EP1675939A2

EP1675939A2 - Method and bioreactor for the cultivation and stimulation of three-dimensional vital and mechanically-resistant cell transplants

Info

Publication number: EP1675939A2
Application number: EP04790613A
Authority: EP
Inventors: Ronny Schulz; Augustinus Bader
Original assignee: Bionethos Holding GmbH; Universitaet Leipzig
Current assignee: BADER, AUGUSTINUS
Priority date: 2003-10-21
Filing date: 2004-10-19
Publication date: 2006-07-05
Also published as: RU2006117360A; JP2007508830A; DE10349484A1; CN1898375A; WO2005040332A3; CA2543374A1; WO2005040332A2; RU2370534C2

Abstract

The aim of the invention is a method and a bioreactor for the cultivation and stimulation of three-dimensional, vital and mechanically-resistant cell cultures which can be cultivated and stimulated within a short time of each other, or simultaneously. The bioreactor should permit a transplant cultivation according to GMP standards under guaranteed sterile conditions. The bioreactor (1) comprises a base body, connected in a pressure-tight and sterile manner to a reactor closure (21), forming at least one reactor chamber in which a deposition surface for a transplant (11) and a mini-actuator (14) are embodied. The bioreactor (1) is further provided with at least two hose coupling connectors (19), for medium introduction and medium withdrawal and for gas introduction. The invention permits a production of three-dimensional, vital and mechanically-resistant cell cultures, according to GMP standards, preferably cartilage cell constructs, which can be cultivated and stimulated simultaneously, serially, or following a controlled time plan in a closed mini-bioreactor. Transplants grown in this manner are useful as tissue replacement materials for the treatment of, for example, connective and support tissue defects, direct joint traumas, rheumatism and degenerative joint diseases and can be used in knee joint arthrosis as an alternative to conventional (operative) therapeutic application, such as, for example, microfracturing or drilling.

Description

DESCRIPTION

Process and bioreactor for cultivating and stimulating three-dimensional, vital and mechanically resistant cell grafts

The invention relates to methods and an arrangement for GMP-compliant production of three-dimensional, vital and mechanically resistant cell cultures, preferably cartilage cell constructs, which can be cultured and stimulated in a novel manner in a closed mini-bioreactor simultaneously, sequentially or according to a time-controlled sequence. These grafts grown in this way are available as tissue replacement material for the therapy of e.g. Connective and supporting tissue defects, direct joint trauma, rheumatism and degenerative joint diseases are available and can, e.g. in the case of knee osteoarthritis an alternative to conventional (operative) therapeutic approaches such as microfracturing or tapping.

Tissue engineering, which is primarily concerned with the in vitro reproduction of autologous cell material, the body tries to grow functional cell and tissue replacement structures that can be inserted into the defective tissue in a transplant step.

For this purpose, cell cultures (eg articular cartilage cells) are routinely propagated in the laboratory. The actual multiplication of these cells (eg chondrocytes) takes place in a monolayer culture on the bottom of a coated cell culture bottle or dish according to standard protocols, which also include the addition of tissue-specific growth factors, mediators and inducers. The aim of these additive factors is, for example, to stimulate the special ability of cartilage cells to synthesize sufficient extracellular matrix components (EZM) to increase the mass ratio of 1% chondrocytes to 99% extracellular matrix components, as is the case in functional articular cartilage (Stockwell RA: The cell density of human articular and costal cartilage. J Anat. 1967; 101 (4): 753-763; Hamerman D, Schubert M: Diarthrodial joints, an essay. Amer J Med. 1962 33 : 555-590).

Since this does not seem possible due to the simple addition of medium supplements, attempts are made to stimulate or stimulate these cartilage cells in a variety of ways in order to be able to cultivate suitable autologous (hyaline) cartilage replacements with a high degree of differentiation in the laboratory.

The described multiplication of cell cultures and cultivation of tissue replacement structures are associated with a number of disadvantages.

This passive cultivation of cartilage cell cultures in a two-dimensional surface culture on a simple culture dish, which is covered with nutrient medium, shows no active stimulation of the differentiable cartilage cells.

From Minuth, WW, Kloth S., Aigner J., Steiner P .: MINUSHEET perfusion culture: stimulation of a tissue-typical environment. Bioscope 1995; 4: 20-25 a concept is known which tries to circumvent this disadvantage by placing the patient cell material in an artificial support structure which resembles cartilage tissue in its biophysical properties and one allows network-like connection between the multi-layered cells and then performs perfusion cultivation in a suitable bioreactor. A large number of experiments indicate an increased ability of the cells to differentiate through increasingly synthesized ECM, which is based on this three-dimensional cultivation of chondrocytes in a wide variety of biocompatible and bioresorbable matrices, such as, for example, the hydrogels, alginates, agaroses (Benya and Shaffer: Dedifferentiated chondrocytes reexpress the differentiated collagen when cultured in agarose gels. Cell. 1982; 30: 215-224.) variable concentrations. This spatial dimension thus simulates the original conditions of the chondrocytes in living tissue, such as in the knee and hip joints, and thus represents an advantageous adaptation of in-vivo conditions.

In the case of adherent surface cultivation of the cells, the adequate supply of medium supplements is relatively simple, since these nutrients are located directly above or on the cells and allow an unimpeded exchange of substances via diffusion. On the other hand, when using three-dimensional matrices with cells embedded in them in a static cultivation scheme, concentration gradients or gradients are formed, which can limit the transport of substances in medial construct regions and thus prevent an optimal supply of nutrients to the cell groups. This impairment in the cultivation of cell material in spatial carrier matrices is countered by the induction of medium perfusion or transfusion by the construct. This active process through this carrier structure ensures a homogeneous nutrient supply to the cells and leads to a constant removal of metabolites from the chondrocytes. In addition, the dynamic cultivation scheme can guarantee a higher gas input and thus mechanically stimulates the cell aggregates depending on the selected medium perfusion flow, by means of a resulting shear force in the μPa. (Raimondi, MT, F. Boschetti, et al.: Mechanobiology of engineered cartilage cultured under a quantified fluid-dynamic environment. Biomechan Model Mechanobiol. 2002; 1: 69 - 82)

Another disadvantage with the multiplication of cells and the production of a transplant is the lack of absolute sterility of the "cell culture bottle" system. Even routine work processes, such as changing the media, cell seeding but also cell harvesting, are associated with an increased risk of infection for the cell culture contained therein. since the associated culture vessel must be opened and working in a laminar flow workbench does not ensure 100% sterility of the working environment in the sense of the "basic rules of the World Health Organization for the production of medicines and the assurance of their quality" (Good Manufacturing Practice guideline of the WHO) can be guaranteed.

Furthermore, this passive system does not allow maximum gas exchange through the diffusion-permeable cover and the nutrient medium to the cell layer on the bottom.

