DE19938518A1 - Tissue culture system for epitheliating or endotheliating and functional examination and delivering of synthetic or natural hollow organs - Google Patents

Abstract

A tissue culture system for epitheliating or endotheliating and functional examination and delivering of synthetic or natural hollow organs in controlled sterile conditions for surgical implantation, is new. A tissue culture system for epitheliating or endotheliating and functional examination and delivering of synthetic or natural hollow organs in controlled sterile conditions for surgical implantation, is new. The hollow organ is arranged in a first tube. One end of the organ is connected to a canule. The first tube is placed in a second tube to form a chamber. The first tube has holes in contact with the chamber. Tubes circulate gases and patient serum through the chamber. Nutrients and cells are supplied to the first tube and metabolized wastes are removed.

Description

The present invention relates to a tissue culture system for epithelialization or endothelialization and for functional examination and delivery of natural or artificial hollow organs or vessels under controllable Sterile conditions for surgical implantation purposes the preamble of claim 1.

In particular, the present invention relates to a Be stratification device for sterile sowing, cultivation, Functional check and delivery of endothelial cells in natural or artificial vascular cylinders - in particular  in cryopreserved and previously deendothelialized blood vessels - and their delivery for the purpose of surgery transplantation or implantation.

Background of the Invention

The use of a functional blood vessel replacement plays a role an ever larger one in all surgical therapy areas Role. Arterial vascular replacement is particularly common in the cardiosurgical field practiced. For example, he this follows in the so-called "bypass operations". If it is somehow possible, the primary case is trying that Patients using autologous vascular material, al for example by using prepared segments of vessels eats (vena saphena magna, vena saphena parva or artery mammaria interna) to treat their function in removal largely from communicating, remaining Ge barrels can be taken over. The situation is developing however difficult in individual cases when such vessels due to insufficient quality (e.g. varicosis or arteriosclerosis) cannot be used. On in any case there is a bottleneck in the surgical ge vascular supply but with the increasing number of aortocoronary Reoperations against the background of the earlier one Patients have already consumed auto log material. Gaining more of the body's own Veins or arteries, such as the gastro-artery piploica or the radial artery is for the patient very complex and risky, and therefore rarely comes in question.

For this reason, there is usually only the alternative of prosthetic vascular replacement. For this stood in the prin zip initially only flexible tubes made of biologically inert Plastic (such as Teflon) are available, their use especially at the heart due to a high surface thrombus  genetics was not very successful. Already before Attempts have been made for 20 years by cryoprä served (i.e. in liquid nitrogen until needed in the case of frozen) donor veins the gap in the required close bypass material. The results after the However, implantation is sobering because there are still many of the previously used vascular prostheses of this type soon thrombosed in the patient. Still, these are "cryopreserved allografts" after the failed Attempt to try autologous vascular replacement to date the most suitable means of choice for aortocoronary Bypass reoperations or in primary bypass patients oh sufficient autologous vascular material.

A promising way out is that surgical vascular prostheses prior to implantation vascular endothelium that immuno to avoid logical rejection reactions from the patient must originate, so-called "autologous endothelium" (lamb P., Juchem G., Weyrich P., Nees S., Reichart B .: New alter native coronary bypass graft: First clinical experience with an autologous endothelialized cryopreserved allograft. J. Thorac. Cardiovasc. Surg. (1999), 117 (6): 1217-1219). Vascular endothelium is due to active development richer antiaggregatory, anticoagulant and pro fibrinolytic activities the long-known hay te but also explainable antithrombogenicity in detail al healthy vascular walls in the cardiovascular system.

The enzymatic isolation of vascular endothelial cells also from human blood vessels is by default today feasible. Even sowing of these cells in fibrin coated PTFE prosthesis was made by rotating a vessel filled with the cell dispersion around its longitu final axis reached. The ent is removed after about 3 hours take the endothelial cells that have become adherent  delten vessels and their conversion into special cultivati on vessels. The outgrowth of the endothelial cells up to the continuous the lining of the vessels ("cultivation up to Confluence ") is in conventional incubators at 37 ° C in saturated water vapor atmosphere reached.

This procedure has already become an autologous one Endothelialization of cryopreserved and first of their native endothelium freed veins used, the Kul tivation phase under constant overpressure perfusion with Endothelial cell populated veins (see above cited Publication).

This applies in principle to all previously known methods elle problem that the blood vessels to be endothelialized for the purpose of their preparation, for sowing the endothelial cells len for the cultivation process and for quality after of the vascular preparation finally obtained several times relocated and reassembled. Apart from the danger of considerable mechanical damage to the sensitive prostheses, for example by collapsing and due to friction effects, especially in the area of the inner wall, stands the inevitably high mi under these conditions risk of contamination of a responsible kli African use of such preparations. From analog Another aspect is the cultivation of the vessels for the medical use in conventional incubators pro blematic, quite apart from the fact that with a series pro endothelialized vessels due to the large space and constant retrofitting needs a lot of incubators, sterile workbenches and large cell culture rooms would be necessary. Furthermore, an acceptable quality control of the dothelialized vessels only possible if the product un indirectly possible before its implantation in the patient is checked using the same geometric alignment, in which it is finally sewn in. It also adds  that the previous logistic measures for this Ge also do not worry about barrel replacement like the ones dothelialized prosthesis over long distances on safest and most gentle in the operating room who is.

