or cartilaginous grafts.
• Tissue engineering aims to apply the principles of biology and engineering to develop functional substitutes for damaged tissues. Technological developments in tissue engineering should make it possible to obtain, from patient cells, tissues cultured in vitro that can be tolerated by the body and to replace damaged or defective tissue. The regenerative perspectives offered by tissue engineering affect many types of tissues including, but not limited to, cardiac tissue, certain tissues of the eye (cornea), liver tissue, pancreatic tissue, blood vessels, and tissues. musculoskeletal: muscular, bony, cartilaginous, but also tendinous and ligamentous tissues.
• tissue or organoid graft In order to obtain a tissue or organoid graft, it is in general necessary to seed the appropriate cells, for example progenitor cells of the targeted tissues, in porous biomaterials that allow the development of a three-dimensional structure.
• This is called cell culture on three-dimensional substrate, or cell culture in three dimensions.
• this culture method consists in immersing the seeded substrate of cells in a nutritive liquid and placing it in an incubator regulating temperature and gaseous mixture.
• the main disadvantage of this technology lies in the fact that the diffusive exchanges, only present here, are generally insufficient to ensure the nutrition of the cells and in particular at the heart of the substrate.
• the porous substrates seeded with cells are subjected to a perfusion flow of nutrient liquid.
• the substrate needs to be held in place in the bioreactor so that the flow can be forced into it. It is therefore imperative that the substrate has a minimum of mechanical strength to withstand the contrary efforts of infusion and maintenance.
• These substrates are mainly useful in the case of bone tissue engineering; in this case, the culture substrate has mechanical performance very close to the bone in culture.
• the main drawback of this technology lies in the fact that some substrates do not necessarily have the mechanical performance sufficient to withstand high transmural pressures (with regard to the size of the substrates provided).
• US Pat. No. 5,320,963 discloses a conical bioreactor for suspension cell culture.
• the device relates to cultures of isolated cells or clusters grown without substrate, the conical portion being put in place to provide a larger deposition area for harvesting cells.
• Porous hydrogels are materials with very high potential in three-dimensional cell culture and they are expected to replace many of the usual materials used in cell culture (coral, hydroxihapatite, titanium ...) because of their similarity with native tissues (rather “soft” materials) and their very large biocompatibility (absorbable polysaccharide).
• these materials have mechanical characteristics insufficient to be placed in the bioreactors known from the state of the art, and in particular the perfusion bioreactors described in the literature (held between "clamps” + application of a transmural pressure).
• the objective of the invention is to overcome the shortcomings of the devices of the prior art.
• the bioreactors according to the invention make it possible to maintain the cells grown on a three-dimensional substrate in suspension because of the equilibrium between different hydrodynamic forces (Stokes drag, gravity and Archimedean thrust).
• Another object of the invention is the development of a device using a reduced volume of fluids while allowing adequate flow for the realization of the first objective.
• Another object of the invention is to develop a device generating a flow of such a nature as to keep the substrates in culture away from the walls, adapted in particular for cell culture on a porous hydrogel-type substrate.
• Yet another object of the invention is to allow perfusion of the substrates so as to obtain optimal and homogeneous growth of the grafts in the bioreactor.
• a further object of the invention is to provide a bioreactor for cell culture on suspension substrate requiring the minimum of human intervention during a culture time of the order of a few days to several weeks.
• bioreactor allows the culture of tissue grafts held in suspension and perfused by the culture medium. It is particularly suitable for the culturing of bone or cartilaginous grafts on soft porous substrates, in particular on porous hydrogels.
• the inventors have made a bioreactor comprising a suitable flow device of the culture medium in the culture chamber.
• the invention as defined in the claims, relates first of all to a bioreactor for cell culture on a three-dimensional substrate, characterized in that it comprises
• a culture chamber whose internal walls form a vertical duct, preferably of frustoconical shape, whose diameter widens in a regular manner from the entrance of the duct (for example the low entrance) to the outlet (for example the high output) of the duct,
• b) means allowing the flow of the culture medium (for example from bottom to top) in said vertical duct.