In order to circumvent these given disadvantages of culture bottles, the development of automated, closed bioreactor systems has been intensified in recent years Generation of tissue replacement structures accelerated. They can thus (Freed and Vunjak-Novakovic: Microgravity tissue engineering. In Vitro Cell Dev Biol Anim. 1997; 33: 381-385) offer the advantage of sterile, controllable cultivation and stimulation of three-dimensional grafts. The combination of tissue engineering with the possibilities of process and biotechnology enables the control and monitoring of selected cultivation parameters such as fumigation with C0 ₂ or 0 ₂ , temperature control, culture media exchange, sampling etc. in the bioreactor systems. (Obradovic, Carrier, Vunjak-Novakovic and Freed: Gas exchange is essential for bioreactor cultivation of tissue engineered cartilage. Biotechnol Bioeng. 1999; 63: 197-205). When designing bioreactors, it is always important to create a well-designed system in which processes can be artificially regulated. When it comes to growing a certain tissue, the bioreactor system must be able to reproduce the physiological conditions and processes in vivo as closely as possible. Each bioreactor system acts on the grown material with at least one type of mechanical stimulus.

The combination of the positive properties of a controlled bioreactor cultivation of autologous tissue replacement materials in a biogenic matrix with perfusion stimulation with culture medium therefore represents the logical consequence for ensuring an automated, sterile or GMP-suitable transplant cultivation for the cultivation of e.g. represents vital cartilage cells with increased EZM synthesis capacity.

From DE 4306661A1 and from Sittinger M, Bujia J, Minuth WW, Hammer C, Burmester GR: Engineering of cartilage tissue using bioresorbable polymer carriers in perfusion culture. Biomaterials. 1994; 15 (6): 451-456 a perfusion reactor is known in which the cells are embedded in a polymer layer and are also surrounded by an agarose capsule. This cylindrical glass reactor is flowed through with artificial nutrient medium at a flow rate of 0.016 ml / min. The reactor itself is located in an appropriate tissue incubator with standardized conditions. Sterile filters on the nutrient tank enable gas exchange with the external environment.

Further experiments with this type of reactor by Bujia J, Rotter N, Minuth W, Burmester G, Hammer C, Sittinger M: Cultivation of human cartilage tissue in a 3-dimensional perfusion culture chamber: characterization of collagen synthesis. Laryngorhinootology. 1995; 74 (9): 559-563 and Kreklau B, Sittinger M, Mensing MB, Voigt C, Berger G, Burmester GR, Rahmanzadeh R, Gross U: Tissue engineering of biphasic Joint cartilage transplants. Biomaterials. 1999; 20 (18): 1743-1749 used copolyτner fabrics made of Vicryl- and Polydioxanon layers, which are impregnated with poly-L-lysine or collagen fibers of type II. Human chondrocytes were embedded in these layers and cultured under perfusion for two weeks. Using a two-phase model of a copolymer of a polyglycolic acid and a poly-L-lactic acid (Ethicon) attached to a calcium carbonate product, the time was increased to 70 days.

Another system very similar to the above-mentioned perfusion bioreactor was developed by Mizuno S, Allemann F, Glowacki J: Effects of medium perfusion on matrix production by bovine chondrocytes in three-dimensional collagen sponges. J Biomed Mater Res. 2001; 56 (3) .368-375. In contrast to the already described reactor, this has a closed area for the artificial nutrient medium. The main part of the cultivated material is in a cylindrical glass column, which is 1 cm wide and 10 cm long. The column is filled with numerous cell / polymer scaffolds, each 7 x 15 mm in size and not additionally encapsulated. The artificial nutrient medium is fed from a reservoir through the column and the entire system at a flow rate of 300 μl / min. Using this system, bovine chondrocyte scaffolds in collagen sponges are examined for their response to perfusion over a cultivation period of 15 days.

A bioreactor device is also known from US Pat. No. 5,928,945, in which adherent cartilage cells are exposed to defined flows or shear forces in a growth chamber and consequently an increased type II synthesis of collagen was detected.

In parallel to the development of perfusion bioreactors, research groups dealt with the construction of bioreactors which exert various mechanical loading processes on explants, cell samples or cell / polymer scaffolds. When designing bioreactors for cartilage cell stimulation, their structure is based on the implementation of mechanical pressure stamps or the like. because they create uniaxial pressure on cartilage grafts to mimic the main form of stress on human cartilage. Many of these printing systems show great similarities in design.

The pressure chamber of one of Steinmeyer J, Torzilli PA, Burton-Wurster N, Lust G: A new pressure chamber to study the biosynthetic response of articular cartilage to mechanical loading. Res Exp Med (Berl). 1993, 193 (3): 137-142 developed system consists of a titanium housing, which is covered on the inside with a polyethylene layer. The test specimen with a maximum diameter of 10 mm can be placed in a container at the bottom of the chamber and covered with about 7 ml of artificial nutrient medium. Since the model does not have a perfusion system for the artificial culture medium, only batches of pressure can be generated with short cultivation times. The load system, which exerts corresponding pressure on the test specimen, consists of a porous pressure crucible that passes through the chamber closure and is moved either by simple weights or by an air cylinder with a pressure piston, which is attached above the chamber. Lee DA, Bader DL: Compressive strains at physiological frequencies influence the metabolism of chondrocytes seeded in agarose. J Orthop Res. 1997; 15 (2): 181-188 published system, which is actuated by a drive, is able to apply pressure to 24 test specimens simultaneously. The drive is mounted on a frame that runs around the incubator and transfers the power down to the loading plate inside the sterile box. The steel loading plate has 24 steel pins with a plexiglass notch of 11 mm in diameter. The drive provides various loads that are made dependent on the degree of deformation. This system is used for the cultivation of bovine chondrocyte / agarose scaffolds over a period of two days. Static and additional cyclic (0.3 to 3 Hz) loads with a maximum voltage amplitude of 15% are generated.

The disadvantage of a variety of pressure stimulation reactors is that the cell culture constructs during a Pressure load cannot be perfused with nutrient medium and therefore the effect of multiple cell irritation cannot be investigated. Furthermore, this lack of nutrients prevents an optimal metabolic exchange and the maximum synthesis of, for example, extracellular matrix components in cartilage cells.

Pressure and perfusion systems, such as are described, for example, in US Pat. No. 6,060,306 and DE Patent 198 08 055, enable simultaneous multiple stimulation with parameters such as perfusion flow, the shear forces induced thereby and a uniaxial pressure load. A disadvantage of reactors that enable pressure stimulation is above all that the pressure mediators, usually driven by servomotors or the like, usually plungers, pistons, etc. into the bioreactor room, where an autologous graft is preferably located, and then exert a defined pressure load on the cell construct. The introduction of this pressure applicator into the sterile system makes the construction of closed pressure application reactors extremely difficult, so that these systems have an increased complexity. The use of these (potentially non-sterile) systems is therefore only possible in basic research, since applications of these devices and methods in the medical field conflict with the guidelines of the existing pharmaceutical law.

