EP2283109A2 - Bioréacteur et procédé pour culture de cellules et de tissus - Google Patents

Bioréacteur et procédé pour culture de cellules et de tissus

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Publication number
EP2283109A2
EP2283109A2 EP09749653A EP09749653A EP2283109A2 EP 2283109 A2 EP2283109 A2 EP 2283109A2 EP 09749653 A EP09749653 A EP 09749653A EP 09749653 A EP09749653 A EP 09749653A EP 2283109 A2 EP2283109 A2 EP 2283109A2
Authority
EP
European Patent Office
Prior art keywords
bioreactor
tissue
medium
lumen
tissue culture
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP09749653A
Other languages
German (de)
English (en)
Inventor
Günther KNEBEL
Sven MÜHLFRIEDEL
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Greiner Bio One GmbH Germany
Original Assignee
Greiner Bio One GmbH Germany
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Greiner Bio One GmbH Germany filed Critical Greiner Bio One GmbH Germany
Publication of EP2283109A2 publication Critical patent/EP2283109A2/fr
Withdrawn legal-status Critical Current

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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
    • 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
    • C12M35/04—Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli
    • 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
    • C12M23/08—Flask, bottle or test tube
    • 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
    • C12M23/40—Manifolds; Distribution pieces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/02—Membranes; Filters
    • 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
    • C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
    • C12M29/14—Pressurized fluid
    • 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
    • C12M29/18—External loop; Means for reintroduction of fermented biomass or liquid percolate
    • 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
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • 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
    • C12M41/12—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature
    • C12M41/16—Means for regulation, monitoring, measurement or control, e.g. flow regulation of temperature by recirculation of culture medium at controlled temperature
    • 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
    • C12M41/26—Means for regulation, monitoring, measurement or control, e.g. flow regulation of pH

Definitions

  • the invention relates to a bioreactor for cell and tissue culture for mechanical stimulation and / or perfusion of tissue or cell cultures comprising an upper part and an associated lower part with a tissue culture chamber, as well as a distribution plate for cell and tissue cultures and a method for cultivation of cells and / or tissues in a bioreactor.
  • tissue reconstruction Skin, metabolism studies on reconstructed liver, and therapy procedures, such as Cartilage transplantation after trauma of the knee cartilage, reconstructed tissues are used.
  • the aim of the tissue reconstruction is the construction of a functional tissue from single cells, whereby the tissue properties should come as close to those in the living organism as possible. Of importance here are the preservation of the degree of differentiation of the cells, the presence of multiple cell layers and an extracellular matrix.
  • Petri dishes, multi-well plates, cell culture inserts and bioreactors are used for tissue reconstruction.
  • Autologous cartilage transplantation is a method of treating cartilage injuries.
  • the main field of application of the ACT is the treatment of cartilage defects in the knee joint, such as may result from sports accidents.
  • autopsy autologous cartilage cells are obtained by biopsy, partly cultivated ex vivo using a carrier material (eg collagen I gel) and reimplanted into the donor to repair the cartilage defect.
  • a carrier material eg collagen I gel
  • cartilage cells can be proliferated, thereby providing the increased number of cells necessary to repair large-area cartilage defects.
  • a well-known problem is the dedifferentiation of cartilage cells, which is associated with the onset of cell proliferation. After reimplantation, dedifferentiated cartilage cells can form atypical cartilage.
  • fibrocartilage after implantation of dedifferentiated cartilage cells into defects of the knee joint.
  • fibrocartilage only withstood the high pressure in the knee joint for a short time. It comes to premature failure of the implant.
  • Subject of the research are methods with which dedifferentiated cartilage cells can be redifferentiated before implantation.
  • the culture in bioreactors in particular with the aid of organically and tissue-specific mechanical stimuli (eg shear forces or rhythmic compressions of the cell-loaded support materials), represents a solution to the problem mentioned. Redifferentiation of dedifferentiated cartilage cells Application of mechanical stimuli has been described several times.
