DE10349484A1 - Method and bioreactor for culturing and stimulating three-dimensional, vital and mechanically resistant cell transplants - Google Patents

Abstract

It is the object to provide a method and a bioreactor for the production of three-dimensional, vital and mechanically resistant cell cultures, which can be cultivated and stimulated in close temporal relation or simultaneously. The bioreactor should enable GMP-compliant graft cultivation under guaranteed sterile conditions. DOLLAR A The bioreactor (1) consists of a base body which is pressure-tight and sterile connected to a reactor closure (21) and forms at least one reactor space in which a shelf for a graft (11) and a mini-actuator (14) are implemented , Furthermore, the bioreactor (1) is provided with at least two hose coupling connections (19) for the medium supply and medium discharge or for gassing. DOLLAR A The invention enables a GMP-compliant production of three-dimensional, vital and mechanically resistant cell cultures, preferably cartilage cell constructs, which can be cultivated and stimulated in a closed mini-bioreactor simultaneously, sequentially or after a timed sequence. These so bred grafts are available as a tissue replacement material for the treatment of z. As connective and supporting tissue defects, direct articular trauma, rheumatism and degenerative joint diseases available and can be such. B. in knee osteoarthritis an alternative to the conventional (surgical) therapeutic approaches such. B. the ...

Description

The 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, hereby in a novel way in a sealed mini-bioreactor simultaneously, consecutively or cultured and stimulated according to a timed sequence can be. These so bred Transplants serve as a tissue replacement material for the treatment of e.g. Connective and supporting tissue defects, direct articular trauma, rheumatism and degenerative joint disease to disposal and can such as. An alternative to the conventional knee joint osteoarthritis (operative) therapeutic approaches such as. the microfracturing or tapping.

At the Tissue engineering, which is mainly the in vitro propagation of endogenous, so-called autologous cell material, one tries functional To grow cell and tissue replacement structures in a transplant step can be introduced into the defective tissue.

For this are routinely cultivated in the laboratory cell cultures (e.g., articular cartilage cells). The actual propagation these cells (e.g., chondrocytes) are in a monolayer culture on the bottom of a coated cell culture flask or dish according to standard protocols, which also includes the addition of tissue-specific Contain growth factors, mediators and inducers.

The The goal of these additive factors is, for example, the special one ability of cartilage cells, sufficient extracellular matrix components (ECM) in order to determine the mass ratio of in vitro propagation of 1 chondrocytes to 99% extracellular matrix components, such as it is given 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).

There this is not possible due to the simple addition of medium supplements, is trying to get these cartilage cells in different ways and Way to stimulate or stimulate to appropriate autologous (hyaline) Growing cartilage replacement with high degree of differentiation in the laboratory can.

The described propagation of cell cultures and breeding tissue replacement structures is associated with a number of disadvantages.

These 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 differentiable cartilage cells.

Out Minuth, W.W., Kloth S., Aigner J., Steiner P .: MINUSHEET Perfusion Culture: Stimulation of a tissue-typical environment. Bioscope 1995; 4: 20-25 is a concept known which tries to circumvent this disadvantage, by placing the patient cell material in an artificial support structure, the resembles the cartilaginous tissue in its biophysical properties and a network-like connection between the multi-layered cells allows and then a perfusion culture in a suitable bioreactor performs. A variety of experiments shows an increased differentiation ability the cells by increasingly synthesized ECM, which on this Three-dimensional cultivation of chondrocytes in a variety of biocompatible and bioresorbable matrices, such as e.g. hydrogels, alginates, Agaroses (Benya and Shaffer: Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell. 1982; 30: 215-224.) Variable concentrations.

These thus created spatial dimension simulates the original conditions chondrocytes in living tissue, e.g. in the knee and hip joint, and thus provides an advantageous adaptation of in vivo conditions represents.

at the adherent Surface cultivation of Cells designed the adequate supply of medium supplements Relatively simple, because these nutrients immediately above or at the cells and an unimpeded mass transfer via diffusion allow.

at the use of three-dimensional matrices with embedded therein Cells in a static cultivation scheme, on the other hand, are produced for the formation of concentration gradients or gradients, which can limit the mass transport into medial construct regions and thus an optimal nutrient supply at the cell aggregates opposes.

These impairment in the breeding of Cellular material in spatial support matrices is induced by the induction of medium perfusion or transfusion encountered by the construct.

This active process by this carrier structure ensures a homogeneous nutrient supply to the cells and leads to a steady metabolite removal from the chondrocytes. In addition, the dynamic cultivation scheme ensure a higher gas input and thus stimulates the cell associations depending on the chosen medium perfusion flow, mechanically induced by a shearing force in 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).

One further disadvantage in the multiplication of cells and a graft production consists in the non-given absolute sterility of the system "cell culture bottle." Even routine operations, such as e.g. the change of media, the cell insemination but also the cell harvest are with increased Danger of infection for the cell culture contained therein connected because the associated culture vessel are opened does not have to and by working in a laminar flow workbench 100% sterility the working environment in the sense of the "World Health Organization Principles for the Producing medicines and ensuring their quality "(Good Manufacturing Practice Policy WHO).

Farther is by this passive system no maximum gas exchange through the diffusion-permeable Lid, as well as the nutrient medium possible to the cell layer located on the ground.

In order to avoid these particular disadvantages of culture bottles, the development of automated, completed bioreactor systems for the generation of tissue replacement structures is increasingly being promoted in recent years. Thus, (Freed and Vunjak-Novakovic: Microgravity tissue engineering, In Vitro Cell Dev Biol Anim., 1997; 33: 381-385) can provide the advantage of sterile, controllable culture and stimulation of three-dimensional grafts. By combining tissue engineering with the possibilities of process and biotechnology, control and monitoring of selected cultivation parameters such as fumigation with CO ₂ or O ₂ , temperature control, culture media exchange, sampling, etc. are provided 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).

at The design of bioreactors is always well thought out To create a system in which artificial Processes can be regulated. If it is a question of cultivating a specific tissue, the system of Bioreactor to be able to understand the physiological conditions and processes as much as possible in vivo to adjust exactly. Each bioreactor system works with at least a kind of mechanical stimulus on the bred material.

The shortcut the positive characteristics of controlled bioreactor cultivation autologous tissue replacement materials in a biogenic matrix below Perfusion stimulation with culture medium therefore represents the logical Consequence to the guarantee an automated, sterile or GMP-suitable graft cultivation for breeding from e.g. vital cartilage cells with increased EZM synthesis performance.