The object of the present invention is therefore to a coating device for a practical endothe lialization or epithelialization of hollow organs, ins special to create prosthetic blood vessels that a practicable and qualitatively acceptable coating of Ge with endothelial or epithelial cells and for operation in standard sterile banks and isolators is suitable to the complex and expensive commissioning to be able to handle clean rooms. Besides, that should Problem of sterile transportability of the finished products dothelialized prosthetic products up to the operations space and that of the sterile removal there.

This task is performed by a coating device solved the features of claim 1.

The coating device according to the invention looks concise trical coating compartments or coating chamber plan to biologically be in their central interior stratifying, natural or non-natural hollow organs can be stored and fixed. Preferred hollow organs show on their inner surface or on the luminal Surface no longer lined with epithelium or endothelium of the donor. A specially designed embodiment the present invention targets such blood vessels and their flaps off, then with patient autologous endothelium to be lined.

This coating device with the ready Be stratification chambers consists of a non-toxic plant  substances that are sufficient in supersaturated water vapor can be autoclaved for long periods at 127 ° C. To this Complete sterilization can thus be achieved.

Another advantage of the present invention is in that this coating device with the Beschich tion chambers firmly on a special rotation device stored and can be rotated by this.

The sterile coating chambers of the Be layering device let over appropriate cannulas, Hoses and 3-way valves provide constant access to the Inside and outside of the hollow organs too. By simple order switching the access routes to the corresponding supply vessels can be optionally used in connection with the switchable rotation device all preparatory work, Sowing measures, cultivation purposes and implementation carry out exact and reliable quality controls, without the central hollow organ in the meantime must be removed from the coating device. This advantageously results in a considerably low level lower risk of contamination. The qualitative care duty is fulfilled.

In addition, the risk of contamination in a be preferred embodiment of the invention also thereby raise Lich reduced that the necessarily at 37 ° C instead seeding and cultivation of the epithelium or ene dothelial cells do not take place in conventional incubators det, but with tempering through its own river liquid heat jacket in a temperature control device can. As tempering liquids according to the invention disinfectant solutions selected in a closed its temperature control circuit can be circulated.  

Another significant advantage of this configuration of the The present invention is that for tempering the coating chambers only a relatively small one technical and spatial effort is required. There this gives the possibility of a lot of coating chambers at the same time or at different times confined space, preferably in a sterile workbench, to operate side by side in a normal air atmosphere.

The construction of the coating chamber according to the invention also includes the possibility of constant breathing gas exchange growth cells while keeping the pH constant Value in the growth medium without major equipment wall.

It becomes through the coating device according to the invention advantageously allows that in the central Coating chamber encapsulated hollow organ while preserving strictly aseptic cautery without problems from the present The coating device can be removed. Au Furthermore, the sterile transport is also carried out beforehand Enables stretching to the operating room. There you can the finished and previously functionally checked hollow organ easy to remove sterile and then immediately in the plant.

The invention can be applied to all artificial and natural Hollow organs and their components advantageously used be, for example in natural blood vessels and ih valves, lymph vessels and their valves, ureters, Vas deferens, bronchi, bile ducts, intestines and with any prosthetic replacement of such hollow organs.

Advantageous embodiments of the invention are shown in the Sub-claims emerge.  

In the following, the inventions are their configurations explained in connection with the figures. That is gene:

Fig. 1a shows an inventive tissue culture system for the endothelialization of vascular prostheses;

Figure 1b is a particularly inventive appropriate design of the encapsulation of the supplied cannulated prosthesis by means of special caps.

Fig. 2 is a schematic representation of the integration of the device according to the invention in a total system;

. Figure 3 is a schematic view showing the structure of a simple rotation assembly;

Fig. 4 is a schematic representation to explain the preferred temperature control of the device according to the invention in a closed circulation circuit, and

Fig. 5 shows a development of the invention.

According to Fig. 1a, the present tissue culture system 100 comprises substantially a first needle 1 and a second Ka Nuele 2, preferably made of steel, a first tube 3, a second tube 4 and an outer tube 9.

The steel cannulas 1 and 2 each extend from the outside into egg nes of the ends of the first tube 3 and are spaced apart from each other in the longitudinal direction of the first tube 3 . The cannulas 1 and 2 are preferably divided into branches, the two parts 1 ′, 1 ″ being in each case screwable into one another ( FIG. 1b). The associated cannula connection points are positioned in the area of the pinch seal 10 and 11 and are sealed circumferentially by this In addition, the end of the cannula 1 or of the part 1 'protruding outward from the seal 10 has a screw thread, by means of which an extension cannula of the same outer diameter can be attached, which is used in the later insertion of the prosthesis to be coated into the lumen of the Tube 3 is threaded through the hole of the pinch seal 10 and then allows the prosthesis fixed to the cannula 1 to be drawn into the tube 3. The cannula extension piece is then removed again (see here and further details which are still to be described FIG. 1b) ). preferential example is the first pipe 3 made of polycarbonate or glass. in the first tube 3, v in a proven design ier longitudinal rows with holes angeord net, each offset by 90 ° to each other in the circumferential direction of the first tube 3 and have a distance of about 15 mm. The holes 5 preferably each have a diameter of approximately 2 mm. In its outer circumference, the first tube 3 has a helical groove 6 extending from one side of the first tube 3 to the other side thereof, which serves to receive a thin-walled, small-lumen line 7 which is wound around the first tube 3 . The line 7 is preferably made of Teflon. Their diameter is approximately 1-1.5 mm. The wall thickness of line 7 is approximately 0.1-0.2 mm.