• the bioreactor further comprises pumping means allowing a pulsed flow of the culture medium.
• the bioreactor comprises means allowing an annular flow of the culture medium in the culture chamber.
• FIG. 1 represents a schematic overview of the different parts of a bioreactor (1).
• Figure 2 shows a detail view of a culture chamber (2).
• Figure 3 shows an exploded detail view of an upstream flow setting device (3).
• FIG. 4 represents a partial sectional view of a bioreactor according to the invention comprising the culture chamber (2), the upstream flow device (3) and the downstream flow device (6).
• Figure 5 shows a detail view of an upstream flow setting device (3) and shows the flow of fluids through the device.
• FIG. 6 represents a partial sectional view of a bioreactor according to the invention and shows the flow of the fluids through the device.
• Figure 7 shows the cell viability revealed by Live / Dead staining of the ADSCs within the matrix, after 5 days of dynamic culture A) on the edges of the porous matrix or B) in the center (objective x10).
• Scale bar 200 ⁇ , as well as the relative expression of mRNA specific marker levels of bone ALP (C), OPN (D), OCN (E) and Cx43 (F).
• bioreactor is meant a device for the growth of biological cells in a preferably sterile medium.
• the biological cells that can be cultivated in bioreactors are prokaryotic or eukaryotic cells, and in particular microorganisms, unicellular eukaryotic or prokaryotic organisms, such as bacteria, archae, yeasts or fungi, or cells of multi-cellular organisms and mammalian cells, especially embryonic or somatic cells, or cells strains, for example mammalian mesenchymal stem cells or their derivatives.
• three-dimensional substrate or “biomaterial” (both terms being used interchangeably), is meant an artificial or natural material, allowing the three-dimensional growth of cells, and in particular the growth of organoids from stem cells comprising the differentiation of cells. strains into different cell types within the biomaterial. These biomaterials must have a porous structure, promoting the growth of cells while allowing a good infusion of nutrient liquids within the structure. For tissue engineering, it is generally considered that an average pore size between 200 and 400 ⁇ is optimal for promoting the penetration of cells into the implant but also the formation after implantation of a vascular network.
• Biomaterials include, but are not limited to, porous biomaterials of polymeric or ceramic nature, titanium, tantalum, or nitinol metal structures.
• porous biomaterials of polymeric type mention may be made, for example, of polylactic acid (PLA), polyglycolic acid (PGA), polylactic co-glycolic acid (PLAGA), hyaluronic acid or polycaprolactone ( PCL).
• PLA polylactic acid
• PGA polyglycolic acid
• PLAGA polylactic co-glycolic acid
• PCL polycaprolactone
• Synthetic ceramics hydroxyapatite and tricalcium phosphates
• natural ceramics coral and mother-of-pearl
• biomaterials known as "resorbable” have been developed and include materials of biological origin (for example, alginate, collagen, or fibrin gels).
• a preferred mode of biomaterial usable in the bioreactors according to the invention are “hydrogels” or “porous hydrogels”. These porous hydrogels are for example based on polymers chosen from Poly-Ethylene Glycol (PEG), polyvinyl alcohol (PVA) and poly (2-hydroxyethyl methacrylate) (pHEMA). These materials may also include various additives, for example different collagens, chitosan, etc., promoting specific phases in the cellular development or modifying the physicochemical properties of the material (see in particular the publication “Tissue Engineering: Fundam entais and applications” , 2006 by Yoshito Ikada in the Interface Science and Technology Ed. Academy Press Series.
• the hydrogels used chosen from polysaccharide-based hydrogels, and in particular those based on Pullulan as described in European Cells and Materials Vol. 13. Suppl. 1, 2007 (page 50).
• organoids or "graft” is meant a three-dimensional structure consisting of at least one biomaterial and biological cells capable of proliferating on the biomaterial, for example to form a graft capable of being transplanted into a patient.
• the grafts or organoids grown in the bioreactor according to the invention may advantageously be of small dimensions, for example having a maximum section of between 2 and 20 mm, for example between 4 and 15 mm, or even between 2 and 10 mm.