  • DE4306661 A1, DE10249903A1, US20060110822A1, DE10208311B4, US006060306A disclose bioreactor systems in which the cell culture medium is moved relative to the culture and thus the nutrient exchange between culture and medium is increased (perfusion bioreactors). In some cases, the formation of hyaline cartilage substance (eg collagen II synthesis) is stimulated by the targeted use of shear forces in these systems (US Pat. No. 5,928,945A, DE10249903A1, US20050095711A1).
  • hyaline cartilage substance eg collagen II synthesis
  • bioreactors allow the application of pressure to the cell-loaded carrier materials by means of a plunger (WO2005040332A2, DE19808055B4, DE102004012010A1 / WO002005087912A3).
  • a bioreactor which consists of a base plate, a cover plate and a commercially available multi-well tissue culture plate.
  • pistons are used, which protrude in the manner of a punch in the well of the underlying tissue culture plate, there, however, a space in which a tissue can be cultured, release.
  • This tissue culture chamber is provided with several inlet and outlet connections for medium and fumigation.
  • An elastic membrane is arranged above the piston, so that forms a sterile area below the membrane.
  • WO2005040332A2 describes a bioreactor which consists of a pressure-tight closable reactor space in which a storage area for a fabric construct and a mini actuator find place.
  • the reactor space is provided with connections for a medium supply and removal and fumigation.
  • Cultured tissue can be removed by deflecting the metallic mini actuator, preferably by application of a magnetic field, are compressed.
  • a method for producing three-dimensional cell transplants in the aforementioned bioreactor is described.
  • Object of the present invention is to provide a bioreactor for cultivation with optimal nutrient supply of cell and / or tissue cultures available.
  • the object of the present invention is in each case independent by a bioreactor, wherein at least one distributor plate is arranged in the tissue culture chamber, and a distributor plate using mechanical stimulation and / or perfusion, the recesses, in particular holes, holes, openings and at least one elevation or depression and a method for cultivating cells and / or tissues in a bioreactor comprising the steps of i) introducing the cells onto a distributor plate in the tissue culture chamber, in particular tissue lumens, ii) supplying the cells with culture medium, iii) mechanical stimulation and / or perfusion, solved.
  • cells or tissue differentiated or dedifferentiated cells can be redifferentiated, because by the application of mechanical stimuli, such as, for example, rhythmic compressions, pressure, tensile, shear forces, etc. are exerted on the cells of the distributor plate and thus the native system, such as Cartilage, bones, tendons, ligaments, skin, endothelia, blood vessels, are imitated in the body of an individual.
  • Perfusion can mimic optimal nutrient supply, as it is effected under native conditions by a vascular system, and can be replicated for many tissues, e.g. Liver tissue and skin, typical.
  • autologous tissue can be bred or prepared for a transplantation.
  • the method and the bioreactor can be used to produce large-area three-dimensional tissue constructs.
  • the bioreactor according to the invention offers both the possibility of medium overflow (for example, suitable for the production of cartilage constructs, cartilage-bone constructs, tendons, ligaments, etc.) as well as the medium flow (for example suitable for the production of liver cells or liver equivalents) and / or the mechanical Stimulation and the combination of these possibilities with continuous process control in a closed system.
  • the bioreactor is also suitable for the reconstruction of endothelia and skin grafts. th, suitable for the production of vascular prostheses and reconstruction of blood vessels. If blood vessels or vascular prostheses are produced with the bioreactor according to the invention, an auxiliary structure in the bioreactor is preferably used to generate the lumen of the vessels.
  • An advantage over the bioreactor described in DE 0200401201 OAl is further in detachment from the concept of protruding into the well of a tissue culture plate punch and the introduction of a large, pressure-applying manifold plate to cultivate large-sized tissue grafts, in particular in the diameter of> 35 mm.