All bioreactor devices that are used for breeding, but also for the stimulation of autologous tissue replacement structures, are therefore subject to the Good Manufacturing Practice Guideline of the WHO ("Basic rules of the World Health Organization for the production of Medicines and ensuring their quality "), the German Medicines Act (AMG), the" Pharmaceutical Inspection Convention "and the GMP Directive 91/356 / EEC. The risk of infection or the sterility of the systems, which cannot be fully guaranteed, therefore do not give rise to a manufacturing license in accordance with Section 13 of the AMG.

The object of the invention is to provide a method and a bioreactor for the production of three-dimensional, vital and mechanically resistant cell cultures, in which cultivation and stimulation can take place in a close temporal connection or at the same time. The bioreactor is intended to enable GMP-compliant graft cultivation under guaranteed sterile conditions.

The object is achieved by the method described in claim 1 and the bioreactor described in claim 13. Advantageous embodiments of the method are set out in claims 2 to 12; the further embodiment of the bioreactor is set out in claims 14 to 57.

The method and the bioreactor according to the invention combine the cultivation and stimulation of GMP-compliant, three-dimensional, vital and mechanically resistant cell cultures, preferably cartilage cell constructs, in one reactor. The stimulation and cultivation can be carried out simultaneously, in succession or according to a time-controlled sequence. These grafts bred in this way are available as Tissue replacement material for the treatment of, for example, connective and supporting tissue defects, direct joint trauma, rheumatism and degenerative joint diseases are available.

The essential feature of the method according to the invention and the bioreactor according to the invention is that there is a transplant in a closed reactor space which can be exposed to in vivo adaptive stimuli in several respects. This includes perfusing the spatial cell culture construct with a conditioned nutrient medium, which on the one hand causes organotypic shear forces on the cell membranes and also allows an increased metabolism. In this closed bioreactor there is a magnetic, piston-like pressure temperature1 above the graft, which acts as a stress applicator on the cell culture. The stamp is controlled contactlessly through the bioreactor room and thereby a directed uniaxial pressure stimulation on the tissue graft is brought about. The contactless control of the mini actuator is carried out by externally arranged control magnets, whose aligned (electro) magnetic field causes a change in the position of the stamp in the bioreactor and results in organotypical dynamic or static pressure stimulation.

The method and the bioreactor have the advantage already mentioned that the cell cultures can also be stimulated during the cultivation. Above all, the cultivation or regeneration of connective and support tissue structures and functional tissue systems (cartilage, bones, etc.) is possible.

With a sterile procedure, the apparatus enables the cultivation of cell transplants, in particular be synchronized, perfused and pressurized and are therefore characterized by an increased production of matrix components (e.g. cartilage cell cultures). Because of his

Degree of automation, this device minimizes the number of operations and thus reduces the risk of infection

Cell culture. Furthermore, the automated breeding and stimulation of the grafts guarantees defined and reproducible processes. Due to the

Design features of the bioreactor according to the invention ensure a closed bioreactor circuit and consequently strictly autologous cultivation or

Stimulation of tissue replacement structures in compliance with the

GMP guidelines possible.

Another area of application for the bioreactor is pharmaceutical active ingredient testing for the characterization of proliferation and differentiation-relevant substances or combinations of substances on transplants.

The method according to the invention and the bioreactor according to the invention are explained below in exemplary embodiments. The accompanying pictures show:

Fig. 1 process for the production of grafts Fig. 2 scheme GMP bioreactor system

Fig. 3 Scheme single-chamber bioreactor

Fig. 4 Scheme two-chamber bioreactor

5 construction and embodiments of the mini

Actuator Fig. 6: Scheme of construct manufacture and

Construct seeds in the bioreactor

Fig. 7: Scheme of the technical device for construct perfusion and media mixing in Single-chamber bioreactor Fig. 8: Scheme of the technical device for construct perfusion and media mixing in the bicameral bioreactor Fig. 9: Fixation scheme of the graft in the bioreactor

Fig. 10: Magnet systems for controlling the mini-actuator. Fig. 11: Stimulation scheme in the two-chamber reactor

example 1

Process for the production of grafts

In FIG. 1 the use of the bioreactor for synchronous cultivation and stimulation of three-dimensional cell transplants is shown using the example of cartilage tissue transplantation.

For this purpose, the patient is the first (I) healthy cell material

(e.g. articular cartilage) and blood taken minimally invasively. Via enzymatic digestion, the cells obtained are separated, counted and, depending on this, either sown using standard methods of tissue engineering in monolayer bottles (II) and reproduced strictly autologously or immediately sent to construct manufacture (III). The cells are inserted into a three-dimensional graft structure made of biocompatible or resorbable carrier material (e.g. hydrogels, agaroses, collagens, hydroxylapatites, polymer complexes, etc.). For this purpose, the suspended cells (eg chondrocytes) are mixed with the biogenic support structure (eg agarose), placed in a seed flask and hardened, for example, in a cylindrical graft shape (eg cartilage-agarose matrix). This in vivo adaptive, three-dimensional In the case of cells of the connective and supporting tissue (eg chondrocytes), structure leads to (re) differentiation and, with it, to a synthesis of tissue-typical substances and matrix components (eg collagens, proteoglycans).

This seeding flask with the internal spatial cell graft is inserted into the bioreactor (IV), the graft is pressed out and positioned in the bioreactor. The GMP-suitable cultivation and stimulation of this cell construct is then carried out either simultaneously, successively or in a time-controlled manner in the newly developed closed bioreactor apparatus (V). In this phase the cell transplant can be brought to an increased differentiation and expression of organotypic markers with this multiple in vivo-like stimulation (stimuli such as shear force, perfusion, deformation, mechanical stress).

After a short time, a highly vital, matrix-rich cell culture construct has regenerated in the bioreactor. This autologous graft is removed (VI), if necessary adapted to the geometry of the tissue defect, and then transplanted into the defective connective or supporting tissue. Example 2 Scheme of the bioreactor system

FIG. 2 illustrates an embodiment of the bioreactor system (with the two-chamber bioreactor) for autologous cultivation and multiple stimulation of cell transplants in a closed reactor structure using a GMP-compliant procedure.

