WO2002040076A1 - Method and device for producing biological prostheses - Google Patents

Abstract

The invention relates to a method for coating biological implants with autologous cells of the recipient, according to which the cell-free allogenic or xenogenic material foreign to the body is colonized in a speed-variable pulsating cell suspension flow close to the natural conditions in the respective organ or part of the organ. The resulting cell conditioning allows a complete, fast and safe coating with cells, the formation of which comes close to the natural conditions and guarantees a trouble-free functioning and a long life of the implant. The device for carrying out the method comprises a bioreactor (2) integrated into a loop (1) and filled with the cell suspension in which the implant (8) is axially and centrally fixated in the direction of flow. The loop (1) is connected to a pump (3) that generates a pulsating flow of medium that flows through the implant (8). The inventive coating with autologous cells can also be applied to artificial prostheses.

Description

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Rinderperi ardi m manufactured prostheses that have a similarly favorable hemodynamics as the homografts and are also available in any size and number.

Due to the required preservation, these bioprostheses do not contain living tissue and cell growth is practically impossible. Such heart valves are therefore not capable of regeneration, not capable of growth and, moreover, are subject to gradual wear. In addition, these prostheses are classified as "foreign" by the immune system of the recipient organism and are combated by immune-competent cells.

To solve this problem, DE 198 28 276 already describes a process for the production of bioartificial grafts, in which a cell-free, loosened collagen matrix has been populated with autologous cells of the transplant recipient or genetically modified cells which are compatible with the recipient , In this method, however, it is difficult to coat the collagen matrix with the body's own cells of the recipient, which is as smooth as possible, and is necessary for a trouble-free blood flow through the heart valves and in the blood vessels or bypass vessels. Trouble-free function and a long lifespan of the implant are therefore not guaranteed.

To eliminate these disadvantages, a method and associated device have already been proposed, in which an acellularized bioprosthesis is located in a container which can be rotated about its longitudinal axis and / or its transverse axis and which is filled with a nutrient solution which contains the body's own or genetically identical cells of the recipient is. By gradually turning with sedimentation breaks or by continuous rotation and The centrifugal forces acting on the cells are intended to ensure a surface-covering coating with as many living cells as possible.

With the known methods and the corresponding devices, however, it is not yet possible to achieve a uniform, flat surface and complete colonization of the collagen matrix with the body's own cells, which is largely adapted to the actual conditions in the human body.

The invention is therefore based on the object of specifying a method of the type mentioned at the outset for producing biological prostheses which approximate the natural conditions with regard to cell coating, in particular heart valves and blood vessels, which improves the durability and functionality of the implant in question and the quality of life of the implant Receiver increased, as well as to develop a device for performing the method.

According to the invention, the object is achieved with a method according to the features of patent claim 1 and a device for carrying out the method characterized by the features of patent claim 8.

Starting from the known solutions for the production of biological prostheses, according to which an acellularized allogeneic or xenogeneic starting product, for example the collagen / elastin matrix of an aortic valve from

Pig, by rotating in a cell suspension formed from cells of the recipient of the prosthesis, is colonized with the body's own cells, the basic idea of the invention is that the coating of the matrix in a cell line approximating the natural flow conditions in the organs, organ parts or vessels concerned suspension current takes place. With such a dynamic coating, the cells to be applied already learn in the coating and first growth phase to adapt to the natural conditions during the later function of the organ (in part), such as flow conditions and wall movements or strains, with regard to their arrangement, shape and adhesion, so that a complete, all-round and fast and safely growing colonization can be achieved and the new body's own cell layer creates resistance and low-friction flow conditions in the later blood-flowed areas. The process is actually characterized by three settlement steps:

1. Distribute the cells on the in one

Bioreactor containing cell suspension, centrically fixed acellular and adhesive-treated implant by rotating the bioreactor with subsequent sedimentation phase;

2. Overflow of the implant surfaces with a cell suspension flow that is largely based on the natural conditions in the organ part concerned for conditioning and further attachment of the cells; and