  • the arrangement of recesses, in particular holes, holes, breakthroughs in the distributor plate is made possible that the cells or the tissue with culture medium not only over-, but also through- or can be flowed through and thus optimal nutrient supply Cells can take place, inter alia, by diffusion.
  • the distribution plate has at least one elevation or depression, which ensures that a permanent supply of the cells or the tissue takes place with culture medium, since even when exerting pressure or during compression, the medium is not entirely the tissue culture chamber, in particular medium and residual medium lumen, is displaced.
  • At least one cover plate is arranged in the tissue culture chamber, preferably between membrane and distributor plate, whereby a uniform transmission of the mechanical stimulation, such as that produced by the pressure chamber on the membrane pressure exerted on the cells or the tissue to be grown is made possible.
  • the cover plate prevents the collapse of the medium lumen if the membrane came to rest directly on the distributor plate.
  • the tissue culture chamber has at least one inflow and outflow device for the culture medium, whereby the nutrient supply of the cells or the tissue is made possible and continuously medium can be transported to and away again.
  • the tissue culture chamber comprises a plurality of lumens, in particular a tissue lumen, a medium lumen and optionally a residual medium lumen, wherein the cells to be cultured or the breeding tissue is contained in the tissue lumen, in the medium lumen for supplying the cells or the tissue Is contained available culture medium and transported away in the residual medium lumen therawtransport Schlude culture medium.
  • the tissue lumen is arranged between an upper and a lower distributor plate, as a result of which the supply of nutrients via the culture medium can be optimized.
  • the culture medium inflow device may open into the medium lumen, the culture medium being distributed uniformly across the cell or tissue to be cultured by the distributor plate, and the biological material either flowed over or through.
  • the effluent for the culture medium flows from the residual medium lumen and / or from the medium lumen, wherein a continuous removal of the culture medium is ensured both in an overflow and in a flow through the tissue or cells to be grown.
  • the pressure chamber has at least one supply device for compressed air, whereby pressure can be exerted on the underlying tissue culture chamber via the pressure chamber.
  • at least one elastic element is arranged in the tissue culture chamber, in particular in the tissue lumen, which allows a compression of the cell and tissue culture and also ensures that the tissue or the cells can expand again after removal of the pressure and the pressure or the shear forces on the tissue or the cells to be cultivated is exerted only intermittently or temporarily, as with native tissue such as in the cartilage of the knee joint or in blood vessels, in particular endothelia, due to the pulsation of the Blood caused by cardiac muscle contractions occurs.
  • At least one abutment is arranged in the tissue culture chamber, which sets the maximum compression, whereby excessive compression and possible damage to the tissue to be cultivated or the cells to be grown is prevented.
  • the elastic element and at least one abutment can be arranged, on the one hand by the elastic element, the above-mentioned restoring behavior is ensured and on the other hand, the maximum compression is determined by the abutment.
  • At least one spacer is arranged in the residual medium lumen, which on the one hand ensures that the lower distributor plate is arranged at a distance from the connection and the circulation of the culture medium can be ensured and, on the other hand, a reserve volume is created in order to increase the variability of the bioreactor increase, by the spacer in the residual medium lumen can be removed and thus the tissue lumen can be increased.
  • the upper and lower part is connected to one another via a detachable connection, whereby the tissue to be cultivated or the cells to be cultivated can be introduced directly into the tissue lumen of the lower part in a simple manner and after connection to the shell under sterile conditions can be further bred.
  • the lower part can be connected via a detachable connection, in particular thread, with a tripod and / or medium reservoir, whereby a continuous supply of culture medium is ensured or the bioreactor spaced from a surface. is maintained in order to ensure, for example, higher temperature stability, which otherwise may not be kept constant by stopping or incubating the bioreactor on a surface, because by direct contact over the surface temperature fluctuations are transmitted much faster to the bioreactor.
  • Another advantage is the safe and user-friendly integration of the medium reservoir in the closed bioreactor system, wherein the bioreactor is secured by means of a thread located in the lower part on a medium bottle, and thus simultaneously acts as a medium reservoir and as a tripod of the system, which thus fully closed view, easy to transport and manipulate.