In this exemplary embodiment, the entire device for ensuring the optimal temperature, air humidity and composition is in a temperature-controlled manner and gas adjustable incubator. Likewise, a separate construction is also possible, in which the bioreactor 1 and the medium in the incubator and the other technical components are arranged outside the incubator for control purposes. The bioreactor 1 itself and the components used therein are biologically and chemically inert and autoclavable. In addition, the bioreactor body and the screw-on lid are made of non-magnetic (e.g. plastics) or weakly magnetic materials (e.g. vanadium-4 steel). The culture medium is fed from the medium reservoir 2 through the hose system 4 with the 3-way valve 6 and the 4-way valve 7 into the bioreactor 1 by means of the circulation pump 5. This culture medium can be enriched with autologous additive factors (for example growth factors, mediators, etc.) obtained from patient blood from the supplement reservoir 3. The medium is fed to the bioreactor 1 and thus to the transplant 11 either in batch, fed-batch or in a continuous process. When the circuit is closed, the medium then passes through the hose system 4 into the medium reservoir 2, which is equipped with measuring probes for checking the physicochemical parameters, such as pH, pC0 ₂ and p0 ₂ . If the medium is considered to be used up, it can be discharged via the hose system 4 into an external, sealed-off waste container. In both cases it is possible to use the valve device 7 to derive a sterile medium sample from the reactor circuit into a sampling section 8 for further analysis. In the bioreactor 1, the graft 11 to be cultivated and stimulated lies in the medial position on the reactor floor. A second, smaller chamber can be located below the graft 11. This flow space is about the Hose system 4 is supplied with medium and can be filled with a highly porous but thin sintered material 16. This lower chamber can be sealed off by a thin clear glass pane 17 and serve as a microscope opening for inverse microscopes.

Inside the upper chamber of the bioreactor 1 there is the mini-actuator 14 next to the biosensors 9 embedded in the bioreactor cover. This mini-actuator 14, which is designed as a magnetic stamp, acts as a pressure applicator without contact and is controlled by the control magnet or the coil 15.

Example 3

Scheme single-chamber bioreactor

FIG. 3 shows a possible embodiment of the bioreactor 1 consisting of a culture chamber which is used to implement the contactlessly controllable mini-actuator 14.

The bioreactor 1, which is designed as a single-chamber bioreactor, consists of a body and the bioreactor closure 21, which is additionally sealed by a squeeze ring 20. Biosensors 9 are embedded in the cover construction, which serve for the on-line measurement of, for example, glucose and lactate concentrations, among other medium components. In the reactor space there is a precisely fitted mini-actuator 14 above the graft 11, which rests on a special reactor floor with a clear glass pane 17 inserted. To supply the transplant 11 with medium, at least one inlet and outlet each via Luer connections 19 lead into the bioreactor 1. A sampling section 8 is integrated into at least one of the outlets 19 via a 3-way valve 6. Example 4

Scheme bicameral bioreactor

FIG. 4 outlines a further embodiment of a bioreactor 1 consisting of two chamber spaces, the upper one containing the pressure stamp 14 and the lower one serving for the flow below the transplant 11. This embodiment does not differ in function, property and requirement of components 1, 6, 8, 9, 14, 19-21 from the bioreactor 1 described in Example 3. In the apparatus there are at least one inlet and outlet 19 in the upper one and lower reactor chamber let in to achieve a valve-controlled flow to the respective chamber and the graft 11.

The dimension of the lower chamber is such that its diameter is less than that of the graft 11. This chamber accommodates a flat, precisely fitting disc made of porous sintered material 16, so that inverse microscopy through the closing glass disc 17 and the membrane 18 to the transplant 11 can take place without being impaired. This disk of sintered material 16 in the lower reactor chamber fulfills another important function in the present apparatus. It prevents an undesired pressing of the gel-like cell construct 11 into the chamber space when the graft 11 is mechanically loaded by the pressure stamp 14. Depending on the support matrix used and its viscosity, the use of a fluid-permeable membrane 18 between the sintered material 16 and the graft 11 is provided in order to prevent the carrier material from being mixed with the sintered material 16. Example 5

Structure and embodiments of the mini actuator 14

FIGS. 5 show the structure, the geometry and the differing shapes of the mini-actuator 14, which slides vertically in the reactor space (here exemplarily in the two-chamber model) and transmits axial compressive forces to the graft 11 lying on the reactor floor. This magnetic pressure applicator 14 is contactless according to the invention (see FIG. 5a) by externally arranged ones

Control magnet 15 in its vertical position in

Bioreactor 1 controlled. An absolutely vertical compression can be ensured on the one hand by the medial positioning of the graft 11 in the bioreactor 1. On the other hand, the outer diameter of the pressure stamp D2 must be dimensioned to fit the inner diameter of the bioreactor D2. This enables the mini-actuator 14 to be guided vertically in the bioreactor 1 without causing the stamp to tilt or incline. In all bioreactor models, this diameter D2 is to be dimensioned larger than the outer diameter D1 of the graft 11. FIG. 5b shows the characteristic structure of this pressure unit 14. It has an extremely powerful permanent magnet 22, preferably made of a neodymium-iron-boron compound even with the slightest magnetic and electromagnetic fields moved in the respective field direction. This permanent magnet 22 is encapsulated in a lacquered or galvanized form in a biologically inert plastic — the enveloping body 23. This, preferably cylindrical, envelope body 23 slides with its precisely fitting outer diameter with little friction and exactly vertically in the bioreactor cylinder. In addition to a flat surface, other organotypical negative shapes can also be embossed as a stamp surface 24 on the underside of the plastic enveloping body 23 in order to reproduce in-vivo adaptive positive shapes (including curvatures, arches, etc.).

The novel actuator geometry present here, without any flow channels 33 in the enveloping body 23, furthermore enables a pump function due to a cyclical magnetic field generation. Due to the existing pressure and valve conditions in bioreactor 1, medium is sucked into the reactor space by an upward movement of mini actuator 14. This medium is pressed out of the bioreactor 1 by downward movement or pressure compression on the graft 11. FIG. 5 c shows a further exemplary embodiment of the mini actuator 14, which is likewise constructed from a strong permanent magnet 22 and an enveloping body 23 with an individual stamp surface 24. To optimize flow, this model has so-called flow channels 33 at the edge of its envelope 23. This enables media to flow around the mini-actuator 14 in the bioreactor space and lower actuating forces are required to overcome the media resistance. The enveloping body 23 must have at least 3 guide lugs with a precisely fitting outer diameter D2 in order to ensure a planar positioning of the entire mini-actuator 14 on the transplant 11.

FIG. 5d shows a modified pressure stamp 14 based on FIG. 5b, which has an extension web 34 to create a spatial distance between the permanent magnet 22 and the cell culture construct 11. The reason for this spacing of the permanent magnet 22 in the upper cylinder head from the graft 11 is the minimization any field influences on cell cultures 11.

FIG. 5e shows a mini-actuator 14 based on FIG. 5d, which has at least 3 flow channels 33 and 3 guide lugs with an outer diameter D2.

Example 6

Scheme of construct production and sowing in

Bioreactor 1

6 shows the method and the device for producing and sowing three-dimensional, preferably cylindrical, cell matrix constructs.

In FIG. 6a (cell matrix seed), increased (see FIG. 1, II) or freshly isolated (see FIG. 1, III) and prepared cells 12 are mixed with the biogenic carrier structure 13, suspended until homogeneous and with the target volume of the cell matrix in injected the plunger 25.