From the DE 4306661 A1 and 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 perfused with artificial nutrient medium, at a flow rate of 0.016 ml / min. The reactor itself is located in a corresponding tissue incubator with standardized conditions. Sterile filters on the culture media allow gas exchange with the outside environment.

Further experiments with this type of reactor from 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. Laryngorhinootologie. 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 copolymer fabrics of Vicryl and Polydioxanone layers coated with poly-L-lysine or collagen fibers of the type II soaked are. Human chondrocytes were embedded in these layers and cultured under perfusion for two weeks. Under use 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 span was increased to 70 days.

Another system very similar to the above-mentioned perfusion bioreactor was described 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 previously described reactor, this has a closed area for the artificial nutrient medium. The majority of the cultured material is contained in a cylindrical glass column which is 1 cm wide and 10 cm long. The pillar is with numerous Filled cell / polymer scaffolds, each having a size of 7 × 15 mm and are not additionally encapsulated. The artificial nutrient medium is passed through the column and the entire system from a reservoir at a flow rate of 300 μl / min. Using this system, bovine chondrocyte scaffolds in collagenous sponges are assayed for their response to perfusion over a 15 day culture period.

Out U.S. Patent 5,928,945 is also a bioreactor device known in the adherent Cartilage cells in a growth chamber defined currents or shear and consequently an enhanced type II collagen synthesis was detected.

Parallel Research groups focused on the development of perfusion bioreactors with the construction of bioreactors, which have various mechanical load operations on explants, cell samples or cell / polymer scaffolds. In the construction of Bioreactors for cartilage cell stimulation is aimed at their construction on the implementation of mechanical stamps or similar out there, these uniaxial pressure on cartilage grafts produce the most important Form of strain to mimic the human cartilage tissue. Many of these printing systems are very similar in construction clear.

The Pressure chamber one by 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, 1993 (3): 137-142 developed system consists of a Titangehäuse, the is covered inside with a polyethylene layer. The trial copy with a maximum diameter of 10 mm can be placed in a container at Placed in the bottom of the chamber and covered with approximately 7 ml of artificial nutrient medium. There the model does not have a perfusion system of the artificial culture medium, are only batchwise pressure generation with short cultivation times possible. The load system, the corresponding pressure on the experimental copy exerts consists of a porous pressure crucible leading through the chamber closure and is made either by simple weights or an air cylinder with pressure piston, the over the chamber is attached, moved.

Also that by 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 System that is set in motion by a drive is in the Able to exert pressure on 24 trial copies at the same time. Of the Drive is mounted on a frame, which leads around the incubator and transmits the power down to the pallet inside the sterile box. The pallet made of steel has 24 steel bolts with a Plexiglas notch of 11 mm diameter. The drive ensures different loads, depending on the degree of deformation be made. This system is used for the cultivation of bovine chondrocyte / agarose scaffolds via a Period of two days used. Static and additional cyclic (0.3 to 3 Hz) loads with a maximum voltage amplitude of 15% are generated.

Of the Disadvantage, a variety of pressure-stimulation reactors is that the cell culture constructs during a pressure load is not perfused with nutrient medium can be and thus did not study the effect of multiple cell challenge can be. Furthermore, this lack of nutrient supply is optimal Metabolic exchange and maximum synthesis of e.g. extracellular matrix components in cartilage cells.

Print- and perfusion systems, as described, for example, in U.S. Patent 6,060,306 and U.S. Pat DE Patent 198 08 055, allow a simultaneous Multiple stimulation with parameters such as perfusion flow, the Shear forces induced thereby and a uniaxial pressure load.

adversely in reactors that allow a pressure stimulation is especially that driven by servomotors or the like Pressure mediators, mostly plungers, pistons et al in the bioreactor room, in which preferably an autologous Transplant is introduced and then a defined pressure load on the cell construct exercise. By introducing this pressure applicator into the sterile system the design of sealed pressure application reactors is extremely difficult so that these systems increased complexity exhibit. One use of these (potentially unsterile) systems is therefore only given in basic research, as an applications these devices and methods in the medical field Contrary to the guidelines of the existing drug law.

Thus, all bioreactor equipment subject to breeding, but also a stimulation of autologous tissue replacement structures are the WHO Good Manufacturing Practice ("Principles of the World Health Organization for the production of medicines and ensuring their quality"), continue to the German Medicines Act (AMG), the "Pharmaceutical Inspection Convention" and the GMP Directive 91/356 / EEC. The risk of infection or the not fully guaranteed sterility of the systems therefore do not give rise to any Manufacturing license according to § 13 of the AMG.

The The object of the invention is a method and a bioreactor for the production of three-dimensional, vital and mechanically resistant cell cultures to create, where cultivated in close temporal context or simultaneously and can be stimulated. The bioreactor should be GMP-compliant Allow graft cultivation under guaranteed sterile conditions.

The The object is achieved by the The method described in claim 1 and that described in claim 13 Bioreactor solved. Advantageous embodiments of the method are set forth in claims 2 to 12; the Further embodiment of the bioreactor is described in claims 14 to 57 executed.

The inventive method and the bioreactor according to the invention combine the cultivation and stimulation of GMP-produced, three-dimensional, vital and mechanically resistant cell cultures, preferably cartilage cell constructs in a reactor. Here can stimulation and cultivation simultaneously, consecutively or after a timed sequence. These so bred grafts are used as tissue replacement material for the therapy of e.g. connective and supporting tissue defects, direct articular trauma, rheumatism and degenerative joint disease to disposal.

The essential feature of the method and the bioreactor according to the invention is that in a closed reactor room a graft which is in several respects in vivo adaptive stimuli can be suspended. Also includes a perfusion of the spatial cell culture construct with a conditioned nutrient medium, which on the one hand causes organotypic shear forces on the cell membranes and about that an elevated Metabolic exchange allowed. Located in this closed bioreactor above the graft a magnetic, piston-like Plunger that acts as a load applicator on the cell culture. The stamp is contactlessly controlled by the bioreactor space and doing a directed uniaxial pressure stimulation on the tissue graft brought about. The non-contact Control of the mini-actuator is done by externally arranged control magnets, their aligned (electric) magnetic field a change in position of the stamp in the bioreactor causes an organotypic dynamic or static pressure stimulation.