  • a tripod and / or medium reservoir can serve a culture medium bottle, whereby a closed bioreactor system consisting of the bioreactor itself and the culture medium bottle can be formed and thus the risk of contamination is reduced when supplying the KuI- turmediums from an external reservoir.
  • the medium culture bottle can be autoclaved before reuse and only the bioreactor can be replaced.
  • the inflow and outflow device is connected via hoses with the media reservoir, whereby a closed connection is made and thereby also the risk of contamination can be excluded.
  • sensors in particular flow sensors, d ⁇ 2 sensors, pH sensors, etc. in the hoses to integrate, thus, different parameters may be determined such as, the flow volume, dissolved the proportion of oxygen or the pH Value of the culture medium that circulates continuously. Once a limit is reached, precautions can be taken to re-establish a defined target value. It is provided that the at least one elevation or depression of the distributor plate is arranged radially, centrally and / or concentrically, whereby the culture medium can flow unhindered over or through the distributor plate even during mechanical stimulation, such as pressure or tension application.
  • a plurality of elevations in the form of webs are arranged radially spaced from each other, whereby a uniform distribution of the culture medium on the tissue to be grown or the cells to be grown is made possible and also a collapse of the medium flow caused by the membrane is prevented ,
  • a survey is arranged centrally and spaced therefrom radial webs are arranged up to the concentric elevation, whereby both an overflow and a flow with culture medium of the tissue to be cultivated is made possible.
  • a survey is arranged centrally and at least one further survey is radially arranged marginally, which in turn can be a continuous supply of the tissue to be cultivated or the cells with culture medium.
  • At least one channel for the supply of the culture medium can be arranged, whereby the supply of the culture medium to the distribution plate and thus to the cells to be cultured and to the tissue to be cultivated can take place.
  • the culture medium is transported via an inflow device to the tissue culture chamber and leaves the tissue via a drainage device, thus ensuring a supply of culture medium of the tissue to be cultivated or of the cells to be cultivated.
  • the supply of the cells and / or tissue with culture medium can be done permanently, with which continuously fresh culture medium can be supplied.
  • the culture medium can either flow through or flow through the distributor plate or flow through it, whereby a uniform supply of nutrients is ensured both by diffusion and by perfusion and thus a uniform growth of the cells to be grown or the tissue to be grown can take place.
  • Fig. 2 shows a bioreactor 1
  • Fig. 2a further embodiment of the bioreactor 1;
  • Fig. 3 is an exploded view of a bioreactor 1;
  • the present invention relates to a bioreactor 1 for the cultivation and growth of cells or tissues with simultaneous mechanical stimulation and / or perfusion.
  • the mechanical stimulation can be effected by pressure, tension or shear forces.
  • three-dimensional cell and tissue cultures are cultivated for tissue reconstruction.
  • the described bioreactor 1 can be used for the production of various artificial tissues, for example for the production of artificial cartilage in the context of autologous cartilage transplantation. Other applications are bone or cartilage bone grafts, tendons, ligaments, skin, vessels, etc.
  • Fig. 1 shows a variant of the bioreactor 1 with a culture medium bottle 2 for the tissue reconstruction in the closed state.
  • This embodiment of the bioreactor 1 consists of an upper part 3 and a lower part 4.
  • the upper part 3 encloses a pressure chamber 5, which is bounded on one side by an elastic membrane 6 and can be acted upon by an opening 7 with compressed air.
  • the lower part 4 encloses a tissue culture chamber 8. It is connected to the upper part 3, preferably by a thread 9, whereby the elastic membrane 6 is clamped and the pressure chamber 5 is sealed tightly against the tissue culture chamber 8 enclosed by the lower part 4.
  • FIG. 2 shows further details of the first embodiment of the bioreactor 1.