On each side of the first tube 3 there is preferably an existing silicone rubber pinch seal 10 , 11 , the cannula 1 or 2 through a central bore 12 or 13 of the pinch seals 10 or 11 and the respective end region of the first tube 3 is inserted into an annular groove 14 or 15 which is located in the side of the respective pinch seal facing the tube 3 . In this way, the first tube 3 is kept concentrically to the cannulas 1 and 2 . The squeeze seals 10 and 11 preferably consist of a silicone rubber from the company Wacker Chemie, Munich (Elastosil 4601 A or B) composed of two components, initially viscous. They are specially cast and hot-polymerized, are completely biologically inert and do not release any toxic additives. Outside the annular groove 14 and 15 , the pinch seal 10 on the left in FIG. 1 has passages 16 and 17 , through which one end region 7 ′ of the line 7 and the other end region 7 ″ of the line 7 are passed.

The first tube 3 is preferably arranged concentrically in the two th, preferably made of glass tube 4 , which engages with its ends in an annular groove 18 or 19 of the pinch seal 10 or 11 . At its end areas, the second tube 4 has an external thread 20 or 21 on the outside, on which the internal thread 22 or 23 of a cap-shaped, autoclavable screw cap 24 or 25 can be screwed, with the pinch seals 10 and 11 between the end wall 26 or 27 of the screw cap 24 or 25 and the respective ends of the first tube 3 and the second tube 4 are squeezed together, the ends of the first tube 3 and the second engaging in the grooves 14 or 15 and 18 or 19 Tube 4 are sealed so that the coating chamber 28 , which comprises the inner space of the first tube 3 and the space 27 'between which he most tube 3 and the second tube 4 , is sealed to the outside. Likewise, the pinch seals 10, 11, the lead-throughs of the Endbe rich 7 are 'and 7 "of the line in the passages 16 and 17 of the left pinch seal 10 sealed. The steel cannula 1 and the end regions 7', 7" upon compression of the pipe 7 pass through a Bore 30 in the end wall 26 of the screw cover 24 to the outside.

The interior of the first tube 3 or the coating chamber 28 is connected via a hose 34 which is passed through a passage 35 of the right pinch seal 11 through to the outside. The hose 34 runs through a bore 36 of the end wall 27 of the right screw cap 25th In a corre sponding manner, a hose line 37 extends from the loading chamber 27 through a passage 33 of the right pinch seal 11 and the bore 36 to the outside.

A design according to the invention of the two pinch seals 10 and 11 with their cannulas 1 and 2 results from FIG. 1b, in which, however, the hose lines 16 and 17 (the pinch seal 10 ) or 34 and 37 (for reasons of clarity) the pinch seal 11 ) have been omitted. There are also outlined the means by which it is possible to shield the inner parts of the two-part cannulas 1 and 2 connected to the prosthesis end pieces from the outside world under sterile cautery, although on the other hand it is necessary to supply the prosthesis from the outside, e.g. B. to perfuse with nutrient medium. Expediently, the pinch seals 10 and 11 are each provided on their outer surface with at least one sealing lip 90 concentrically encircling the central cannula guide hole, the stamp-like steel parts 91 which can be pressed on by means of the screw caps 24 and 25 and have a central bore 92 on their bearing surface to the seals 10 and 11 to their respective cannula. The cylinder-like end of both steel parts 91 is provided with an external thread 93 which enables the screwing of a perforated cover 94 provided with a silicone seal 95 , with the aid of which the part of the cannulas 1 and 2 leading to the seals 10 and 11, respectively, leads to the outside world can be shielded. After finishing the desired coating of the prosthesis (see below), the outer parts of the cannula can be unscrewed and the remaining cannula pieces in the area of the seals 10 and 11 1 ", 2 " with the attached prosthesis by screwing on an end cap 96 with unperforated device 97 on End of the steel parts 91 are closed sterile for transport. It is pointed out that in Fig. 1b only the left side with the cannula parts 1 'and 1 "is shown. The right side is designed accordingly.

The outer tube is arranged concentrically to the arrangement described so far, which preferably consists of a glass tube which has an access piece 38 , 39 at each end region and each has an annular holding bead 40 or 41 in front of this on the outer surface of the outer tube 9 . On the cap-shaped screw bendeckel 24 and the cap-shaped screw cap 25 , which hold the first tube 3 and the second tube 4 in the manner described, a cap part 42 and 43 are each set on, the end regions of the outer tube 9 each in an annular groove 44 or 45 of the cap part 42 or 43 engage and the retaining beads 40 or 41 engage in corresponding recesses 46 or 47 of the cap parts 42 or 43 . In this way, the outer tube 9 is held by the flexi ble and taut cap parts 42 and 43 concentric to the second tube 4 , the space 48 between the second tube 4 and the outer tube 9 being sealed to the outside.