The precisely fitting seed piston 25 corresponds in its inner diameter D1 to the future outer diameter of the graft 11 and in its outer diameter D2 to the inner diameter of the bioreactor 1.

FIG. 6b (stamp insert) shows the stamp insert 26 in the seed piston 25. During the curing or polymerisation of the respective cell matrix in the reactor piston 25, the precisely fitting planar punch 26 with the outer diameter D1 is inserted into the hollow piston cylinder on the flat sliding plate 27.

The underside of this stamp 26 can be organotypically analogous to the stamp surface 24 of the mini actuator 14 Structures.

FIG. 6c (stamp attachment) shows the attachment of the stamp 26 on the transplant 11 in the seed piston 25. The stamp 26 is placed on the cell structure with a slight pressure in order to counteract meniscus formation or curvature of the matrix top of the transplant 11, e.g. to get a cylindrical graft shape. If an in vivo adaptive surface is to be imprinted on the graft 11, this stamp attachment 26 must take place during the curing or polymerization phase.

In FIG. 6d (removal of the sliding plate), the attached stamp 26 is lifted after the graft 11 has been shaped and the, preferably hydrophobic, sliding plate 27 located at the bottom of the seed piston 25 is removed. In order to prevent a gel-like cell construct 11 from adhering to the sliding plate 27 and the seed piston, e.g. an inert film or an inert polymer fleece is used to line the surfaces.

FIG. 6e (constructing seeds in the bioreactor) shows the sowing of a cylindrical construct using the example of the two-chamber bioreactor. In this case, the precisely fitting seed piston 25 is implemented in the bioreactor 1, then the cell construct 11 is introduced medially into the prepared reactor by means of a pressure stamp 26 and the sowing device is removed from the bioreactor 1. This prepared bioreactor 1 contains the porous sintered material 16 and optionally a diffusion-permeable membrane 18. Example 7

Scheme of the technical device for construct perfusion and media mixing in the single-chamber bioreactor

7 shows the construction and construction of the single-chamber reactor body and its effect on the diffusion and perfusion in the graft 11.

In the embodiment shown in FIG. 7a, four inlets and outlets with an integrated Luer coupling 19 open into the bioreactor 1. These can differ both in their position and position for flow optimization, i.e. also e.g. Enter the bioreactor body 1 tangentially. The number of inlets and outlets 19 leading into bioreactor 1 is at least two. At each flowing Luer connector 19, e.g. a 3-way valve 6 a sampling section 8 can be installed.

With a static cultivation method in the bioreactor, the medium diffuses primarily into the upper and lateral edge areas of e.g. cylindrical tissue graft 11 and supplies the cell culture and others. with nutrients and at the same time removes metabolic end products from the carrier matrix.

FIG. 7b shows a continuous supply of nutrient medium from the medium reservoir 2 with an optional one behind it

Supplement reservoir 3 (not shown) by means of a meterable circulation pump 5 through the hose system 4 into at least one inlet 19 of the bioreactor 1.

Carried out the outflow of the medium through at least one outlet 19 in the tube system ^'4, to which at least one point on a 3 -way valve 6 is a decoupled sampling route

8 can be integrated.

The medium used can, as shown here, in The circuit remains in that it enters the medium reservoir 2 and from there is used again for the continuous perfusion of the graft 11. Otherwise it can be completely removed from the circuit. The graft 11 is then cultivated in a batch or fed-batch process.

A targeted, continuous supply of culture medium into the reactor space can significantly improve the inflow and throughflow of the graft 11 in comparison to the static scheme of FIG. 7a. Due to the induced perfusion, deeper construct regions are flushed out with medium. As a result, the metabolism is optimized, which can lead to increased cell differentiation. Furthermore, this design of the flow of the construct exerts a shear force stimulation on the embedded cells.

Example 8

Scheme of the technical device for construct perfusion and media mixing in the two-chamber bioreactor

FIGS. 8 show a bicameral bioreactor which allows an optimized flow, diffusion and perfusion of the graft and thus helps to improve the quality of the tissue replacement.

A variant with static cultivation and diffusion is shown in FIG. 8a. The inlets and outlets 19 opening into the bioreactor 1 are at least two, at least one each having to open into the lower and upper reactor chambers. The two inlets and outlets 19 per chamber listed here can differ in terms of their position, position and thickness for flow optimization. The sampling section 8 can be installed on each outflow-oriented Luer connector 19 of both chambers, for example with a 3-way valve 6 or the like.

In addition to media diffusion into the upper and lateral graft areas, the chamber newly created in this construction below the graft 11 during the static cultivation phase leads to a diffusion of the culture medium from the porous sintered material into the regions of the support structure near the bottom, resulting in an improved metabolism in the entire graft 11 results.

FIG. 8b shows the continuous supply of nutrient medium from the medium reservoir 2 with an optional supplementary reservoir 3 (not shown) arranged behind it by means of a meterable circulation pump 5 or the like. through the hose system 4 into at least one inlet 19 of the lower and upper chamber of the bioreactor 1. The medium is discharged through at least one outlet 19 per chamber into the hose system 4, at which at least one point via a 3-way valve 6 uncoupled sampling section 8 can be integrated.

As shown here, the used medium can remain in the circuit by entering the medium reservoir 2 and from there being used again for the continuous perfusion of the transplant 11. Otherwise it can be completely removed from the circuit. The graft 11 is cultivated in a batch or fed-batch process. The integration of a second chamber according to the invention, here below the transplant 11, shows its positive properties, above all when the biological construct is subjected to a targeted flow. Accordingly, if the 3- Directional valve 6 of the media flow from the medium reservoir 2 into the lower chamber, an induced upward perfusion of the graft 11 takes place with the lower drain closed, since the medium can only leave the reactor space through the upper drain.

Analogous to this scheme, by switching the 3-way valves 6, a flow of grafts from the upper to the lower chamber through the construct 11 can be achieved. The result of this device shown, in addition to complete perfusion, is a further cell stimulation via an induced shear force, which acts on the cells and can be adjusted via the volume flow of the circulation pump 5. Partial or total opening of the 3-way valves 6 is also possible in order to achieve a faster medium exchange in the bioreactor 1.

Example 9

Fixation scheme of the graft 11 in the bioreactor 1

9 shows the fixation scheme of the graft 11 in the bioreactor 1 either in the single-chamber or two-chamber version.

FIG. 9a shows the graft 11 to be stimulated, which is fixed medially above the clear glass 17 in the single-chamber bioreactor 1. With at least 3 of these fixing walls 28, a horizontal movement of the graft 11 on the reactor base caused by the inflowing medium is to be prevented in order to be able to carry out an optimal perfusion and pressure stimulation. These biocompatible fixing walls 28 let into the reactor 1 must have a height below the pressure amplitude to be applied to the graft 11 be dimensioned.