• a culture chamber (2) whose internal walls form a vertical duct, preferably of frustoconical shape, whose diameter widens regularly from the inlet
• a bioreactor (1) according to the invention may comprise in particular the five elements as represented in FIG.
• the bioreactor may include several culture chambers, or a culture chamber divided into several compartments, for example by grids or semi-sealed walls whose porosity allows
• the duct whose geometric axis is vertical, has a preferably circular cross section, the diameter of which widens in a regular manner from the entrance of the duct to the outlet of the duct.
• the inlet and the outlet of the duct are determined by the direction of flow of the culture medium in the culture chamber.
• the advantageous form of the vertical duct of the culture chamber of the bioreactors according to the invention contributes to self-regulation of the lift of the biomaterials or grafts by making it possible to establish an equilibrium between the drag forces (flow of the fluid around the hydrogels), of gravity (weight of the substrate) and the resultant of the Archimedes force (buoyancy related to the difference in density between the solid and the fluid).
• This equilibrium has been described for example in industrial applications of flow measurement (flowmeter type "rotameter") but in such applications, the single object in levitation has dimensions very close to those of the duct.
• the conduit has a cross section whose diameter widens regularly from the low inlet duct to the top outlet of the duct.
• the biomaterials grown are driven vertically by the high speeds present in the small section of the cone until they arrive in a zone of lower speeds (in the larger section) where the drag forces are proportionally lower (gravity becomes the dominant force again) which lowers the biomaterials grown towards the higher speed zone where ascents to the smaller area speed start again.
• This is followed by a three-dimensional vertical alternating circulation phenomenon in the body of the culture chamber.
• This movement promotes the renewal of the culture medium on the surface of the cultured biomaterials, and consequently, permanently ensures a concentration gradient between the inside and the surface of the most favorable support possible (maximized diffusive effects).
• the porosity of the cultured biomaterial allows the passage of a fluid.
• the convection of the fluid in the biomaterial is then ensured by the existence of a gradient of pressure between the lower and upper faces of the substrate. This convection is reinforced on the one hand by the alternating circulation movements described above and on the other hand by the application of a pulsed flow in the culture chamber.
• the cultured biomaterial is then advantageously perfused (in proportion to its permeability and to the pressure gradient between the lower and upper walls of each biomaterial).
• a device comprising a vertical duct having a cross section whose diameter widens in a regular manner from the upper entrance of the duct towards the low outlet of the duct.
• This embodiment is more particularly suitable for the culture of porous substrates whose density is lower than that of the fluid (for example when the substrate contains air bubbles).
• the culture chamber comprises a cylindrical body pierced with a frustoconical duct, in the form of a truncated cone.
• the frustoconical duct is for example reversed as shown in FIG.
• the cultivated biomaterials are in general of small dimensions relative to the culture chamber, for example of dimension less than 20 mm, for example between 4 and 15 mm, for a inlet diameter of the vertical duct (smaller section) which may for example be between 3cm and 10cm.
• a culture chamber will be chosen comprising a frustoconical shaped conduit whose apex angle does not exceed 8 °.
• the diameter of the inlet cross-section of the duct may for example, without being limiting, be between 3 cm and 10 cm, the height of the vertical duct between 5 cm and 30 cm and the diameter of the outlet cross-section. duct (larger section), between 3cm and 15cm.
• the culture chamber may contain a culture volume of about 35 ml to 4 liters.
• the most suitable material for the culture chamber especially from those known from the state of the art for the production of bioreactor culture chambers. These materials include glass, transparent polymers such as PE, PET, PVC, PS, PP, PMMA, PEI and ABS. It is preferably a sterilizable material.
• the cylindrical body pierced with a conical conduit of the culture chamber is made of a sterilizable PolyEtherimide material.
• the material is a transparent material. It thus makes it possible to adjust visually the conditions allowing the sustenance of the cultured biomaterials. Their position in the culture chamber is thus controlled. This is an advantageous aspect of the invention since it enables non-specialists to establish an adequate flow and to correct it throughout the evolution of the cell culture (in the case of the manufacture of bone grafts, the cells make an extracellular matrix and calcification that will increase the force of gravity).