The cap parts 42 , 43 preferably also consist of the specially cast and hot-polymerized 2-component silicone rubber from Wacker Chemie, Munich, already described above.

The cannulas 1 , 2 run through bores 49 and 50 of the left and right cap parts 42 and 43, respectively. Through the bore 49 of the left cap part 42 , the end regions 7 ', 7 "of the hose 7 also extend outwards. Through the bore 50 of the right cap part 43 , the hose lines 34 , 37 also run outwards.

For its endothelialization, a blood vessel 51 is arranged approximately centrally in the first tube 3 , the cannulas 1 and 2 running into the ends of the blood vessel 51 . For sealing, the ends of the blood vessel 51 are fixed to the end areas of the cannulas 1 , 2 with a thread.

In the recognizable from FIG. 1 example, are located at the ends of the needles 1, 2, the ends of the end regions 7 ', 7 "of the hose 7 and the ends of the hose lines 34, 37 coupling pieces 52 which preferably already mentioned, also of the 2-component silicone rubber be available and serve for tight connection with external connection hoses, not shown in FIG. 1.

Due to the selection of the materials described above can the entire present facility in saturated Steam autoclaved at a temperature of 121 ° C and therefore absolutely reliable even in all cavities be sterilized.

Referring to FIG. 2, the present tissue culture device 100 of FIG. 1 ver with various supply units connected. Details of FIG. 2, which have already been explained in connection with FIG. 1, are designated accordingly. In the left part of Fig. 2 it is shown that the blood vessel 51 via the cannula 1 , 3-way valves 53 , 54 , 55 and various hose lines 56 , 57 , 58 , 59 contacted with different solutions: from a vessel 60 via line 59 with culture medium, via line 58 with autologous serum from a vessel 61 , via line 57 also with other solutions or suspensions. The gas-permeable line 7 made of thin-walled Teflon allows an adequate supply of the blood vessel cells in the otherwise completely closed system with oxygen and, because of the admixture of 5% CO ₂ in the blown atmospheric air, ensures a constant pH of the inside of the tissue culture direction used bicarbonate-buffered culture medium. For this purpose, the end region 7 ′ of the line 7 is connected to a gas inlet 62 for atmospheric air and 5% CO ₂ and the end region 7 ″ of the line 7 is connected to a gas outlet 63 .

With the help of the line 7 and with a corresponding switching device of the 3-way valves 53 , 54 , 55 and further 3-way valves 64 , 65 shown on the right in FIG. 2, the exterior of the blood vessel 51 can also be used Hose line 56 or 34 are supplied with culture medium, the culture medium being passed through a passage through the coating chamber 28 via the hose line 37 and the 3-way valves 64 , 65 to a drip vessel, not shown. On the one hand, this has the advantage that the unstable, mostly thin-walled blood vessel 51 does not rest with its entire weight on the support tube, but rather floats in the culture medium, and on the other hand, when a slight excess pressure is applied (ΔP = 10-20 cm H ₂ O) for filling and perfusion of the blood vessel 51 with culture medium, there is also a slight transmural filtration of this medium through the wall layer of the blood vessel 51 if the path for this filtrate quantity is released via the 3-way valves 64 , 65 to the drip vessel. This procedure promotes the mass exchange of the wall cells. With long-term flow (perfusion) of the blood vessel 51 with the - because of the admixture of auto logical patient serum - valuable culture medium, it is advisable to guide the culture medium dripping from the cannula 2 via a further 3-way valve 66 under the control of a periodically opened Solenoid valve 67 or a periodically activated peristaltic hose pump.

Fig. 3 shows the side view of the onseinrichtung on a Rotati 68 positioned the present tissue culture system. A continuous and alternating preferably at least 180 ° in one and then at least 180 ° in the other direction, 15-20 times per 1 hour rotating drive roller 69 , expediently with a silicone coating, causes a passive rotation of the coating device. The also passively rotating roller 70 together with the drive roller 69 serves as a support for the tissue culture system. An easy-to-lock bracket 72 provided with an easily rotatable wheel 71 holds the coating device preferably securely from above between the rollers 69 , 70 . The longitudinal dimensioning of the rollers 69 , 70 is preferably chosen so that it lies - in a side view - between the two access ports 38 , 39 of the outer tube 9 of the present coating device. This eliminates the risk of breakage of the access port 38 , 39 .

In connection with FIG. 4, the preferred temperature control of the present coating device in the recirculation principle will now be explained. Since the bath fluid should also have disinfectant properties and may therefore also contain organic solvents (e.g. ethanol), the circulation is expediently carried out in a closed system. The temperature control device 73 has two shear spiral 74 connected via an intermediate heat exchanger and the space 67 between the tube 4 and the outer tube 9 , storage surfaces 75 , 76 connected in series. Storage bottle 75 is located together with the heat exchanger coil 74 in the 37 ° C warm bath or water of a standard circulating thermostat. Storage bottle 76 is preheated to 37 ° C. by means of its own heating jacket 77 with the circulated temperature control water. The liquids in the storage bottles and in the heat jacket of the endothelialization unit communicate via the hose lines 78 , 79 , while the gas spaces 80 , 81 of the two bottles 75 , 76 communicate via the air exchange hose 82 .