FIG. 9b shows the use of at least 3 of these fixation walls 28 in the two-chamber bioreactor in order to achieve a horizontal fixation of the graft 11 in the various flow conditions and to enable ideal vertical perfusion and mechanical pressurization.

Example 10

Magnet systems for controlling the mini actuator 14

FIGS. 10 (shown on the single-chamber bioreactor) represent characteristic devices and apparatus arrangements for the contactlessly controllable stimulation method of the mini-actuator 14 on the transplant 11.

FIG. 10a (magnetic control effect of magnetic attraction) shows the characteristic device and the principle for contactless control of the magnetic

Mini-actuator 14 in bioreactor 1 for pressure deformation of the

Transplant 11 listed. The orientation of the

Permanent magnets in the mini actuator 14 take place according to the predominant magnetic field direction, which is generated by externally arranged control magnets 15. Through this

Control magnet 15, e.g. is at least one permanent magnet or at least one coil, a defined one

(Electro-) magnetic field generated, which extends with its field lines into the entire bioreactor room 1 and a field direction-dependent movement of the pressure stamp of the

Mini actuator 14 triggers. In the example in FIG. 10a, the control magnet 15 shows the principle of magnetic attraction. In the exemplary embodiment shown in FIG. 10 b (magnetic control effect - magnetic repulsion), the magnetic repulsion represents the second magnetic control effect between the magnetic control system 15 and the mini actuator 14. By changing the magnetic field direction of the control magnet 15, the direction of movement of the mini-actuator 14 changes, which is now directed downward onto the transplant 11. Increasing the power or magnetic flux density starting from the control magnet 15 also increases the pressure load on the transplant 11 up to the target value of the in vivo adaptive stimulation.

The figures 10c-10e show arrangements of control elements with which it is possible to control the mini-actuator 14 cyclically and at high frequency in a closed bioreactor 1. FIG. 10c (control of the mini-actuator 14 by means of a control magnet guide plate) represents an embodiment of a permanent magnetic control system. In this magnetic field variant, an arrangement of several permanent magnets 32 of different sizes, polarities and thus field strength and direction works on a linearly controlled guide plate 31 which is positioned here as an example above the prototype reactor.

A linear rail 29 drives a guide rail 30 with the permanent magnets 32 inserted in the magnet holder 31. This mobile phase of the magnet system makes movement of the bioreactor 1 unnecessary.

The control system in FIG. 10d (control of the mini actuator 14 by means of rotating permanent magnets) is based also on a control of the magnetic pressure stamp 14 by means of a permanent magnet arrangement on a rotating disc.

Here, an actuator 29 drives a magnet holder 31 with fitted permanent magnets 32 of alternating polarities in a rotating manner. This rotating magnet holder can be occupied, for example, with four alternately polarized magnets 32 and, as a result, causes two complete pressurizations on the graft 11 during a full revolution. The combination of this magnet occupation of the rotating disc with the rotational speed of the servomotor 29 results in a higher frequency magnetic field change and consequently a highly dynamic one Stimulation scheme for the graft 11. The front view illustrates both magnetic effects of the rotation system using two bioreactors 1. The embodiment of this device is suitable for a large number of bioreactors 1, as long as they can be placed exactly above or below the control magnet center.

FIG. 10e (control of the mini-actuator 14 by means of an iron core coil 35) represents a magnet device based on a coil arrangement. This magnet coil system works with an induction coil 35, which is fixed here above the bioreactor 1, producing a defined electromagnetic field which is distributed over the supplied electrical power can be regulated continuously and thus enables any positions of the mini-actuator 14 in the bioreactor body. By reversing the polarity of the current direction, the existing field direction and the electromagnetic effect are reversed. The iron core coil 35 used builds up its electrical field perpendicular to the coil winding and acts on it static permanent magnets of the mini actuator 14 attracting and repelling one.

An automated station of this system consists of a powerful coil 35 with low heat development with a connected adjustable transformer, the performance of which is monitored by means of a multimeter. In addition, the use of a microcontroller ensures the activation of a relay, which switches the current in the desired direction, the desired effect of an intermittent pressure application on the cell construct.

Example 11 Stimulation scheme in the two-chamber reactor

11 shows the entire stimulation scheme of the novel GMP-capable bioreactor 1. Here, the mechanical pressure stimulation, the perfusion and the shear force-induced flow in the three-dimensional graft 11 run in parallel.

In FIG. 11 a (perfusion stimulation without mechanical stress), the cell construct 11 is stimulated only via a directed flow of media, which leads to a perfusion of the construct with exerting a shear force on the

Cell membrane leads in the μPa range. This process example shows a continuous supply of nutrient media in two inlets 19, so that each reactor chamber is supplied, first a concentration compensation in the graft 11 is established and then, depending on the selected volume flows, an upper and lower perfusion zone is formed in the construct. This used medium leaves the reactor space via two further outlets 19. During this Flow is not stimulated here, since the pressure stamp 14 is held in an upper position in the bioreactor 1 by the control magnet system 15. FIG. 11 b (perfusion stimulation and stamp attachment) shows the second step of multiple stimulation of tissue replacement materials 11 in the bioreactor 1. First, as in this example, the flow conditions are modified. Via the 3-way valves 6, the nutrient medium flow is directed only into the lower reactor chamber, from where it perfuses through the graft 11, brings about the mass transfer and can leave the upper reactor chamber via a drain. By reversing the polarity of the control magnet system, here an iron core coil 35 with low power induction, the magnetic mini-actuator 14 is attached to the cylindrical tissue replacement 11, for example. This stamp attachment with 0% construct deformation marks a reversal point of the mini-actuator 14 at one dynamic high-frequency deformation of the cell matrix 11. In the next step of the stimulation method, as shown in FIG. 11c (perfusion stimulation and mechanical stress), the magnetic field strength emanating from the coil 35 is increased. The result of this increased magnetic flux density is an increased compression of the graft 11 to the desired target deformation, which preferably imitates in vivo-like processes. After this pressure stimulation has taken place, an intermittent procedure can be used to switch between cell stimulation and stamp attachment. Static compression of the replacement material is also possible with the apparatus and the method described. Basically, a can during this mechanical load directed construct perfusion are introduced through the carrier matrix, which supplies the cells with the necessary nutrients and removes metabolites, which are exchanged especially during metabolically active phases, such as during proliferation and differentiation (extracellular matrix synthesis).

After the pressure loading protocol has been processed, the stamping device is returned to its initial state, the cell culture is perfused, for example, continuously and the transplant 11 is removed, for example if the extracellular matrix is sufficiently synthesized.