  • This embodiment variant of the bioreactor 1 further comprises an abutment 10, for example an upper spacer, in order to determine the maximum compression in the tissue culture chamber 8.
  • An elastic element 11, in particular an elastic ring or a spring ensure the return to the starting position.
  • at least one distributor plate 12, preferably with recesses 13, is arranged.
  • the distributor plate 12 may have a perforated bottom. Between the distributor plate 12 and the cover plate 14, the tissue lumen 15 is arranged.
  • the tissue culture chamber 8 comprises, in addition to the tissue lumen 15 where the tissue to be cultivated or the cells to be cultured are located, a medium lumen 16 where culture medium is transported and optionally a residual medium lumen 17 where culture medium is transported away.
  • the tissue lumen 15 is formed between an upper and a lower distributor plate 12. Adjacent to the tissue lumen 15, the residual medium lumen 17 is arranged.
  • a spacer 18 is arranged.
  • the medium lumen 16 is arranged, in which the culture medium passes.
  • culture medium located in the tissue lumen 15 can be flowed through.
  • this operating mode of bioreactor 1 closed the medium-discharging channels 22 with blind plugs and the port 23 to the medium discharge. It is also possible to switch over between the two operating modes (overflow and throughflow) with continuous process control and can be used for fabrics whose mechanical properties (eg flowability) change during maturation in the cell culture.
  • the embodiment shown allows the bioreactor 1 to be screwed onto a culture medium bottle 2 by means of a thread 9 (eg GL45 thread).
  • the culture medium bottle 2 thus serves as a medium reservoir and as a base of the closed system.
  • the medium-supplying and -abblockden channels 19, 22 of the bioreactor 1 may be connected to hoses 20 which dip into the medium reservoir 24 of the culture medium bottle 2.
  • a gas-permeable filter, in particular sterile filter 25, can be used for gas exchange between the closed bioreactor system and the ambient atmosphere (for example: interior of a cell culture incubator). Continuously operating as a closed system and ensuring sterile tissue culture conditions, Bioreactor 1 meets the requirements of the WHO Good Manufacturing Practice Directive.
  • the hose connections in the system of the medium-supplying and -leaking channels 19, 22 can be used for the integration of sensors (for example: flow sensors, dO 2 sensors, pH sensors) while maintaining the closed character of the medium circuit in the bioreactor.
  • the illustrated embodiment of the bioreactor allows the application of pressure pulses and shear forces on the tissue cultivated in the tissue culture chamber 8, in particular in the tissue lumen 15.
  • an overpressure can be generated by introducing compressed air into the pressure chamber 5, which are transmitted through the elastic membrane 6 via the cover plate 14 to the upper distributor plate 12 and from there to the tissue located in the tissue culture chamber 8.
  • the support of the upper distributor plate 12 on an elastic element 1 1, in particular ring allows their movement downwards, towards the cultured tissue, but only so far, until they on the acting as a stop, solid upper abutment 10, in particular upper spacer, rests. Less the strength of the built over pressure (eg. 0.2 - 0.5 bar), but rather the height difference between elastic ring and upper spacer determines precisely to what extent the cultured tissue is compressed.
  • the illustrated embodiment of the Bioreactor 1 allows the application of pressure pulses and shear forces without interrupting the medium flow.
  • the tissue to be cultured or the cells to be cultured may also be arranged on a distributor plate 12 and not between two distributor plates 12 as shown in FIGS. 1 and 2.
  • the tissue lumen 15 connect directly to the medium lumen 16 and be limited by a cover plate 14.
  • the arrangement of the pressure chamber 5 is omitted because, for example, via a punch intermittently pressure can be exerted on the cover plate 14 and this is transmitted to the tissue or cells in the tissue lumen 15.
  • the mobility of the cover plate 14 can be achieved for example by a seal made of elastic material in the connection region with the bioreactor 1. By applying pressure, shearing forces automatically act on the cells.
  • FIG. 2a the embodiment is shown without lower spacer 18 below the distributor plate 12 in the residual medium lumen 17.