If the storage bottle 76 is now alternately raised from level II to level I or lowered again to level II with the aid of a mechanical device (not shown) which is as simple as possible, etc., there are corresponding compensating flows of temperature-control liquids in lines 77 , 78 and 81 and thus for uniform temperature control of the tissue culture system or the blood vessel 51 positioned therein. This temperature control system is expediently mounted on the side outside of the sterile workbench used to shield the coating unit and is only connected to the interior thereof via the hoses 77 and 78 .

The described coating device can be used in a geous manner in a confined space also for series coating of several blood vessels at the same time, if a corresponding number of tissue culture systems are arranged next to one another on their individual or series-connected rotating devices 68 and are provided with bath fluid. Expensive incubators are no longer required, the space requirement is relatively small.

In the following the function of the invention direction based on the autologous endothelialization of a a patient's cryopreserved vein.  

Cryopreservation, thawing and deendothelia vein and isolation from autologous serum or autologous endothelial cells and their cultivation before the actual endothelialization due to in the Technical literature on published methods.

For a better understanding of all the processes described below, reference is made to FIGS. 1 to 4.

The autoclaving of the outer tube 9 of the present loading coating device with the attached cap parts 42 , 43 is carried out separately from the tubes 3 , 4 held together via the pinch seals 10 , 11 or the screw caps 24 , 25 . The end regions 7 ′, 7 ″ of the line 7 or the hose lines 35 , 37 are also previously threaded into the pinch seals 10 , 11 designed in accordance with FIG. 1b and provided with their couplings 52 .

After the parts of the present coating device have cooled, a thawed and de-endothelialized donor vein freed from their native endothelium is connected at its ends to the two-part cannulas 1 , 2 (see FIG. 1 b). Then the separately autoclaved pipe system consisting of pipes 3 and 4 is opened from the right end by unscrewing the screw cap 25 with the inserted steel part (see FIG. 1b) and removing the pinch seal 11 . At closing, the two-part cannula 1 is screwed on extension cannula (see. Fig. 1b and text of the ge technical description on pages 9 and 11/12) through the corresponding bore 12 of the opposite pinch seal 10 and the bore 30 of the screw cap 24th guided. This measure is facilitated by slightly unscrewing the screw cover 24 with an inserted steel part, so that the pinch seal 10 can relax. In this way, the blood vessel 51 finally reaches its full length in the interior of the first tube 3 and the right, two-part cannula 2 can now through the central bore 13 of the previously removed but already positioned pinch seal 11 and the bore 36 of the Screw cap 25 are introduced. Now the United circuit of the double pipe system with the tubes 3 and 4 is also possible at its right end by seal 11 and screwing the screw cap 25 . The two ends of the cannula len 1 , 2 now receive coupling elements 52nd Finally, the entire construction can be encased with the help of the outer tube 9 and the associated cap parts 42 , 43 .

The fully assembled coating device is connected via the various lines 56 , 57 , 58 and the 3-way valves 53 , 54 , 55 , 64 , 65 , 66 according to FIG. 2 and positioned on a rotating device 68 according to FIG. 3. The line 7 is flowed through to the end of all the steps described below by an air mixture with 5% by volume carbon dioxide.

To prepare the blood vessel 51 for sowing the patient autologous endothelial cells, it is first filled with autologous serum from the storage vessel 61 via the appropriately placed 3-way valves 53 , 54 and 55 ( FIG. 2) 3-way valve 66 must briefly release the displaced air from the blood vessel. There is an incubation for 12 to 24 hours at 37 ° C with the help of the tempering device shown in Fig. 4.

The serum is then expressed by injecting sterile air through line 57 and with the 3-way valve 55 closed and 3-way valves 54 , 53 and 66 open. Immediately afterwards, the patient's endothelial cells are injected in an analogous manner by cultivation and then suspended in a concentration that has been precisely determined by microscopic counting (120,000 per cm ^{2 of} the inner blood vessel wall). The extravascular space is filled with culture medium via the hose line 34 , the air escaping from the tissue culture system which is inclined from left to right at the top in FIG. 2 via the corresponding 3-way valve 64 and the hose line 37 . Since after the device shown in Fig. 3 Rotationseinrich 68 is activated for three hours (15 to 20 alternating de half-turns / h). During this period, the sedimenting endothelial cells adhere evenly to the inner wall of the vessel. The temperature control at 37 ° C remains all the time and also during all subsequent work steps. After switching off the rotation and then expressing the contents of the blood vessel 51 (it can be checked microscopically for endothelial cells still suspended!) With injected air via line 57 and the 3-way valve 66 , the blood vessel 51 is opened accordingly 3-way valves 53 , 54 , 55 and 66 filled with culture medium that flows from the vessel 60 - in a particularly simple manner using a Boyle-Marriott bottle, but of course possibly also using electronic or other constant pressure transmitters - is offered under slight overpressure (approx. 10-20 cm H ₂ O).

The culture medium emerging from the blood vessel 51 or the vein lumen is guided with a suitably switched 3-way valve 66 over a hose section which is only periodically (e.g. every hour for about 10 minutes) by activating a peristaltic pump or of a solenoid valve 67 is released. This results in a particularly economical, discontinuous perfusion of the blood vessel 51 with the valuable culture medium (medium 199 with the addition of 20% autologous serum and recombinant basic fibroblasts - growth factor in the concentration 2 ng / ml). In order to achieve a possible physiological flushing of the vascular wall layers due to the slight hydrostatic excess pressure in the blood vessel 51 by transmural filtration, the 3-way valves 64 and 65 are switched so that the path from the extravascular space to a sterile shielded from the outside world Drainer is released.