The Method and the bioreactor have the advantage already mentioned, that while of breeding also a stimulation of the cell cultures can take place. It is before especially the breeding or regeneration of connective and supporting tissue structures and functional Tissue systems (cartilage, bone, etc.) possible.

The Apparatus allows in a sterile procedure, the growth of cell transplants which, in particular synchronously, perfused and pressure-loaded and thus by an increased Production of matrix constituents (e.g., cartilage cell cultures). Due to its degree of automation, this device minimizes the Number of operations and thus reduces the risk of infection of the cell culture. Still guaranteed the automated breeding and stimulating the transplants defined and reproducible Processes. Due to the design features of the bioreactor according to the invention a completed bioreactor circulation is guaranteed and consequently a strictly autologous cultivation or stimulation of tissue replacement structures in compliance with the GMP guidelines possible.

For the bioreactor results as another application, the pharmaceutical drug testing for characterization of proliferation and differentiation relevant substances or Substance combinations on transplants.

in the The following is the method according to the invention and the bioreactor according to the invention in exemplary embodiments explained. The accompanying pictures demonstrate:

1 : Method of making transplants

2 : Scheme GMP bioreactor system

3 : Scheme single chamber bioreactor

4 : Scheme two-chamber bioreactor

5 : Construction and embodiments of the mini-actuator

6 : Scheme of construct fabrication and construct seed in bioreactor

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

8th : Scheme of the technical device for construct perfusion and media throughput in the two-chamber bioreactor

9 : Fixation scheme of the graft in the bioreactor

10 : Magnetic systems for controlling the mini-actuator

11 : Stimulation scheme in the two-chamber reactor

example 1

method for the production of transplants

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

To becomes the patient's first (I) healthy cell material (e.g., articular cartilage) and blood taken in a minimally invasive manner. About enzymatic digestion the cells obtained are isolated, counted and dependent of it either over Standard Methods of Tissue Engineering in Monolayer Bottles (II) seeded and increased autologous or immediately added to construct preparation (III). Here, the cells are transformed into a three-dimensional graft structure, made of biocompatible or absorbable carrier material (e.g., hydrogels, Agaroses, collagens, hydroxyapatites, polymer complexes, etc.), brought in. For this, the suspended cells (e.g., chondrocytes) with the biogenic support structure (e.g., agarose), placed in a seed flask and e.g. in a cylindrical graft form (e.g., cartilage-agarose matrix) hardened. This in vivo adaptive, three-dimensional structure performs especially in cells of connective and supporting tissue (e.g., chondrocytes) to a (re) differentiation and, consequently, to a synthesis tissue-specific substances and matrix components (e.g., collagens, proteoglycans).

This Seed piston, with the internal spatial cell graft, is introduced to the bioreactor (IV), the graft is squeezed out and positioned in the bioreactor. Subsequently GMP-compliant either concurrent, consecutive or timed cultivation and stimulation of this cell construct in the newly developed closed bioreactor apparatus (V). In this Phase can be similar to the cell graft with this multiple in vivo Irritation (stimuli such as shear, perfusion, deformation, mechanical stress) to an increased Differentiation and expression of organotypic markers are brought.

To In a short time a highly vital, matrix-rich cell culture construct has developed regenerated in the bioreactor. This autologous transplant will taken from (VI), optionally to the geometry of the tissue defect adjusted and then into the defective connective or supporting tissue transplanted.

Example 2

Scheme of bioreactor system

2 Figure 1 illustrates one embodiment of the bioreactor system (with the dual chamber bioreactor) for autologous culture and multiple stimulation of cell transplants in a closed reactor structure with GMP-compliant procedure.

In this embodiment, the entire device is to ensure the optimum temperature, humidity and composition in a temperature-controlled and gastregulierbaren incubator. Likewise, a separate structure is possible, in which the bioreactor 1 and the medium in the incubator and the other engineering components are arranged outside the incubator for control.

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 consists of non-plastics (eg plastics) or weak-magnetic materials (eg vanadium-4 steel).

The culture medium is from the medium reservoir 2 through the hose system 4 with the 3-way valve 6 and the 4-way valve 7 by means of the circulation pump 5 in the bioreactor 1 guided. This culture medium can be used with autologous additive factors (eg growth factors, mediators, etc.) derived from the patient's blood from the supplement reservoir 3 be enriched. The medium is either the batch, fed-batch or continuous process the bioreactor 1 and thus the graft 11 fed.

The medium then passes through the hose system when the circuit is closed 4 in the medium reservoir 2 which is equipped with measuring probes for controlling the physicochemical parameters, such as pH, pCO ₂ and pO ₂ . If the medium is considered to be depleted, it may be via the tubing system 4 be discharged into an external, sealed waste container. In both cases, there is the possibility of the valve device 7 a sterile medium sample from the reactor loop into a sampling line 8th for further analysis.

In the bioreactor 1 lies the graft to be cultivated and stimulated 11 in medial position on the reactor floor. Below the graft 11 There may be a second, smaller chamber. This flow space is via the hose system 4 supplied with medium and can with a highly porous but thin sintered material 16 filled out. This lower chamber can pass through a thin clear glass 17 be sealed off and serve as a microscope opening for inverse microscopes.

Inside the upper chamber of the bioreactor 1 Located next to the biosensors embedded in the bioreactor lid 9 the miniature actuator 14 , This designed as a magnetic stamp mini-actuator 14 acts as a pressure applicator contactless, and is by the control magnet or the coil 15 controlled.

Example 3

Scheme single chamber bioreactor

3 shows a possible embodiment of the bioreactor 1 consisting of a culture chamber, the implementation of the contactless controllable mini-actuator 14 serves.

The single-chamber bioreactor bioreactor 1 consists of a body and the bioreactor closure 21 which also has a squeeze ring 20 is sealed. In the lid construction are biosensors 9 which are used for online measurement of, for example, glucose and lactate concentrations and other medium components. In the reactor room is a precisely inserted mini-actuator 14 above the graft 11 , which on a special reactor floor with inserted clear glass pane 17 rests.

For medium supply of the transplant 11 lead at least one each inlet and outlet via Luer connections 19 in the bioreactor 1 , At least one of the processes 19 is via a 3-way valve 6 a sampling route 8th integrated.