Reference character list

Bioreactor Medium reservoir Supplementary reservoir Hose system Circulation pump 3-way valve 4-way valve Sampling section Biosensors Glu / Lac measuring probes pH, pC02, pθ2 Transplant cells / tissue Support matrix Mini-actuator Control magnet Porous sintered material Clear glass Permeable membrane Hose coupling connector Luer ring connector Luer ring connector Seeding piston Stamp Sliding plate Fixing wall Actuator Guide rail Magnetic holder Permanent magnet Flow channels Extension bar Coil

Claims

claims

1. A method for cultivating and stimulating three-dimensional, vital and mechanically resistant cell transplants in a GMP-suitable bioreactor, with method steps a) the explant cells (12) taken from the organism and prepared using known methods for bioreactor cultivation and from commercially available biocompatible, carrier matrices (13) consisting of resorbable or autologous or homologous materials, after they have been mixed as a cell matrix suspension b) are introduced into an optionally foiled seed flask (25) with any cross-section adapted to the later graft and cured or polymerized there, c) if appropriate, by means of this a precisely fitting, inert, optionally structured or foiled stamp (26) is loaded with minimal pressure, d) the seed piston with the graft (11) is introduced into the chamber space of the bioreactor body, e) where the graft (11) from the seed piston (25 ) on applied medially to the bioreactor bottom and the biorector (1) is closed after removal of the seed flask, f) where the graft is further cultivated by the introduction of perfusion flow and g) the tissue replacement material is removed for further use after completion of the cultivation, characterized in that the Graft is subjected to pressure during the cultivation phase on the surface opposite the bioreactor floor.

2. The method of claim 1, d a d u r c h g e k e n n z e i c h n e t that the graft is loaded by a pressure-imparting stamp.

3. The method according to claim 1 and 2, so that the mixing of the bioreactor space by the entry of perfusion flow and the pressure imparting pressure on the graft can be controlled as a function of the cultivation conditions, in terms of time and in quantity or strength, both.

4. The method according to claim 1 to 3, d a d u r c h g e k e n n z e i c h n e t that the graft is flowed through at intervals with conditioned nutrient medium and is loaded cyclically with the pressure-imparting stamp.

5. The method according to claim 1 to 4, d a d u r c h g e k e n n z e i c h n e t that the pressure loading of the graft takes place during perfusion.

6. The method according to claim 1 to 5, so that the graft is irritated, depending on the use, with static, preferably with in vivo-like pressure loads or construct deformations, or is continuously loaded with intermittent or dynamic pressure forces.

A method according to claim 1 to 6, characterized in that the mechanical loads take place at a frequency of more than 0.1 Hz.

Method according to Claims 1 to 7, that the mechanical pressure stimulation is in the form of a symmetrical or asymmetrical cosine or sine half-wave.

9. The method according to claim 1 to 8, d a d u r c h g e k e n e z e i c h n e t that the pressure stamp consisting of a magnetic material is moved longitudinally to the surface of the graft in the bioreactor by an (electro-) magnetic field generated outside the bioreactor.

10. The method according to claim 1 to 9, d a d u r c h g e k e n n z e i c h n e t that the magnetic field is generated by at least one permanent magnet.

11. The method according to claim 1 to 10, so that at least two permanent magnets with alternating polarity are arranged on a mobile holder above the bioreactor and cyclically driven by a servomotor, change their position, whereby the pressure stamp alternately presses on the graft.

12. The method according to claim 1 to 8, characterized in that the coil of an electromagnet high-frequency changes their current direction and voltage and thus field direction and magnetic flux density via the bioreactor, whereby the pressure stamp alternately presses on the graft.

13. Bioreactor for cultivating and stimulating three-dimensional, vital and mechanically resistant cell transplants in a GMP-suitable bioreactor, characterized in that the bioreactor (1) is connected in a pressure-tight and sterile manner from a base body to a reactor closure (21) and forms at least one reactor space, in which a storage area for a transplant (11) and a mini actuator (14) is implemented and the bioreactor (1) is provided with at least two hose coupling connections (19) for the medium supply and medium discharge or for gassing.

14. The apparatus of claim 13, so that the cell culture constructs (11) can be cultured and stimulated directly or indirectly on the bioreactor base of a single-chamber bioreactor (1).

15. The apparatus according to claim 13, characterized in that the cell culture constructs (11) lies directly or indirectly, at least partially, on a bottom surface of the upper reaction chamber of a two-chamber bioreactor for cultivation and stimulation, while a second reactor space is located below this graft (11).

16. The apparatus of claim 14 or 15, so that the bioreactor (1) is provided with a graft insert on the bottom of the upper reactor chamber into which the cell constructs (11) can be inserted.

17. Device according to one of claims 13 to 16, so that the container of the bioreactor (1) has a hollow cylindrical body which is closed on the top by a reactor closure (21).

18. The apparatus according to claim 13 to 17, characterized in that the reactor closure unit or units (21) and the bioreactor (1) are connected to one another by a threaded connection and at least one sealing ring (20) such that either the threaded connection between the reactor closure (21) and the container (1) by means of an internal thread in the container (1) and a cooperating external thread in the reactor closure (21) or the threaded connection between the reactor closure (21) and the container (1) by an external thread in the container (1) and an internal thread cooperating therewith is produced in the reactor closure (21).

19. The apparatus of claim 13 to 18, so that the reactor closure (21), which is designed as a cover, is provided with biosensors (9) and / or measuring probes (10).

20. The apparatus of claim 13 to 19, dadurchge indicates that the cover (21) is provided with a sampling section (10).

21. The device according to claims 13 to 20, so that the base body of the bioreactor (13) is designed as a single-chamber bioreactor with at least one inlet and outlet bore for the establishment of hose coupling connections (19).

22. The device according to claims 13 to 20, so that the basic body of the bioreactor (1) in the design as a two-chamber bioreactor is provided with at least two bores for the establishment of hose coupling connections (19).

23. The device according to claim 22, so that at least one hose coupling connection (19) is let into the lower and upper reaction chamber in the two-chamber bioreactor.

24. The device according to claim 21 to 23, characterized in that the inlet connections (19) and outlet connections (19) opening into the bioreactor chambers are provided with a 3-way valve (6) or with a 4-way valve (7) with a non-return function are.

25. The device according to claim 24, characterized in that on at least one of the drain connections (19) are provided with a sampling section (10).

26. The device according to at least one of claims 13 to 25, that the bioreactor (1) on the reactor base is made entirely or partially of a transparent material, preferably a clear glass pane (17), for monitoring the transplant production.

27. The device according to claim 13 to 26, characterized in that a film, fleece or membrane (18) made of an antistatic or inert material for supporting the graft (11) is arranged above the reactor bottom of the bioreactor (1) and this material is preferably wide-meshed, is permeable to light, fluid and gas.

28. The device according to at least one of claims 13 to 27, characterized in that the upper reactor chamber of the bioreactor (1) corresponds to the base surface of the graft when it is designed as a two-chamber reactor, while the dimensions of the lower chamber are below those of the graft (11). lie, so that with a medial cell culture overlay, this construct is largely above the lower chamber and partly on the reactor floor of the upper chamber.