After 5 to 7 days of discontinuous perfusion of the blood vessel 51 and the resulting confluent lining of its inner surface with patient-autologous endothelium, various quality controls can in principle be carried out to demonstrate the completeness and quality of the endothelial layer obtained. The required filling of the blood vessel 51 with special reaction solutions or substrates is again achieved via the line 57 and by accordingly switching the 3-way valves 53 , 54 , 55 or opening the 3-way valve 66 .

Finally, the blood vessel 51 is carefully rinsed again with culture medium and filled under a slight excess pressure. All coupling pieces 52 are clamped, then all hose connections are pulled off at the distal end of all coupling pieces 52 . The overpressure in the blood vessel 51 is thus maintained and the interior of the tissue culture system is sterile. Now the cap parts 42 , 43 and the heat jacket of the coating device can be removed. Now the still closed central double tube system (tubes 3 , 4 ) can be removed with vein 51 , rinsed on its outer walls with sterile PBS solution and then sent to the operating room. Only there who removed the screw cap 24 , 25 . Then the blood vessel 51 is carefully removed and immediately implanted.

Fig. 5 shows a preferred development of the inven tion, in which the cover 42 of Fig. 1a, which holds the screw cover 24 and seals to the tube 9 , is replaced by a cap pen member 198 , the 197 with a hollow cylindrical portion Screw cap 24 overlaps and lies with a flange 196 on a support flange 195 of the tube 9 . The flange 196 also bears against a circular plate 194 , preferably a metal plate, which has an inclined edge 193 on the outside. The end region of the tube 9 also has an inclined edge 192 on the outside. The oblique edges 192 , 193 form a "V" or the same, which can be overlapped on the outside by a complementarily formed clamping ring 190 . When clamping the known clamping ring 190 by actuating an eccentric lever, the oblique edges 192 , 193 are pulled towards each other and the plate 194 and the flange 196 of the cap member 198 are pressed against the bearing flange 195 of the tube 9 to produce a tight seal.

The cap member preferably consists of a hot polyme The two-component silicone rubber material.

In the plate 194 and in the cap member 198 there are preferably holes 199 , 2000 in the center , through which the end regions 7 ′, 7 ″ of the hose 7 , the cannulas 1 and 2 or the hose lines 34 , 37 can extend to the outside.

Claims (24)