Example 4

Scheme two-chamber bioreactor

4 outlines another embodiment of a bioreactor 1 consisting of two chamber chambers, the upper one being the plunger 14 includes and the lower of the flow below the graft 11 serves. This embodiment differs in function, property and requirement of the components 1 . 6 . 8th . 9 . 14 . 19 - 21 not from the bioreactor described in Example 3 1 ,

In the apparatus are at least one each inlet and outlet 19 embedded in the upper and lower reactor chamber to a valve-regulated flow of each chamber and the graft 11 to reach.

The lower chamber is dimensioned so that its diameter is less than that of the graft 11 is. This chamber takes a flat, accurately fitting disc of porous sintered material 16 on, giving an inverse microscopy through the final glass pane 17 and the membrane 18 to the graft 11 undisturbed. This disc of sintered material 16 in the lower reactor chamber fulfills another important function in the present apparatus. It prevents, with mechanical load of the transplant 11 through the plunger 14 undesired inward compression of the gel-like cell construct 11 in the chamber space. 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 provided to a mixing of the carrier material with the sintered material 16 to prevent.

Example 5

Construction and embodiments of the mini-actuator 14

The 5 show the structure, the geometry and differing shapes of the miniature actuator 14 , which precisely slides in the reactor space (here exemplarily in the two-chamber model) vertically and axial compressive forces on the lying on the reactor bottom graft 11 transfers.

This magnetic pressure applicator 14 is according to the invention (see 5a ) Non-contact by externally arranged control magnet 15 in its vertical position in the bioreactor 1 controlled. Absolutely vertical compression can be achieved on the one hand by the medial positioning of the transplant 11 in the bioreactor 1 be ensured. On the other hand, a tailor-made dimensioning of the plunger outer diameter D2 must be made to the bioreactor inner diameter D2. This allows vertical guidance of the mini-actuator 14 in the bioreactor 1 without causing tilting or skewing of the punch. For all bioreactor models, this diameter D2 must be dimensioned larger than the outside diameter D1 of the graft 11 ,

5b represents the characteristic structure of this printing unit 14 He has an extremely powerful permanent magnet 22 , preferably of a neodymium-iron-boron compound, which is already at the lowest magnetic and elec tromagnetischen fields after the respective field direction moves. This permanent magnet 22 is in a painted or galvanized form in a biologically inert plastic - the envelope body 23 - encapsulated. This, preferably cylindrical, enveloping body 23 slides with its precisely fitting outer diameter friction and exactly vertical in the bioreactor cylinder. On the underside of the plastic envelope 23 In addition to a flat surface, other organotypical negative forms can also be used as a stamp surface 24 be imprinted to adapt in vivo adaptive positive forms (including vaults, arches, etc.).

The present novel actuator geometry, without any flow channels 33 in the envelope 23 , also allows a pumping function due to a cyclic magnetic field generation. Due to the existing pressure and valve conditions in the bioreactor 1 is due to an upward movement of the miniature actuator 14 Suction medium in the reactor space. By downward movement or pressure compression on the graft 11 This medium is removed from the bioreactor 1 forced out.

5c shows another exemplary embodiment of the mini-actuator 14 also made of a strong permanent magnet 22 and an enveloping body 23 with individual stamp area 24 is constructed. This model features flow optimization at the edge of its envelope 23 so-called flow channels 33 on. This is a media flow of the mini-actuator 14 in Bioreaktorraum possible and it will be required lower actuating forces to overcome the media resistance. The envelope body 23 must be at least 3 Guide tabs with a matching outside diameter D2 to a planar positioning of the entire mini-actuator 14 on the graft 11 sure.

5d represents a modified printing stamp 14 based on 5b which is to create a spatial distance of permanent magnets 22 and cell culture construct 11 an extension bar 34 having. Cause of this distancing of the permanent magnet 22 in the upper cylinder head to the graft 11 is the minimization of any field effects on cell cultures 11 ,

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

Example 6

Scheme of construct preparation and construct seed in the bioreactor 1

The 6 illustrate the method and apparatus for making and seeding three-dimensional, preferably cylindrical, cell matrix constructs.

In the 6a (Cell matrix seed) are propagated (see 1 , II) or freshly isolated (see 1 , III) and prepared cells 12 with the biogenic carrier structure 13 blended, suspended to homogeneity and with the target volume of the cell matrix in the seeding flask 25 injected.

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

In the 6b (Stamp insert) is the punch insert 26 in the sowing flask 25 shown. During the curing or polymerizing of the respective cell matrix in the reactor flask 25 gets on the flat sliding plate 27 the perfectly fitting planar stamp 26 introduced with the outer diameter D1 in the piston hollow cylinder.

The bottom of this stamp 26 can be analogous to the stamp surface 24 of the miniature actuator 14 be influenced by organotypical structures.

In the 6c (Stamp attachment) is the attachment of the stamp 26 on the graft 11 in the sowing flask 25 demonstrated. The Stamp 26 is placed on the cell scaffold with a slight pressure to meniscus or bulge the matrix top of the graft 11 counteract, for example, to obtain a cylindrical graft form.

Intended to be an in vivo adaptive surface on the graft 11 be stamped, so this stamp attachment 26 take place during the curing or polymerization.

In the 6d (Removing the sliding plate) is the attached punch 26 after shaping the graft 11 lifted off and at the bottom of the sowing flask 25 located, preferably hydrophobic, sliding plate 27 away. To prevent a gel-like cell construct 11 to the sliding plate 27 and adheres the seeding flask, for example, an inert film or an inert polymer fleece is used to line the surfaces.

The 6e (Construct seed in the bioreactor) shows the sowing of a cylindrical construct using the example of the two-chamber bioreactor. Here, the tailor-made Einsaatkolbens 25 in the bioreactor 1 implemented, then by means of plunger 26 the cell construct 11 introduced medially into the prepared reactor and the sowing device from the bioreactor 1 away. This prepared bioreactor 1 includes 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 Einkammerbioreaktor

In the 7 is the construction and construction of the single chamber reactor body and its effect on diffusion and perfusion in the graft 11 shown.

In the in the 7a illustrated embodiment open four inlets and outlets with integrated Luer coupling 19 in the bioreactor 1 , These can differ for flow optimization both in their position and position, that is also, for example, tangentially into the bioreactor body 1 enter. The number of in the bioreactor 1 leading inflows and outflows 19 is at least two. At each outflowing Luer connection 19 can with eg a 3-way valve 6 a sampling route 8th be installed.