29. The device according to claim 28, characterized in that the space of the lower reactor chamber fills a flat disc (16) made of biologically inert, translucent, wide-pored material, preferably made of porous sintered material, so that a flat reactor bottom end from this disc (16) and the bottom of the upper reactor chamber is formed.

30. The device according to claim 28 and 29, characterized in that above the through the disc (16) filled lower reactor chamber on the reactor bottom of the two-chamber bioreactor (1) forming in the upper reactor chamber, a film, fleece or membrane (18) made of an antistatic or inert material for supporting the graft (11) is arranged and this material is preferably wide-meshed, light, fluid and gas permeable.

31. The device according to claim 28 to 30, characterized in that the components, which are located in the two-chamber bioreactor (1) below the graft (11), such as the transparent disc (17), the lower chamber with inserted porous material (16) and one wide-meshed membrane (18), do not exceed a total height within the focal length of commercially available microscope and camera lenses.

32. Device according to claim 13, characterized in that a magnetic, preferably piston-like, mini-actuator (14) is arranged in the bioreactor (1), which is controlled by one or more externally arranged control and control magnets (15) through the bioreactor (1 ) is moved in a controlled manner.

33. Device according to claim 13 to 32, characterized in that the mini-actuator (14) in the single-chamber bioreactor (1) above the membrane (18) and the graft (11) is arranged in the bioreactor chamber.

34. Device according to claim 13 to 32, so that the mini-actuator (14) in the two-chamber bioreactor (1) is arranged above the porous material (16), the membrane (18) and the graft (11) in the upper reactor space.

35. Device according to claims 32 to 34, characterized in that the magnetic mini-actuator (14) to be implemented is constructed from a magnetic core body (22), preferably from a permanent magnet, which is located in a biologically inert envelope body (23) , preferably plastic, is encapsulated.

36. Device according to claim 32 to 35, characterized in that the magnetic core body (22) is oriented such that the field forming between the poles runs perpendicular to the graft (11), so that the magnetic north pole of the entire mini-actuator (14 ) is oriented upwards.

37. Device according to claim 32 to 36, so that the biocompatible enveloping body (23) surrounding the core magnet (22) corresponds in its outer shape to the cross-sectional shape of the reactor chamber of the bioreactor (1).

38. Apparatus according to claim 32 to 37, characterized in that the total height of the enveloping body (23) is selected such that it is in an implementation of the mini-actuator (14) in the reactor space to a vertically oriented guide of the pressure stamp of the mini-actuator ( 14) on that Graft (11) is coming.

39. Apparatus according to claim 32 to 38, characterized in that the piston-shaped mini actuator (14) consists of more than one envelope cylinder, so that an envelope cylinder, preferably the upper one, contains the encapsulated permanent magnet and a further cylinder is used for stamping (24) , The spatially separate cylinders being connected to one another via a web (34) or a functionally identical connection.

40. Device according to claim 32 to 39, so that the planar stamp surface (24) formed by the enveloping body (23) on the underside of the mini-actuator (14) runs perpendicular to the guide direction in the bioreactor chamber (1).

41. Apparatus according to claim 32 and 40, d a d u r c h g e k e n e z e i c h n e t that on the stamp surface (24) of the mini-actuator (14) organotypical negative forms, such as e.g. a bulge, are embossed.

42. Apparatus according to claim 32 and 41, so that the planar or shaped stamp surface (24) has an embossing in the form of a lattice structure to enlarge the stamp surface, preferably with a small-mesh structure.

43. Device according to claim 32 to 42, characterized in that the enveloping body (24) of the mini-actuator (14) is provided with bores and / or .flow channels (33) such that over At least 3 places of the outside diameter, a precise, vertical guidance of the mini actuator (14) is further guaranteed.

44. Apparatus according to claim 32 to 43, so that the stamp surface (24) of the mini-actuator (14) is provided with at least one guide lug, which slides vertically in its precisely inserted guide rail in the bioreactor body.

45. Device according to claim 32 to 44, characterized in that the control and control magnet (15) arranged outside the bioreactor (1) with the (electro-) magnetic field emanating from it, a directional movement of the implemented mini-actuator (14), with its upward magnetic north pole of the permanent magnet (22).

46. Apparatus according to claim 32 to 45, characterized in that the control and control magnet (15) in a vertical axis to the pressure stamp (14) is arranged medially, preferably above the pressure stamp (14), and depending on the polarity the mini Actuator (14) moves up and down, resulting in a change in pressure on the graft (11).

47. Device according to claim 32, characterized in that the control and control magnet (15) consists of at least two permanent magnets (32) with different vertical magnetic pole directions, which are inserted into a cuboid magnet holder (31) and are cyclically displaced in their horizontal position by an actuator (29) and a guide rail (30) via the bioreactor (1).

48. Device according to claim 32, characterized in that the control and control magnet (15) consists of at least two permanent magnets (32) with different vertical magnetic pole directions, which are inserted into a disk-shaped magnet holder (31) and by the rotary drive of a servomotor (29). be cyclically passed over the bioreactor (1).

49. Apparatus according to claim 32, characterized in that the bioreactors fixed in their horizontal position are brought up by means of a vertical movement to the magnet holder (31) with the permanent magnets (32) by means of a stepping motor in order to increase the respective field effect and to exert higher pressure forces on the Generate graft (11).

50. Apparatus according to claim 49, so that at least two bioreactors (11) are arranged in a station in such a way that their mini-actuators (14) are driven contactlessly by only one permanent magnetic control system.

51. Device according to claim 32, characterized in that the control and control magnet (15) is designed as an electromagnet with at least one induction coil (35), the field of which is infinitely variable by means known per se.

52. Apparatus according to claim 32 and 51, so that the induction coil (35) is actuated at high frequency with a variable frequency in order to produce highly dynamic magnetic field changes and movements of the mini-actuator (14) on the graft (11).

53. Device according to claim 13 to 52, characterized in that a, preferably cylindrical, seed piston (25) with an inner diameter which corresponds to the outer diameter of the graft is provided for injecting the cells (12) and the carrier matrix (13) onto a movable one Sliding plate (27).

54. Device according to claim 13 to 53, so that the movable sliding plate (27) and the interior of the seed piston (25) are covered with an inert membrane, film or polymer fleece.

55. Device according to claims 53 and 54, so that a precisely fitting stamp (26) with a planar stamp surface or an organotypical negative shape is easily placed on the cell suspension in the seeding flask (25).

56. Apparatus according to claim 53 to 55, so that the outer diameter of the seed piston (25) corresponds exactly to the inner diameter of the bioreactor (1).

57. Device according to claim 13 to 56, dadurchge indicates that at least 3 fixation walls (28) are embedded in the reactor bottom of the bioreactor (1), the dimensions of which correspond to a graft insert and do not hinder pressure compression.