• the use of a transparent material for the culture chamber may also be an advantage in the case of using the bioreactor in other applications than cell culture for tissue engineering, in particular those requiring activation of photosynthesis.
• the bioreactor according to the invention comprises:
• a culture chamber (2) whose inner walls (21) define a vertical duct whose diameter widens in a regular manner from the entrance of the duct towards the outlet of the duct, preferably of conical shape,
• an outlet grid (23) placed in the downstream part (for example the upper part) of larger diameter of the vertical duct preventing the grafts from circulating in the rest of the device,
• the bioreactor according to the invention comprises an inlet gate (22) placed upstream of the body.
• This is for example a perforated disk that promotes the establishment of a flow having an annular type of speed profile.
• annular flow is meant a flow in which the flow is greater through elementary surfaces located at the periphery compared to the flow generated through elementary surfaces located in the center of the duct.
• the type of flow thus generated contrary to the case of parabolic velocity profiles, makes it possible to maintain the substrates in the center of the conical part of the culture chamber. Indeed, in the case of parabolic velocity profiles, the substrates tend to migrate towards the walls because of the velocity gradient between the center and the periphery of the section of the flow.
• the diameter and distribution of the perforations ensures the maintenance of the substrates during the establishment or start of the bioreactor but also more generally in case of stopping the pumping system.
• the generation of an annular flow can be achieved using concentric cylindrical pipes replacing the perforated disk.
• the inlet gate is a perforated grid having orifices, preferably with a diameter of less than 6 mm, for example from 2 to 5 mm, distributed in such a way that the flow is faster in the near regions. walls only in the center.
• the bioreactor according to the invention may also comprise an upper gate (23) placed downstream of the cylindrical body pierced with a conical conduit. It is mainly a safety device designed to prevent the accidental passage of substrates in the rest of the hydrodynamic circuit.
• it may consist of a perforated disk but without particular distribution of perforations.
• the diameter of the perforations is, however, adapted to the size of the substrates placed in culture in order to prevent their possible circulation in the rest of the installation.
• the pumping means (4)
• the bioreactor comprises pumping means for obtaining a vertical flow, from the smallest section to the largest section of the vertical duct of the culture chamber, for example a pulsed flow from bottom to top in the vertical duct. Any type of pump conventionally used in dynamic bioreactors can be envisaged.
• a pump will preferably be chosen to prevent heating of the culture medium for the control of the optimum culture temperature.
• a pump is chosen to obtain a pulsed flow.
• pulsed flow is meant a flow with a short and regular time interval, an acceleration phase followed by a deceleration phase.
• the frequency of the pulses is between 0.05 and 10 Hz depending on the size of the bioreactor, for example of the order of 1 Hz, thus reproducing the frequencies observed in the cardiovascular flows of the adult.
• the pulsed character of the flow makes it possible to intermittently apply large values of infusion rate.
• the second phenomenon is related to the generation of a wake downstream of objects in relative displacement in a fluid. The pulsed flow makes it possible not to maintain the same wake over time and therefore prevents the appearance of these stasis zones.
• a check valve at the outlet of the pump or any means to avoid backflow, especially at the start of the culture process. . Indeed, at startup, the three-dimensional substrates are placed on the lower grid 22 and are then likely to be sucked at the start of the pump for a pulsed flow.
• It is a device allowing the generation of favorable conditions for the annular flow produced in the culture chamber. It is placed upstream of the culture chamber.
• the device according to the present invention consists of a series of geometrical singularities (variations of sections and changes of direction of the flow) making it possible to transform a tangential flow at the inlet of the device to make it axial with an axially symmetrical speed profile. (90%) at the input gate of the culture chamber.
• the upstream flow setting device (3) comprises:
• the lower, inner and outer sides are formed of watertight walls with the exception of one or more orifices (34) allowing the flow of the culture medium in the first flow zone in a horizontal tangential direction,
• the upper side (312) is perforated so that the culture medium arriving in the first flow zone, engages through the perforations in a substantially vertical direction from bottom to top to a second flow area (32) ,
• the diameters of the first and second cylinders are very close so that the second cylinder can fit into the first cylinder leaving a space, for example of the order of a few millimeters for a cylinder diameter of the same order of magnitude as the lower inlet diameter of the culture chamber (2).