In a static culture in the bioreactor, the medium diffuses mainly in the upper and lateral edge regions of, for example, cylindrical tissue graft 11 and supplies the cell culture, inter alia, with nutrients and at the same time carries out metabolic end products from the carrier matrix.

The 7b shows a continuous supply of nutrient medium from the medium reservoir 2 with optional supplement reservoir behind 3 (not shown) by means of a metered circulating pump 5 through the hose system 4 in at least one inlet 19 of the bioreactor 1 ,

The outflow of the medium is carried out by at least one flow 19 into the hose system 4 , in which at least one point via a 3-way valve 6 a decoupled sampling route 8th can be integrated.

The spent medium can, as shown here, remain in the circulation by placing it in the medium reservoir 2 and from there back to the re-continuous perfusion of the graft 11 is used. Otherwise, it can be completely removed from the circulation. The cultivation of the transplant 11 then takes place in a batch or fed-batch process.

A targeted continuous supply of culture medium in the reactor space, the arrival and flow through the graft 11 compared to the static scheme of 7a improve significantly. The induced perfusion rinses deeper construct regions with medium. As a result, mass transfer is optimized, which can lead to increased cell differentiation. Furthermore, this embodiment of the construct flow exerts shearing force stimulation on the embedded cells.

Example 8

Scheme of the technical Device for construct perfusion and media mixing in the Two-chamber bioreactor

The 8th show a dual compartment bioreactor that allows optimized flow, diffusion and perfusion of the graft, thus improving the quality of tissue replacement.

In the 8a a variant with static cultivation and diffusion is shown.

The in the bioreactor 1 opening inflows and outflows 19 are at least two, with at least one each in the lower and upper reactor chamber must open. The two inflows and outflows listed here 19 per chamber can differ for flow optimization both in their position, position and thickness.

The sampling route 8th can be attached to any outflowing Luer connector 19 Both chambers with eg a 3-way valve 6 etc. be installed.

In addition to a media diffusion into the upper and lateral graft areas leads the chamber newly created in this construction below the graft 11 during the static culture phase to a diffusion of the culture medium from the porous sintered material in the ground-level regions of the support structure, so that therefrom an improved metabolism throughout the graft 11 results.

The 8b shows the continuous supply of nutrient medium from the medium reservoir 2 with optional supplement reservoir behind it 3 (not shown) by means of a metered circulating pump 5 etc. through the hose system 4 in at least one inlet 19 the lower and upper chamber of the bioreactor 1 ,

The outflow of the medium is carried out by min at least one process 19 per chamber in the hose system 4 , in which at least one point via a 3-way valve 6 a decoupled sampling route 8th can be integrated.

The spent medium may, as shown here, remain in the circulation by placing it in the medium reservoir 2 and from there back to the re-continuous perfusion of the graft 11 is used. Otherwise, it can be completely removed from the circulation. The cultivation of the transplant 11 takes place in a batch or fed-batch process.

The integration according to the invention of a second chamber, here below the graft 11 , shows its positive properties especially with a targeted flow of the biological construct on. Is therefore by means of the 3-way valve 6 the media stream from the medium reservoir 2 switched into the lower chamber, there is an induced upward perfusion of the graft 11 when the lower outlet is closed, since the medium can leave the reactor space only through the upper outlet.

Analogous to this scheme can be achieved by switching the 3-way valves 6 a graft perfusion from the upper to the lower chamber through the construct 11 obtain. Result of this device shown next to the complete perfusion another cell stimulation via an induced shear force acting on the cells and the volume flow of the circulation pump 5 can be adjusted. Also possible is a partial or total opening of the 3-way valves 6 to a faster medium exchange in the bioreactor 1 to achieve.

Example 9

Fixation scheme of the transplant 11 in the bioreactor 1

The 9 show the fixation scheme of the graft 11 in the bioreactor 1 either in the one-chamber or two-chamber version.

The 9a shows the graft to be stimulated 11 , the medial above the clear glass 17 in the single chamber bioreactor 1 is fixed. With at least 3 of these fixation walls 28 should be caused by inflowing medium, horizontal movement of the graft 11 be prevented at the reactor bottom to perform an optimal perfusion and pressure stimulation can. This in the reactor 1 embedded, biocompatible fixation walls 28 must be lower in height than the pressure amplitude to be applied to the graft 11 be dimensioned.

In the 9b is the use of at least 3 of these fixation walls 28 outlined in the two-chamber bioreactor to a horizontal fixation of the graft 11 to achieve at the different flow conditions and to allow an ideal vertical perfusion and a mechanical pressurization.

Example 10

Magnetic systems for controlling the mini-actuator 14

The 10 (illustrated at Einkammerbioreaktor) characteristic devices and apparatus arrangements for the contactlessly controllable stimulation method of the mini-actuator 14 on the graft 11 represents.

In the 10a (Magnetic Control Effect - Magnetic Attraction) are the characteristic device and principle for non-contact control of the miniature magnetic actuator 14 in the bioreactor 1 for pressure deformation of the graft 11 listed. The orientation of the permanent magnets in the mini-actuator 14 occurs after the prevailing magnetic field direction, by externally arranged control magnet 15 is produced. Through this control magnet 15 , for example, is at least one permanent magnet or at least one coil, a defined (electro) magnetic field is generated, which with its field lines in the entire bioreactor space 1 in ranges and a field direction-dependent movement of the plunger of the mini-actuator 14 triggers. In the example of 10a is determined by the, here exemplarily above, control magnet 15 the principle of magnetic attraction shown.

In the in the 10b (Magnetic Control Effect - Magnet Repulsion), the magnetic repulsion becomes the second magnetic control effect between the magnetic control system 15 and the mini-actuator 14 shown. By changing the magnetic field direction of the control magnet 15 changes the direction of movement of the mini-actuator 14 , which now points downwards on the graft 11 is controlled. By increasing the power or magnetic flux density starting from the control magnet 15 the pressure load of the graft also increases 11 to the target value of in vivo adaptive stimulation.

The 10c - 10e show arrangements of control elements with which it is possible to use the mini-actuator 14 cyclic and high frequency in a sealed bioreactor 1 to control.