  • the tissue lumen 15 can be increased and the residual medium lumen 17 can be reduced.
  • FIG. 3 shows a variant embodiment of the bioreactor for tissue reconstruction in the opened state during the introduction of a cell-loaded collagen I sol into the bioreactor 1.
  • the cells can also be introduced without carriers, ie in suspension, or on other carriers.
  • a cell-loaded collagen I-sol are filled. After passing the collagen I sol into the gel state, it fills the entire tissue lumen 15.
  • the tissue culture chamber 8, in particular the tissue lumen 15, can be tightly closed by placing a sterile upper distributor plate 12 and a sterile elastic membrane 6 and by screwing on the bioreactor upper part 3.
  • Other application examples include the introduction of cell-loaded nonwoven and fiber materials, cell-loaded porous structures and cell-loaded composites of gel, nonwoven, fibrous and / or porous support materials into the tissue culture chamber 8.
  • the abutment 10 By varying the dimensions of the abutment 10, in particular the upper spacer, the elastic member 11, such as the ring, and the lower spacer 18, taking advantage of the existing volume in the Restmediumlumen 17 both thickness and diameter of the cultured tissue, as well as the strength of a possibly applied mechanical stimulation, eg Compression, shear force, etc., can be adjusted.
  • a possibly applied mechanical stimulation eg Compression, shear force, etc.
  • 4a-e show variants of the embodiment of the upper distributor plate 12 of the bioreactor 1 according to the invention.
  • inflowing culture medium can be taken up by a channel 27 and discharged through the recesses 13 in the ground down.
  • Webs 28 are arranged radially to achieve a uniform distribution of the culture medium. A flow through the cultured tissue located on the distributor plate 12 or between the distributor plate 12 is made possible.
  • the densely disposed webs 28 prevent the collapse of the medium-flowed medium lumen 16 of the distributor plate 12 during mechanical stimulation, e.g. during the pressure pulse.
  • the webs 28 also prevent the elastic membrane 6 when applying mechanical stimulation close to the distributor plate 12 and the tissue located thereon and thus closes the recesses 13, whereby the supply of culture medium would be interrupted.
  • culture medium can be taken through a channel 27 and discharged through a second channel 27 again. Nutrient and metabolite diffusion between the culture medium and the tissue below the plate are made possible by recesses 13 in the bottom of the plate. Both variant a and variant b can absorb pressure pulses and transmit them to the underlying tissue. In variant b in Fig. 4b, this function is taken over by a solid disc 29 with support points on the edge and in the center, so the elevations 30, the distributor plate 12.
  • a third variant c of the distributor plate 12 is shown in FIG. 4c. The elevations 30 are arranged spirally. In addition, a central elevation 30 is shown, wherein the distributor plate 12 works without this elevation 30, because the spirally arranged webs 28 can take over their function.
  • FIG. 4 d shows a further embodiment variant of the distributor plate 12, with optionally an elevation 30 being arranged centrally and individual webs 28 spaced therefrom.
  • FIG. 4e shows a laminar arrangement of the elevation 30 on a distributor plate 12.
  • the elevations can be arranged evenly over the distributor plate 12 or in the edge region denser and less close to the center.
  • distributor plate 12 Further variants of the distributor plate 12 are conceivable, whereby these direct the medium flow over and / or through the cultivated tissue and at the same time can transmit mechanical stimulation, such as pressure pulses, tensile and / or shear forces, to the cultivated tissue.
  • mechanical stimulation such as pressure pulses, tensile and / or shear forces
  • primary chondrocytes are obtained from the knee cartilage of a young pig and cultured for 14 days in a 3% collagen 1 gel in DMEM / Ham's F12 medium with 50 ⁇ g / ml ascorbate-2-phosphate and 10% FCS in the bioreactor , During the entire culture period is overflowed with culture medium. After culturing, collagen II and aggrecan are detected by immunocytochemistry.
  • Proteins are marker proteins for hyaline cartilage.