3. Recovery and growth of the conditioned cells in a resting phase following the overflow phase.

According to a further feature of the invention, the conditioning and resting phase can be carried out in two or more substeps, each with an increased flow rate compared to the previous phase. The device according to the invention for carrying out the method comprises a bioreactor that can be handled separately, rotatable in the longitudinal and transverse directions by hand or by motor, which is filled with the cell suspension and in which the implant to be coated is fixed tightly axially to the flow direction. For the flow-through or cell conditioning process, the bioreactor is integrated in a ring line which is connected to a diaphragm pump in order to generate a pulsating, variable-speed liquid flow.

Further features and advantageous developments of the invention result from the subclaims and the following description of an exemplary embodiment.

With the method according to the invention and the corresponding device, it is possible to produce implants coated with the body's own cells from xenogeneic or allogeneic or artificial foreign material which, in addition to the known advantages of using the body's own cells, also largely correspond to the natural conditions in terms of their dynamic effect, and have a long service life exhibit and ensure a high level of comfort.

An embodiment of the invention is explained in more detail for the production of a scaffold-free aortic valve with reference to the drawing. Show it:

Fig. 1 is a schematic view of a device according to the invention for coating an acellularized porcine aortic valve in a flow circuit from a body with endothelial cells of the intended recipient

nutrient solution; and

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a screw cap 23 with an internal thread, which engages in an external thread on the hollow cylinder 9, can be clamped. A second seal 19 is provided for liquid-tight fastening. The hose section of the ring line 1 connected downstream of the bioreactor consists of flexible

Material (silicone) so that the flow is guaranteed due to the pulsating pumping action. All parts of the device that come into contact with the cell material during the coating process are made of biologically repellent material to which the cells cannot adhere.

Due to the previously described design and arrangement of the inserts 10, 11, a correspondingly prepared, that is, decellularized, treated and cut with the natural adhesive Pronectin + L-Laminin can be cut outside the hollow cylinder 9 on the opposite fastening sections 14, 15 of the Inserts 10, 11 are sewn on. In this form, the aortic valve 8 is introduced into the hollow cylinder 9 in such a way that the direction of flow in the device corresponds to the natural direction of flow through the implant in the human body. Due to the fact that two quasi-identical valves are connected in series with the outlet valve 6 and the aortic valve 8, the outlet valve 6 of the diaphragm pump 3 could be dispensed with in this case. However, if blood vessels are coated with body cells in the same way, the outlet valve 6 in the diaphragm pump 3 is still required.

For the colonization of the aortic valve, the endothelial cells proven to be viable are available from the cell cultivation described above in a number of at least 8 million in the medium also used in cell cultivation, which is 20% from the serum of the recipient commercially available cell growth agents DMEM and bFGF as well as antibiotics, L-glutamine and possibly a HEPES buffer. This cell suspension is filled into the bioreactor 2 via one of the connecting pieces 12, 13 detached from the ring line 1 or a fill and drain valve 24, 25. After the bioreactor 2 detached from the ring line 1 is closed at the connecting piece 12, 13 with plugs or cover caps (not shown), the bioreactor 2 held in a rotating device (not shown) is rotated both around its longitudinal axis and around as a first step of the settlement its transverse axis and intermediate settling phases distribute the endothelial cells on the surfaces of the aortic valve 8, both under the effect of gravity and centrifugal forces. The turning can preferably also be carried out manually with simultaneous visual observation. In the case of a ring line 1 connected to the bioreactor 2, a first cell conditioning step then takes place in that a pulsating cell suspension flow is generated via the membrane pump 3 at a flow rate of 11 / min in the direction of the arrow. The direction of flow of the cell suspension corresponds to the direction of flow of the blood in the implanted state of the bioprosthesis. With the impulsive flow of the cell suspension over the