The 10c (Control of the mini-actuator 14 by means of a control magnetic guide plate) represents an embodiment of a permanent-magnetic control system. In this magnetic field variant, an arrangement of a plurality of permanent magnets operates 32 different size, polarity and thus field strength and direction on a linearly controlled guide plate 31 , which is positioned here by way of example above the reactor prototype.

This is done by a linear motor 29 a guide rail 30 with those in the magnet holder 31 used permanent magnets 32 driven. This mobile phase of the magnet system makes a movement of the bioreactor 1 unnecessary.

The control system in 10d (Control of the mini-actuator 14 by means of rotating permanent magnets) is also based on a control of the magnetic plunger 14 by means of a permanent magnet arrangement on a rotating disk.

This drives a servomotor 29 a magnetic holder 31 with fitted permanent magnets 32 alternating polarities in a rotating manner. This rotating magnet holder can, for example, with four alternately poled magnets 32 be busy and causes as a result of a full turn two complete pressurizations on the graft 11 , The combination of this magnetic occupation of the rotating disk with the rotational speed of the servomotor 29 gives a higher frequency magnetic field change and consequently a highly dynamic stimulation scheme on the graft 11 , The front view illustrates both magnetic effects of the rotation system using two bioreactors 1 , The embodiment of this device is for a variety of bioreactors 1 suitable, as long as they can be placed exactly above or below the control magnetic center.

The 10e (Control of the mini-actuator 14 by iron core coil 35 ) represents a magnetic device based on a coil arrangement.

This magnet coil system works with an induction coil 35 that's here above the bioreactor 1 is fixed, generating a defined electromagnetic field, which can be controlled continuously via the supplied electric power and thus any position of the mini-actuator 14 in the bioreactor corpus. By reversing the direction of the current, there is a reversal of the existing field direction and of the electromagnetic effect. The iron core coil used 35 builds up their electrical field perpendicular to the coil winding and acts on the static permanent magnet of the mini-actuator 14 attractive and disgusting.

An automated station of this system consists of a powerful coil 35 with low heat generation with a connected adjustable transformer whose performance is monitored by means of a multi-meter. In addition, the use of a microcontroller ensures the control of a relay, which switches the current in the desired direction, the desired effect of an intermittent Druckapplizierung on the cell construct.

Example 11

stimulation scheme in the two-chamber reactor

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

In the 11a (Perfusion stimulation without mechanical stress) takes place stimulation of the cell construct 11 only via a directional media flow, which leads to a construct tethering with a shearing force on the cell membrane in the μPa range. Shown in this process example is a continuous nutrient media feed in two feeds 19 So that each reactor chamber is supplied, first a concentration balance in the graft 11 and thereafter, depending on the selected volume flows, an upper and lower perfusion zone is formed in the construct. This spent medium leaves via two further processes 19 the reactor room. During this flow stimulation takes place here no pressurization, since the plunger 14 from the control magnet system 15 in an upper position in the bioreactor 1 is held.

In the 11b (Perfusion Stimulation and Stamp Attachment) is the second step in multiple stimulation of tissue replacement materials 11 in the bioreactor 1 demonstrated. First, as in this example, the flow conditions are modified. About the 3-way valves 6 The nutrient media stream is directed only into the lower reactor chamber from where it passes through the graft 11 perfused, causes the mass transfer and can leave the upper reactor space via a drain. By reversing the polarity of the control magnet system, here an iron core coil 35 with low power induction, it comes to the attachment of the magnetic mini-actuator 14 on the eg cylindrical tissue replacement 11 , This stamp attachment with a 0% construct deformation marks a reversal point of the miniature actuator 14 at a dynamic high frequency Deformation of the cell matrix 11 ,

In the next step of the stimulation procedure, as in 11c (Perfusion stimulation and mechanical stress) shown by the coil 35 increased outgoing magnetic field strength. The result of this increased magnetic flux density is increased compression of the graft 11 to the desired target formation, which mimics preferably in vivo-like processes. After this pressure stimulation has taken place, a change between cell irritation and stamp attachment can be brought about in an intermittent procedure.

Also a static compression of the replacement material is with the listed equipment and the method set out.

Basically while This mechanical stress is a directed construct perfusion through the carrier matrix introduced which needed the cells Nutrients provides and dissipates metabolites, especially during metabolic activity Phases, e.g. in proliferation and differentiation (extracellular matrix synthesis), be replaced.

After the pressure load protocol has been processed, the stamper is returned to its original state, for example, it continues to perfuse the cell culture continuously and, for example, with sufficiently synthesized extracellular matrix, removes the transplant 11 ,

1

bioreactor

2

medium reservoir

3

Supplement reservoir

4

hose system

5

circulating pump

6

3-way valve

7

4-way valve

8th

Sampling distance

9

biosensors Glu / Lac

10

Measuring probes pH, pCO ₂ , pO ₂

11

graft

12

Cells / tissue

13

support matrix

14

Mini-actuator

15

control solenoid

16

porous sintered material

17

clear glass

18

Permeable membrane

19

Hose coupling / Luer connector

20

crimp

21

reactor closure

22

permanent magnet

23

enveloping body

24

stamp surface

25

Einsaatkolben

26

stamp

27

sliding plate

28

fixing wall

29

servomotor

30

guide rail

31

magnetic holder

32

permanent magnet

33

flow channels

34

extension web

35

Kitchen sink

Claims (57)