• the outer diameter of the first flow zone (31) is greater than the inlet diameter of the vertical duct, for example 1.5 to 2 times greater;
• the inside diameter (delimited by the first cylinder (36)) is substantially greater than the inlet diameter of the vertical duct forming the culture chamber (2);
• the inside diameter of the second cylinder (38) is equal to the inlet diameter of the vertical duct forming the culture chamber (2).
• FIG. 3 An example of such a device is shown in Figures 3, 4 and 6.
• the path of the culture medium in the device is shown in Figures 5 and 6.
• the device (3) is described above in the context of a vertical flow of the culture medium from bottom to top in the culture chamber. Of course, a similar device can be used in a top-down flow embodiment.
• the essential element for transforming a horizontal centrifugal / tangential flow into a vertical homogeneous flow over a short distance is the coupled use of a perforated ring (allowing fluids to pass only on the inner radii where the fluid rotates at lower speed) and a baffle (formed by two concentric cylinders). These elements achieve speed variations and directional changes that allow the proper reorientation of the fluid.
• the bottom wall 311 of the first flow zone has a helical shape so that said flow zone passes from a maximum section to the right of the fluid inlet orifice 34 (corresponding to the distance between the lower wall 311 and the upper wall 312) at a zero section in a revolution around the vertical axis of the device.
• Other variants are of course conceivable leading to a reduction in the volume of the first flow zone in the main flow direction of the culture medium.
• This device is placed downstream of the output grid. It participates in maintaining the symmetry of the flow.
• this device comprises an axial output of small diameter (for example connected to a buffer tank) and does not pose a problem of pre-rotation of the flow (which would be the case in the event of a tangential exit).
• the reservoir makes it possible to ensure the conditions of gas exchange and renewal of the nutritive liquid (culture medium).
• the reservoir must meet the usual constraints of sterile cell culture.
• it may include means for measuring physical quantities such as pressure, temperature or flow. It may also include catheters or other devices for sterile delivery of products into the device (culture medium, etc.).
• One of the reservoirs may also be withdrawn from the circuit for, for example, transferring the medium into another device, for example, a device that would extract the active principles if the cultured cells are biomolecule-producing cells of interest (proteins, antibodies therapeutic, antiviral, ...) Uses of the bioreactor according to the invention and method of implementation
• bioreactors according to the invention are particularly advantageous for the culture of cells on a three-dimensional support, in particular on porous hydrogels.
• they can be used for:
• tissue graft such as bone grafts, in particular vascularized bone grafts, cartilaginous grafts or any other type of tissue / cell (epithelial cells, hepatocytes, granulocytes, erythrocytes, ... without being limiting) or,
• biopharmaceutical molecules for example for the production of antibodies or proteins.
• tissue graft bioreactor has been described in the prior art for example in the book by Lanza, Langer and Vacanti "Principle of Tissue Engineering", published by Elsevier.
• the bioreactor according to the invention can be used in a process for producing a tissue graft, in particular a bone or cartilaginous graft, comprising the following steps:
• the porous biomaterial (s) are seeded with cells capable of regenerating a tissue, for example a bone or cartilaginous tissue, in order to obtain one or more organoids,
• the one or more organoids is cultured in the bioreactor under conditions suitable for the formation of said tissue graft, for example of said bone or cartilaginous graft.
• it will be chosen to cultivate a plurality of organoids of greater section between 2 and 20 mm, for example between 4 and 15 mm, or even between 2 and 10 mm, for example each organoid being constituted by a porous hydrogel fragment of a size of a few millimeters, for example between 2 and 20mm, for example between 4 and 15mm, or between 2 and 10mm, for their largest section.
• a plurality of grafts are cultured in the bioreactor, preferably at least 5 grafts, for example at least 10 grafts.
• the number and size of grafts in culture that can be adapted, in particular according to the dimensions of the bioreactor used.