  • Part of the tissue constructs is subjected to a one-week mechanical stress program after one week of unloaded culture.
  • the unloaded culture shows low immunoreactivity to collagen II (collagen II red, nuclei blue) and aggrecan (aggrecan red, cell nuclei blue).
  • the compression requires the formation of collagen II (collagen II red, nuclei blue) and aggrecan (aggrecan red, cell nuclei blue).
  • a cartilage construct can be generated whose extracellular matrix corresponds to that of a hyaline cartilage. The dedifferentiation of the primary chondrocytes is counteracted.
  • primary chondrocytes are obtained from the knee cartilage of a young pig, dedifferentiated in a monolayer culture for 3 days and then incubated for 3 weeks in a 3% collagen I gel in DMEM / Ham's F12 medium with 50 ⁇ g / ml ascorbate-2. phosphate and 10% FCS cultured in the bioreactor. During the entire culture period, the collagen gel is overflowed with culture medium and rhythmically compressed in the interval of 12 hours for two hours at a frequency of 0.5 Hz. In the control experiment, a collagen gel is cultured for three weeks, overflowed with medium but not subjected to any mechanical compression. After culturing, collagen II and aggrecan are detected by immunocytochemistry.
  • Both proteins are marker proteins for hyaline cartilage.
  • the unloaded culture of the control experiment shows low immunoreactivity to collagen II and aggrecan. In contrast, compression promotes the formation of collagen II and aggrecan.
  • dedifferentiated primary chondrocytes can be redifferentiated and form hyaline cartilage features.
  • FIGS. 4b, 4c, 4d and 4e show further and, if appropriate, separate embodiments of distributor plate 12, again using the same reference numerals or component designations for identical parts as in the preceding FIG. 4a. To avoid unnecessary repetition, reference is made to the detailed description in the preceding figures.
  • FIGS. 1, 2, 2a, 3, 4a-e can form the subject of independent solutions according to the invention.
  • the relevant objects and solutions according to the invention can be found in the detailed descriptions of these figures.

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Abstract

L'invention concerne un bioréacteur (1) pour culture de cellules et de tissus, pour la stimulation mécanique et/ou la perfusion de cultures de tissus ou de cellules, comprenant une partie supérieure (3) et une partie inférieure (4) reliée à la précédente, présentant une chambre de culture de tissus (8), caractérisé en ce qu'au moins un plateau de distribution (12) est disposé dans la chambre de culture de tissus (8). L'invention concerne en outre un procédé de culture de cellules et de tissus dans un bioréacteur (1).
EP09749653A 2008-05-23 2009-05-25 Bioréacteur et procédé pour culture de cellules et de tissus Withdrawn EP2283109A2 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
AT0082308A AT506826B1 (de) 2008-05-23 2008-05-23 Bioreaktor und verfahren zum kultivieren von zellen und geweben
PCT/EP2009/003682 WO2009141163A2 (fr) 2008-05-23 2009-05-25 Bioréacteur et procédé pour culture de cellules et de tissus

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EP2283109A2 true EP2283109A2 (fr) 2011-02-16

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US (1) US20110111504A1 (fr)
EP (1) EP2283109A2 (fr)
AT (1) AT506826B1 (fr)
WO (1) WO2009141163A2 (fr)

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DE102010039229A1 (de) * 2010-08-11 2012-02-16 Universität Potsdam Perfusionsvorrichtung
KR20130044912A (ko) * 2011-10-25 2013-05-03 삼성전기주식회사 배양 장치 및 배양액 교체 방법
WO2014111518A1 (fr) * 2013-01-17 2014-07-24 Medibiome Ab Bioréacteur pour tissu
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AT506826B1 (de) 2010-03-15
US20110111504A1 (en) 2011-05-12
WO2009141163A2 (fr) 2009-11-26
WO2009141163A3 (fr) 2012-01-12
AT506826A1 (de) 2009-12-15

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