Method for cultivating and stimulating three-dimensional, vital and mechanically resistant cell transplants in a GMP-suitable bioreactor, with the method steps a) the explant cells removed from the organism and processed using methods known per se for bioreactor culturing ( 12 ) and from commercially available biocompatible, resorbable or autologous or homologous materials existing carrier matrices ( 13 ) after mixing them as a cell matrix suspension b) in a, optionally foiled, sown flask ( 25 ) is introduced with any desired cross section adapted to the later graft and hardens or polymerizes there, c) if appropriate in this case by means of a precisely fitting, inert, optionally structured or foiled stamp ( 26 ) is loaded with minimal pressure, d) the sowing flask with the graft ( 11 ) is introduced into the chamber space of the bioreactor body, e) where the graft ( 11 ) from the sowing flask ( 25 ) is applied medially to the bioreactor bottom and the biorector ( 1 f) where the graft is further cultured by the introduction of perfusion flow, and g) after the completion of the cultivation, the tissue replacement material is removed for further use, characterized in that the graft is cultivated on the Bioreactorboden opposite surface is loaded by pressure. Method according to claim 1, characterized in that that the graft through a pressure-imparting stamp is charged. Method according to Claims 1 and 2, characterized that both the mixing of the bioreactor space by the entry of perfusion flow as well as the pressure on the graft mediating stamp dependent on of the conditions of cultivation in time and in quantity or Strength are controllable. Method according to Claims 1 to 3, characterized that the graft intermittently with conditioned nutrient medium flows through is cyclically loaded with the pressure-imparting stamp. Method according to Claims 1 to 4, characterized that the pressure load of the graft occurs during the perfusion. Method according to Claims 1 to 5, characterized that the graft is dependent on of use with static, preferably with in vivo-like Pressure loads or construct deformations is irritated or continuously with intermittent or dynamic pressure forces is charged. Method according to Claims 1 to 6, characterized that the mechanical loads with a frequency of more than 0.1 Hz. Method according to Claims 1 to 7, characterized that the mechanical pressure stimulation takes the form of a symmetrical or unbalanced cosine or sine half-wave. Method according to Claims 1 to 8, characterized that of a magnetic material existing plunger through an outside the bioreactor generated (electro) magnetic field along the surface of the graft in the bioreactor is moved. Method according to Claims 1 to 9, characterized that the magnetic field by at least one permanent magnet is produced. Method according to claims 1 to 10, characterized in that that at least two permanent magnets with alternating polarity arranged on a mobile bracket above the bioreactor are cyclically driven by a servomotor, their position change, causing the plunger alternating on the graft suppressed. Method according to Claims 1 to 8, characterized that the coil of an electromagnet high frequency their current direction and voltage and thus field direction and magnetic flux density over the Bioreactor changes, whereby the plunger alternately presses on the graft. Bioreactor for culturing and stimulating three-dimensional, vital and mechanically resistant cell transplants in a GMP-suitable bioreactor, characterized in that the bioreactor ( 1 ) from a base body with a reactor closure ( 21 ) is pressure-tight and sterile connected and forms at least one reactor space in which a storage area for a transplant ( 11 ) and a mini-actuator ( 14 ) and the bioreactor ( 1 ) with at least two hose coupling connections ( 19 ) is provided for the medium supply and medium removal or for gassing. Device according to claim 13, characterized in that the cell culture constructs ( 11 ) directly or indirectly on the bioreactive bottom of a single-chamber bioreactor ( 1 ) are cultivable and stimulable. Device according to claim 13, characterized in that the cell culture constructs ( 11 ) is directly or indirectly, at least partially, on a bottom surface of the upper reaction chamber of a two-chamber bioreactor for breeding and stimulation, while below this graft ( 11 ) is a second reactor space. Device according to claim 14 or 15, characterized in 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. Device according to one of claims 13 to 16, characterized in that the container of the bioreactor ( 1 ) has a hollow-cylindrical body, which on the upper side of a reactor closure ( 21 ) is completed. Apparatus according to claim 13 to 17, characterized in that the or the reactor closure unit ( 21 ) and the bioreactor ( 1 ) by a threaded connection and at least one sealing ring ( 20 ) are connected to one another in such a way that either the threaded connection between the reactor closure ( 21 ) and the container ( 1 ) by 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 a cooperating internal thread in the reactor closure ( 21 ) is made. Apparatus according to claim 13 to 18, characterized in that the lid designed as reactor closure ( 21 ) with biosensors ( 9 ) and / or measuring probes ( 10 ) is provided. Apparatus according to claim 13 to 19, characterized in that the lid ( 21 ) with a sampling line ( 10 ) is provided. Device according to claims 13 to 20, characterized in that the basic body of the bioreactor ( 13 ) in the embodiment as a single-chamber bioreactor with at least one each inlet and Ablaufboh for the installation of hose coupling connections ( 19 ) is provided. Device according to claims 13 to 20, characterized in that the basic body of the bioreactor ( 1 ) in the version as a two-chamber bioreactor with at least two holes for the installation of hose coupling connections ( 19 ) is provided. Apparatus according to claim 22, characterized in that in the two-chamber bioreactor at least one hose coupling connection ( 19 ) is embedded in the lower and upper reaction space. Apparatus according to claim 21 to 23, characterized in that the opening into the bioreactor chambers inlet connections ( 19 ) and drain connections ( 19 ) with a 3-way valve ( 6 ) or with a 4-way valve ( 7 ) are provided with kickback function. Apparatus according to claim 24, characterized in that at least one of the drain connections ( 19 ) with a sampling line ( 10 ) are provided. Device according to at least one of claims 13 to 25, characterized in that for monitoring the transplant production of the bioreactor ( 1 ) at the bottom of the reactor completely or partly from a transparent material, preferably a clear glass pane ( 17 ) consists. Apparatus according to claim 13 to 26, characterized in that above the reactor bottom of the bioreactor ( 1 ) a film, fleece or membrane ( 18 ) of an antistatic or inert material for supporting the transplant ( 11 ) is arranged and this material is preferably weitmaschig, light, fluid and gas permeable. Device according to at least one of claims 13 to 27, characterized in that the upper reactor chamber of the bioreactor ( 1 ) when designed as a two-chamber reactor in its base area of the graft base area, while the dimensions of the lower chamber below those of the graft ( 11 ), so that in a medial cell culture overlay, this construct is largely overlying the lower chamber and partially on the reactor bottom of the upper chamber. Apparatus according to claim 28, characterized in that the space of the lower reactor chamber a flat disc ( 16 ) of biologically inert, translucent, wide-pore material, preferably of porous sintered