Inner surfaces (flow surfaces) of the aortic valve 8 are gradually adapted in shape and orientation of the endothelial cells in shape and orientation to the flow direction and forces as well as the movements of the implant walls, ie the cells are conditioned and learn, at initially low flow velocity to be designed and arranged in such a way that in the growth phase a smooth, single-layer endothelial cell surface which opposes the flow as little as possible arises. That is, in the resting phase following the first cell conditioning step phase, which can be connected with a discontinuous rotation of the bioreactor 2, the endothelial cells have the opportunity to grow on the collagen matrix in the shape and direction learned by the previously entered flow stimuli. Cells that may have been detached when flowing through are resettled in this phase.

In the subsequent second cell conditioning step with a flow rate of 2 l / min, the endothelial cells become correspondingly stronger

Information stimuli are provided to develop and grow in accordance with these changing flow conditions.

After a further resting and growth phase (intermediate incubation) without flow movement of the cell suspension, but optionally with a rotary movement of the bioreactor 2, there follows a third cell conditioning step with a flow rate of 4 1 / min, which is followed by another post-incubation phase (recovery and growth - phase).

The entire previously described process of cell coating in the bioreactor 2 takes place in an incubator (not shown) in which the temperature is constantly 37 ° C. and an air humidity of 98% and a CO ₂ content of 5% are maintained , The access of this atmosphere or the corresponding media to the bioreactor 2 is realized via at least one semi-permeable membrane valve 26 which is gas-permeable from the outside, but is not liquid-permeable in the opposite direction. The membrane valves 26 can be arranged at a suitable point on the bioreactor 2, for example in the inserts 10, 11 or also in the ring line 1. As a result of this process of gradual colonization and formation of the endothelial cells with a gradually increasing and at the same time gentle and with a learning function pulsating flow rate acting on the cells with intermediate and subsequent recovery and

Growth phases are created under physiological flow conditions corresponding to the functions of the human heart on the collagen matrix of the bioprosthesis (aortic valve 8) provided with a submatrix of fibronectin -Recipient largely corresponds to the shape, size and location of the cells and the strength and flatness of the cell layer.

Implants for cardiac and vascular surgery are thus provided, the use of which is not associated with the risk of immune reactions and viral infections. Of essential importance compared to the closest state of the art is the fact that the cell layer applied to the implant is largely designed to approximate the physiological processes in the human organism, here the specific dynamics in the area of the heart and vessels. The formation of the endothelial cell layer achieved with the method according to the invention and the corresponding device ensures, in the implanted state of the bioprosthesis, flow conditions which correspond to the natural conditions, so that the signs of wear are significantly minimized and the life of the implant can thus be increased.

The invention is not restricted to the exemplary embodiment described above. Rather, various modifications of the are within the scope of the claims Process and the device conceivable. Instead of the porcine aortic valve treated as an example, pulmonary or mitral valves or vessels, generally from allogeneic, xenogeneic or artificial starting material, can also be produced in the manner described.

LIST OF REFERENCE NUMBERS

loop

bioreactor

diaphragm pump

compensation chamber

Hose line (for 3)

outlet valve

intake valve

Aortic valve (bioprosthesis)

Hollow cylinder first insert (in the flow direction at the front) second insert (in the flow direction at the rear)