• a flow mode selected from the culture chamber will preferably be selected.
• the flow rate of the culture medium can be advantageously controlled so that the organoid (s) cultured in the bioreactor are in suspension in the culture medium at an average vertical position, in particular which makes it possible to prevent the organoids from come in contact with the high and low parts of the duct.
• the bioreactor allows the generation of vascularized tissues, for example vascularized bone tissue.
• vascularized tissues for example vascularized bone tissue.
• vascularized bone tissue can be cultivated in co-culture on a porous biomaterial, endothelial progenitor cells and osteoprogenitor cells capable of regenerating vascularized bone tissue (Unger et al 2007, Biomaterials No. 28 3965-3976).
• a porous hydrogel for example porous hydrogels based on polysaccharides as described in European Cells and Materials Vol. 13. Suppl. 1, 2007 (page 50).
• the culture medium will be selected according to the intended purpose.
• Various culture media suitable for culture on a three-dimensional substrate, in particular for obtaining bone tissues, are described for example in Lanza, Langer and Vacanti "Principle of Tissue Engineering” published by Elsevier.
• the cells grown on a three-dimensional support are biomolecule-producing cells of interest, for example proteins, in particular therapeutic antibodies.
• the bioreactor comprises
• FIG. 2 represents a detailed view of the culture chamber comprising the cylindrical type culture chamber (2) whose internal walls (21) form an inverted cone. At the entrance to the culture chamber is disposed a perforated grid (22) promoting the annular flow of the culture medium in the culture chamber. At the outlet, a perforated grid (23) is also placed, preventing the grafts from circulating in the rest of the device.
• FIG. 3 represents a detail view of the device for establishing the upstream flow and comprises, in particular, a perforated ring (35) allowing the culture medium to flow in an inner radius (31), two concentric cylinders ( 36) and (38) and a perforated disk (37) and a cap ring (39), all forming the baffle promoting axial flow limiting the horizontal velocity gradient. All of these elements are located in the axis of the culture chamber upstream of the inlet gate (see Figure 4 for the arrangement of these elements in a sectional view).
• this device makes it possible to transform the flow, over a short distance, from a horizontal centrifugal / tangential inlet to a vertical homogeneous flow.
• the fluids engage in the inner radius of the ring in a horizontal tangential direction, in a first zone delimited by the inner walls of the perforated ring and the first concentric cylinder. It then passes into a second zone in a vertical direction through the perforations of the perforated disk and then back down between the walls of the first concentric cylinder and the second concentric cylinder to arrive in a third zone to go back to the inlet gate of the chamber of culture.
• FIG. 6 shows the arrangement of the constituent elements
• the bioreactor was the subject of a first test campaign during which mesenchymal stem cells derived from the bone marrow were cultured on porous hydrogels based on polysaccharides such as described in 2007 by European Cells and Materials Vol. 13. Suppl. 1, 2007 (page 50).
• the nutrient medium (culture medium) used was EVIDM (Iscove's Modified Dulbecco's Medium) with 10% fetal calf serum (commercially available).
• a 5% CO 2 air mixture was maintained above the only free surface of the bioreactor circuit (buffer tank).
• the bioreactor according to the invention was used to apply dynamic stresses to 3D hydrogel matrices seeded with adult adipose tissue derived stem cells (ADSCs) and to modulate the osteoblastic differentiation of ADSCs in 3D in the absence of osteoinductive factors.
• the cellularized hydrogels (or substrates) were placed in the bioreactor after 48 hours of static culture. The size of the substrates is about 6mm in diameter and 2mm thick.
• the hydrodynamic conditions of the pulsed flow generated in the culture chamber of the bioreactor ensure adequate sustenance of the 12 porous cellularized substrates placed in this room. These flow conditions result in dynamic perfusion of the porous substrates with an average flow rate of approximately 6.10 -4 4 mL / min After 5 days of dynamic culture the substrates were harvested for analysis and comparison with substrates grown only in static in the same hydrogels and in the same culture medium.

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  • 2011-02-04 FR FR1150906A patent/FR2971255B1/fr not_active Expired - Fee Related
• 2012
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