material, so that a flat reactor bottom conclusion of this disc ( 16 ) and the bottom of the upper reactor chamber is formed. Apparatus according to claim 28 and 29, characterized in that above the through the disc ( 16 ) filled lower reactor chamber on the forming in the upper reactor chamber reactor bottom of the two-chamber bioreactor ( 1 ) a film, fleece or membrane ( 18 ) of an antistatic or inert material for supporting the transplant ( 11 ) is arranged and this material is preferably weitmaschig, light, fluid and gas permeable. Apparatus according to claim 28 to 30, characterized in that the components which are in the two-chamber bioreactor ( 1 ) below the graft ( 11 ), like the transparent pane ( 17 ), the lower chamber with inserted porous material ( 16 ) and a wide-meshed membrane ( 18 ), do not exceed an overall height within the focal length of commercially available microscope and camera lenses. Apparatus according to claim 1, characterized in that a magnetic, preferably piston-like, miniature actuator ( 14 ) in the bioreactor ( 1 ) arranged by one or more externally arranged control and control magnets ( 15 ) through the bioreactor ( 1 ) is moved under control. Apparatus according to claim 13 to 32, characterized in that the miniature actuator ( 14 ) in the single-compartment bioreactor ( 1 ) above the membrane ( 18 ) and the graft ( 11 ) is arranged in the bioreactor space. Apparatus according to claim 13 to 32, characterized in that the miniature actuator ( 14 ) in the two-chamber bioreactor ( 1 ) above the porous material ( 16 ), the membrane ( 18 ) and the graft ( 11 ) is arranged in the upper reactor space. Device according to claims 32 to 34, characterized in that the magnetic mini-actuator ( 14 ) from a magnetic core body ( 22 ), preferably of a permanent magnet, constructed in a biologically inert envelope ( 23 ), preferably plastic, is encapsulated. Device according to claim 32 to 35, characterized in that the magnetic core body ( 22 ) is oriented so that the field forming between the poles perpendicular to the graft ( 11 ), so that the magnetic north pole of the entire miniature actuator ( 14 ) is oriented upward. Apparatus according to claim 32 to 36, characterized in that the core magnet ( 22 ) surrounding biocompatible enveloping bodies ( 23 ) in its outer shape of the cross-sectional shape of the reactor chamber of the bioreactor ( 1 ) corresponds. Apparatus according to claim 32 to 37, characterized in that the total height of the envelope body ( 23 ) is selected in an implementation of the mini-actuator ( 14 ) in the reactor space to a vertically oriented guide of the plunger of the mini-actuator ( 14 ) on the graft ( 11 ) comes. 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 body cylinder, preferably the upper, the encapsulated permanent magnet includes and another cylinder of the stamp imprint ( 24 ), wherein the spatially separated cylinder via a web ( 34 ) or a functionally identical connection are connected to each other. Apparatus according to claim 32 to 39, characterized in that the by the envelope body ( 23 ) formed planar stamp surface ( 24 ) on the underside of the miniature actuator ( 14 ) perpendicular to the guiding direction in the bioreactor space ( 1 ) runs. Apparatus according to claim 32 and 40, characterized in that on the stamp surface ( 24 ) of the miniature actuator ( 14 ) organotypic negative forms, such as a curvature, are imprinted. Device according to claim 32 and 41, characterized in that the planar or shaped stamp surface ( 24 ) has an embossing in the form of a lattice structure for enlarging the stamp surface, preferably with a small-meshed structure. Apparatus according to claim 32 to 42, characterized in that the enveloping body ( 24 ) of the miniature actuator ( 14 ) with bores and / or flow channels ( 33 ) is provided so that over at least 3 points of the outer diameter of a precisely fitting, vertical guidance of the mini-actuator ( 14 ) is guaranteed. Apparatus according to claim 32 to 43, characterized in that the stamp surface ( 24 ) of the miniature actuator ( 14 ) is provided with at least one guide lug, which slides vertically in their accurately inserted guide rail in Bioreaktorkorpus. Apparatus according to claim 32 to 44, characterized in that the outside of the bioreactor ( 1 ) arranged control and control magnet ( 15 ) with the outgoing from him (electro) magnetic field, a directed movement of the implemented mini-actuator ( 14 ), with its upward magnetic north pole of the permanent magnet ( 22 ). Apparatus according to claim 32 to 45, characterized in that the control and control magnet ( 15 ) in a vertical axis to the plunger ( 14 ) medially, preferably above the plunger ( 14 ), and depending on the polarity of the miniature actuator ( 14 moved up and down, resulting in a change in the pressure on the graft ( 11 ) follows. Apparatus according to claim 32, characterized in that the control and control magnet ( 15 ) of at least two permanent magnets ( 32 ) with different vertical magnetic pole direction, in a cuboid magnetic holder ( 31 ) are used and by a servomotor ( 29 ) and a guide rail ( 30 ) over the bioreactor ( 1 ) are cyclically offset in their horizontal position. Apparatus according to claim 32, characterized in that the control and control magnet ( 15 ) of at least two permanent magnets ( 32 ) with different vertical magnetic pole direction, which in a disc-shaped magnetic holder ( 31 ) are used and by the rotational drive of a servomotor ( 29 ) cyclically via the bioreactor ( 1 ) are carried away. Apparatus according to claim 32, characterized in that the fixed in its horizontal position bioreactors via a vertical movement of the magnetic holder ( 31 ) with the permanent magnets ( 32 ) are introduced by means of a stepper motor in order to increase the respective field effect and higher pressure forces on the graft ( 11 ) to generate. Apparatus according to claim 49, characterized in that at least two bioreactors ( 11 ) are arranged in a station so that their mini-actuators ( 14 ) are driven without contact by only one permanent magnetic control system. Apparatus according to claim 32, characterized in that the control and control magnet ( 15 ) as an electromagnet with at least one induction coil ( 35 ) is carried out, the field is infinitely variable by means known per se. Device according to claim 32 and 51, characterized in that the induction coil ( 35 ) high frequency with variable frequency to high-dynamic magnetic field changes and movements of the mini-actuator ( 14 ) on the graft ( 11 ). Apparatus according to claim 13 to 52, characterized in that a, preferably cylindrical, seed flask ( 25 ) having an inner diameter corresponding to the outer diameter of the graft, is provided for injecting the cells ( 12 ) and the carrier matrix ( 13 ) on a movable sliding plate ( 27 ). Apparatus according to claim 13 to 53, characterized in that the movable sliding plate ( 27 ) and the interior of the sowing flask ( 25 ) are coated with an inert membrane, film or polymer fleece. Device according to claims 53 and 54, characterized in that in the sowing flasks ( 25 ) a precise stamp ( 26 ) is easily placed on the cell suspension with a planar stamp surface or an organotypic negative mold. Apparatus according to claim 53 to 55, characterized in that the outer diameter of the sowing piston ( 25 ) precisely to the inner diameter of the bioreactor ( 1 ) corresponds. Apparatus according to claim 13 to 56, characterized in that at the reactor bottom of the bioreactor ( 1 ) at least 3 fixing walls ( 28 ) are admitted, which meet in their dimensions of a graft insert and not hinder the pressure compression.