connecting branch

connecting branch

attachment section

attachment section

mounting holes

Attachment holes

BundSteg from 11 first seal

Ring bridge from 9

screw ring

Bunch of 10

Screw Caps

Filling and drain valve

Filling and drain valve

Me branventi1

Second seal

Claims

claims 1. Process for the production of biological prostheses, in particular implants for cardiac and vascular surgery, in which a collagen / elastin matrix is produced from an allogeneic or xenogeneic foreign material by removing the cells and pretreated with an adhesive, and then in a container is populated with cells obtained from the respective recipient of the implant and located in a cell suspension, characterized in that the pretreated collagen / elastin matrix of the implant for conditioning the recipient cells in the natural flow direction from one with respect to frequency and speed to the natural flow conditions in the flows through the human body adapted pulsating cell suspension flow, which is followed by a cell recovery and growth phase. 2. The method according to claim 1, characterized in that the flow rate of the cell suspension stream is between 0.5 1 / min and 5 1 / min. A method according to claims 1 and 2, characterized in that two or more through-flow phases are carried out with a flow rate which is respectively higher than the previous one. 4. The method according to claim 1 to 3, characterized in that the cell conditioning is preceded by a first step for cell distribution on the collagen matrix by rotating the container with intermediate and downstream resting and weaning phases. 13 5. The method according to any one of claims 1 to 4, characterized in that the cell distribution and conditioning and cell growth is carried out at 37 ° C and at 98% humidity and a C0 ₂ content of 5%. 6. The method according to any one of claims 1 to 4, characterized in that the cell suspension contains about 8 million cells and a serum portion of the recipient of 20% and consists of cell growth agents, antibiotics, L-glutamine and a Hepes buffer. 7. The method according to any one of claims 1 to 6, characterized in that an artificial prosthesis is used as the starting product for the cell coating. 8. An apparatus for performing the method according to claim 1, characterized by a ring line (1) with this connected to generate a pulsating liquid flow variable speed membrane pump (3) and a detachably integrated in the ring line (1) bioreactor (2), in which the allogeneic or xenogeneic implant material (8) to be treated is arranged axially in the flow direction of the cell suspension and can be fixed under prestress. 9. The device according to claim 8, characterized in that the bioreactor (2) from a hollow cylinder (9) with releasably and sealingly held insert pieces (10, 11) is formed on its opposite end faces, the insert pieces (10, 11) each one closable or to the ring line (1) connectable connecting piece (12, 13) and a Fastening section (14, 15) for the central have fix the bioprosthesis (8) in the hollow cylinder (9). 10. The device according to claim 9, characterized in that the fastening sections (14, 15) are annular and are provided with fastening holes (16, 17) distributed around the circumference for sewing on the bioprosthesis (8) to be coated. 11. The device according to claim 9 and 10, characterized in that an insert (10) from the inside and the other insert (11) from the outside with a fastening means (21, 23) on the hollow cylinder (9) is held and both inserts (10 , 11) can be inserted into or removed from the hollow cylinder (9) in the same direction. 12. The apparatus according to claim 11, characterized in that the fastening means as a screw ring (21), whose internal thread engages with an external thread provided on the insert (10), or as a screw cap (23) screwable on the circumference of the hollow cylinder (9) ) are executed. 13. The apparatus of claim 8 and 9, characterized in that in the ring line (1) filling and drain valves (24, 25) for supplying the cell suspension in the bioreactor (2) for coating the collagen matrix with the recipient's own cells or for withdrawing the medium from the bioreactor (2) and the ring line (1). 14. Device according to one of claims 8 to 13, characterized by its arrangement in an incubator, wherein in the bioreactor (2) and / or the ring line (1) a gas-permeable from the outside and from a semipermeable membrane valve (26) impermeable to liquids or a membrane for gas exchange with the atmosphere in the incubator is provided on the inside. 15. Device according to one of claims 8 to 14, characterized in that in the flow direction behind the bioreactor (2) in the ring line (1) a compensation chamber (4) is integrated to compensate for the pressure peaks of the pulsating liquid flow. 16. The apparatus according to claim 8, characterized in that the pump (3) is a diaphragm pump and has an outlet and an inlet valve (6, 7), the outlet valve (6) being omitted in the manufacture of heart valve prostheses. 17. Device according to one of the preceding claims, characterized in that the components coming into contact with the body's own cells consist of a biologically repellent material which opposes cell adhesion. 18. The apparatus according to claim 17, characterized in that the inserts (10, 11) consist of Teflon and the hollow cylinder (9) is made of piacryl. 19. The apparatus according to claim 17, characterized in that the ring line (1) consists of flexible material, for example silicone.