1. tissue culture system for epithelialization or endothelialization and for functional examination and delivery of natural or artificial hollow organs or vessels under controllable sterile conditions for surgical implantation purposes, characterized in that a hollow organ ( 51 ) can be arranged in a first tube ( 3 ), wherein one end of the hollow organ ( 51 ) with egg ner from one side of the tube ( 3 ) tightly lead out first or second cannula ( 1 , 2 ) is connectable, that the first tube ( 3 ) is arranged in a second tube ( 4 ) , A chamber ( 77 ) sealed on the sides of the second tube ( 4 ) is formed between the first tube and the second tube, so that the first tube ( 3 ) has holes ( 5 ), the connections between that of the first tube ( 3 ) enclosed space ( 28 ) to the chamber ( 77 ) ago that around the outer circumference of the first tube ( 3 ) around a gas-permeable line ( 7 ), the E nd areas ( 7 ', 7 ") are led out of the chamber ( 77 ) so that a patient's autologous serum can be filled through a cannula ( 1 ) into the blood vessel ( 51 ) and incubated for a predetermined first period of time and then suspended cells of the patient are injectable in a predetermined concentration, which remain in the blood vessel ( 51 ) for a second predetermined time, such that the line ( 7 ) can be flowed through by an air mixture with a predetermined volume fraction of carbon dioxide, that into the chamber ( 77 ) and through the holes ( 5 ) in the space ( 28 ), a culture medium can be filled in via a line ( 34 ) inserted from one side of the second tube ( 4 ) into the chamber ( 77 ) and that after removing the injected, suspended cells of the patient from the blood vessel ( 51 ) a culture medium can be introduced into the blood vessel ( 51 ), which precedes during the growth of cells sedimented on the vessel wall during a third The given period of time in the blood vessel ( 51 ) remains under hydrostatic excess pressure or is perfuned by this. 2. tissue culture system according to claim 1, characterized in that the first tube ( 3 ) is arranged concentrically in the second tube ( 4 ). 3. tissue culture system according to claim 1 or 2, characterized in that on each side of the first tube ( 3 ) and the second tube ( 4 ) a first or second pinch seal ( 10 , 11 ) is arranged, that an end portion of the first Tube ( 3 ) engages in a first groove ( 14 , 15 ) of the first or second pinch seal ( 10 , 11 ), that one end region of the second tube ( 4 ) in a second groove ( 18 , 19 ) of the first or second Pinch seal ( 10 , 11 ) engages that on the outer circumference of the end region of the second tube ( 4 ) each has a fastening device for a cover ( 24 , 25 ) which can be arranged over the first or second pinch seal ( 10 , 11 ), such that when the lid ( 24 , 25 ) is fastened, the first or second pinch seal ( 10 , 11 ) is compressed, and that the first or second cannula ( 1 , 2 ) is sealed through a bore ( 12 , 13 ) of the first or second pinch seal ( 10 , 11 ), the end regions ( 7 ', 7 ") of the line ( 7 ) tightly through a passage ( 16 , 17 ) of a pinch seal ( 10 ) and the lines ( 34 , 37 ) tightly through passages ( 35 , 36 ) of a pinch seal ( 11 ). 4. tissue culture system according to claim 3, characterized in that the fastening device is formed by a Außenge thread ( 20 , 21 ) of an end region of the second tube ( 4 ) and an internal thread ( 22 , 23 ) of the lid designed as a screw cap ( 24 , 25 ) is. 5. tissue culture system according to one of claims 1 to 4, characterized in that the holes ( 4 ) in the longitudinal direction of the first tube ( 3 ) extending rows of holes are arranged, which are spaced apart in the circumferential direction of the first tube res ( 3 ). 6. tissue culture system according to one of claims 3 to 5, characterized in that the end regions ( 7 ', 7 ") of the line ( 7 ) and the first cannula ( 1 , 2 ) through a passage ( 33 ) of a cover wall ( 26 ) a cover ( 24 ) are guided. 7. tissue culture system according to one of claims 1 to 7, characterized in that the leading to the chamber ( 77 ) lines ( 34 , 37 ) through passages ( 35 , 36 ) of a pinch seal ( 11 ) are guided. 6. tissue culture system according to claim 7, characterized in that the chamber ( 77 ) leading lines ( 34 , 37 ) and the second cannula ( 2 ) through a passage ( 32 ) of a cover wall ( 27 ) of a cover ( 25 ). 9. tissue culture system according to one of claims 1 to 8, characterized in that the ends of the first cannula ( 1 ), the second cannula ( 2 ), the end regions ( 7 ', 7 ") of the line ( 7 ) and the chamber ( 77 ) leading lines ( 34 , 37 ) are each connected to a coupling piece ( 52 ) for connection to external lines. 10. Tissue culture system according to one of claims 1 to 9, characterized in that the second tube ( 4 ) is surrounded by an outer tube ( 9 ) which in the vicinity of its ends each has an access port ( 38 , 39 ) that the end regions the outer tube ( 9 ) for sealing the between the second tube ( 4 ) and the outer tube ( 9 ) formed space ( 67 ) tightly in a groove ( 44 , 45 ) of a tight on the first or second cover ( 24 , 25 ) put on first or second cap part ( 42 , 43 ) and that a heat transfer liquid can be introduced into the space ( 67 ) via the access ports ( 38 , 39 ). 11. tissue culture system according to claim 10, characterized in that the first or second cap part ( 42 , 43 ) on the respective end region of the outer tube ( 9 ) is held by a further fastening device ( 40 ). 12. Tissue culture system according to claim 11, characterized in that the further fastening means comprises a first or second retaining bead ( 40 , 41 ) arranged on the outside at the respective end region of the outer tube ( 9 ), which in the inner circumference of the first or two th Cap part ( 42 , 43 ) engages tightly. 13. Tissue culture system according to one of claims 1 to 12, characterized in that the first pinch seal ( 10 ) and the second pinch seal ( 11 ) and / or the first lid ( 24 ) and the second lid ( 25 ) and / or the first cap part ( 42 ) and the second cap part ( 43 ) and / or the coupling pieces ( 52 ) consist of a hot-polymerized two-component silicone rubber material. 14. Tissue culture system according to one of claims 1 to 13, characterized in that it comprises a rotation device ( 70 ) which rotates the outer tube ( 9 ) and the elements connected therewith alternately in one and in the other direction. 15. Tissue culture system according to claim 1, characterized in that the rotation device ( 70 ) comprises a driving roller ( 71 ) which engages on the outer circumference of the outer tube ( 9 ). 16. Tissue culture system according to claim 15, characterized in that the outer tube ( 9 ) with its region between the outlet ports ( 38 , 39 ) arranged on the drive roller ( 71 ) and a roller ( 72 ) spaced apart from this. 17. tissue culture system according to claim 15 or 16, characterized in that above the on the drive roller ( 71 ) and the roller ( 72 ) lying on the outer tube ( 9 ) on the outer tube ( 9 ) engaging wheel ( 73 ) is provided, the wheel ( 73 ) in an adjustable bracket ( 74 ) is kept ge. 18. Tissue culture system according to one of claims 15 to 17, characterized in that the length of the drive roller ( 71 ) and the roller ( 72 ) corresponds approximately to the distance between the two access ports ( 38 , 39 ) of the outer tube ( 9 ) from each other. 19. Tissue culture system according to one of claims 1 to 18, characterized in that a temperature control device ( 80 ) is provided which has two vessels ( 82 , 83 ), each containing a heat transfer liquid, the heat transfer liquids via a heat exchange device ( 81 ) and the space ( 67 ) between the second tube ( 4 ) and the outer tube ( 9 ) are connected in series, the air volumes of the vessels ( 82 , 83 ) above the bath fluids being connected to one another by an air exchange line ( 87 ), and that a mechanical cal device is provided which raises or lowers the vessels ( 82 , 83 ) alternately in opposite phase between alternating levels (I, II), so that through the space ( 67 ) between the second tube ( 4 ) and the outer tube ( 9 ) and the heat exchanger device ( 81 ) flows alternately between the vessels. 20. Tissue culture system according to claim 20, characterized in that the heat exchanger device ( 81 ) is arranged together with the one vessel ( 82 ) in a tempering bath kept at a given temperature. 21. Tissue culture system according to claim 20, characterized in that the outer tube ( 9 ) at its ends each has a support flange ( 195 ) that a cap pen member ( 198 ) can be fitted that the cap member ( 198 ) has a flange ( 196 ) , the flange on the Aufla ( 195 ) of the outer tube ( 9 ) rests that a plate ( 194 ) on the side facing away from the cover part ( 24 ) can be arranged and placed on the flange ( 196 ), and that the plate ( 194 ) and the end of the outer tube res ( 9 ) each run obliquely outwards and form a bulge, preferably a "V", on which a complementarily designed clamping ring ( 190 ) can be placed, which clamps the plate ( 198 ) against the flange ( 196 ) and presses it against the support flange ( 195 ). 22. Tissue culture system according to claim 21, characterized in that the cap member ( 198 ) consists of a hot poly merized two-component silicone rubber material. 23. Tissue culture system according to one of claims 1 to 22, characterized in that the cannulas ( 1 , 2 ) are designed in a two-part manner, and in each case comprise two mutually bindable cannula parts ( 1 ', 1 ", 2 ', 2 ") that the junction of the cannula parts ( 1 ', 1 ", 2 ', 2 ") in the area of the pinch seals ( 10 , 11 ) positionable and circumferentially sealable, that with the cover part ( 24 , 25 ) a stamp-like part ( 91 ) with it Contact surface against the pinch seal ( 10 , 11 ) can be pressed, a cylindrical area running through the opening ( 30 , 36 ) of the cover part ( 24 , 25 ) and that the cannula part ( 1 ', 2 ') through a central bore ( 92 ) of the cylindrical portion of the stamp-like Tei les ( 91 ), and that on the free end of the cylin drical area, a cover ( 94 ) with an opening for passing the cannula part ( 1 ', 2 ') or from a cover plate ( 96 ) can be placed tightly . 24. A method for coating a blood vessel ( 51 ) with cells in a coating device according to one of claims 1 to 23, characterized by the following steps:

• a) Tightly connecting a be from their native endothelium free blood vessel ( 51 ) of a donor at its ends with the first cannula ( 1 ) and the second cannula ( 2 ).
• b) arranging the blood vessel ( 51 ) and the associated first, cannula ( 1 ) and second cannula ( 2 ) in the first tube ( 3 ) such that the second cannula ( 2 ) through the passage ( 13 ) of the second pinch seal ( 11 ) runs that the first cannula ( 1 ) and the end regions ( 7 ', 7 ") of the line ( 7 ) through the passage ( 12 ) and the passages ( 16 , 14 ) of the first pinch seal ( 10 ) and that the passages leading to the chamber ( 77 ) run through the second pinch seal ( 11 ).
• c) Attach the first cover ( 24 ) and the second Dec kels ( 25 ) on the second tube ( 4 ).
• d) arranging and fastening the outer tube ( 9 ) over the second tube ( 4 ) with the first cap part ( 42 ) and the second cap part ( 43 ) or with cap members ( 198 ), plates ( 194 ) and clamping levers ( 190 ).
• e) flowing through the line ( 7 ) with an air mixture with a predetermined volume fraction of carbon dioxide.
• f) introducing patient autologous serum into the blood vessel ( 51 ) via a cannula ( 1 ).
• g) alternating flow through the space ( 67 ) between the second tube ( 4 ) and the outer tube ( 9 ) with a tempering liquid temperature in the temperature control device ( 80 ) during a first predetermined period of time.
• h) removing the patient autologous serum from the blood vessel ( 51 ).
• i) Filling the blood vessel ( 51 ) with the patient's epithelial cells increased and subsequently suspended in a precisely determined concentration.
• j) filling the chamber ( 77 ) with a culture medium.
• k) Alternating rotation of the outer tube ( 9 ) in the Rotati onseinrichtung ( 70 ) for a second predetermined period of time, wherein the sedimented endothelial cells adhere to the inner wall of the vessel.
• l) Continuous rotation device ( 70 ) and delivery of the contents of the blood vessel ( 51 ) via a cannula ( 2 )
• m) filling the blood vessel ( 51 ) with culture medium and continuous discontinuous perfusion of the blood vessel ( 51 ) with the culture medium.
• n) removing the outer tube ( 9 ) and the cap parts ( 42 , 43 ) or the cap members ( 198 ), plates ( 194 ) and tensioning lever ( 190 ) after a discontinuous perfusion with the culture medium for a third predetermined period of time and sealing the first cannula ( 1 ) and the second cannula ( 2 ) and transport of the blood vessel ( 51 ) together with the first tube ( 3 ) and the second tube ( 4 ), via the first pinch seal ( 10 ) and the first cover ( 24 ) and the second pinch seal ( 11 ) and the second cover ( 25 ) are tightly connected to one another in an operating room and there the blood vessel ( 51 ) is removed.