EP1414295A2 - Method for the devitalisation of natural organs and/or for the preparation of extracellular matrices for tissue engineering - Google Patents

Abstract

The invention relates to methods for devitalising and conserving human and animal organs and tissue, preferably natural hollow organs and the complete components thereof, in particular blood vessels and coronary valves. The invention further relates to methods for production of matrices for the partial and full construction of organs and tissues and furthermore to organs and tissue, in particular natural and artificial hollow organs, which may be obtained by said method. The invention also relates to the clinical application and use of organs thus produced in clinical and veterinary medicine, preferably in coronary and vascular surgery and novel culture media.

Description

 Process for devitalizing natural organs and / or

Provision of extracellular matrices for "tissue engineering"

The invention relates to methods for the devitalization and preservation of human and animal organs and tissues, but preferably natural hollow organs and all of their components, in particular of blood vessels and heart valves. Furthermore, the invention relates to methods for the production of matrices for the partial and new structure of organs and tissues. In addition, the invention relates to organs and tissues, in particular natural and artificial hollow organs, which can be obtained by the processes according to the invention. Furthermore, the invention relates to the clinical use and use of the organs and tissues produced in medicine and veterinary medicine, preferably in cardiac and vascular surgery. The method according to the invention provides organs and tissues which have a higher mechanical stability and a better suitability for further processing by the methods of "tissue engineering" compared to the organs and tissues produced with conventional methods. In particular, the immunological tolerance and antithrombogenicity of the processed organs and tissues is significantly improved by the washing-out process according to the invention of undesired cell degradation products and cell debris. Such organs and tissues show a significantly reduced thrombogenicity and immunogenicity in comparison to the original starting organs and tissues and also in comparison to organs or tissues that were only partially produced according to the invention, that is to say without the washout process.

To date, various preservation techniques have been used to store organs and body tissues that are to be reused in humans:

The short-term storage of human heart valves for 3 to 4 days in an antibiotic cocktail at 4 ° C is carried out when the

Donor organ already has a recipient available for the affected heart valves stands and due to the severity of the disease, the operation can no longer be delayed. This technique is called "fresh wet" transplantation. Prolonged storage of the organs leads to their decay.

Furthermore, the preservation of organs after preservation with so-called "cross-linking agents", such as Glutaraldehyde, formaldehyde, polyether oxides (“polyepoxy compound”), hexamethylene or acylazides are known. An advantage of this technique is the possibility of long-term storage after pretreatment with this technique. A disadvantage, however, is the principle unsuitability of such pretreated tissue for use in body systems that are subject to high mechanical stress, such as the arterial tract. Veins and arteries pretreated in this way have so far been characterized by increased early occlusion rates and a high mechanical error rate. Attempts to detoxify "cross-linking agents" and then to rebuild them using "tissue engineering" methods have so far been unsuccessful.

The clinically most important preservation technique for organs and tissues is cryopreservation.

The cryopreservation and storage of organs and body tissues for preservation and later use, for example in the context of a transplant, are known and clinically established. The techniques used differ only slightly (Brockbank KGM. Basic Principles of Viable Tissue Preservation. In: Transplantation Techniques and Use of Cryopreserved Allograft Cardiac Valves and Vascular Tissue. DR Clarke (ed.), Adams Publishing Group Ltd., Boston. S 9-23.American Association of Tissue Banks Standards for Tissue Banking (1995), AATB, McLean, VA, USA European Association of Tissue Banks General Standards for Tissue Banking (1995), EATB, Vienna, Austria). Cryopreservation is used primarily in the storage of human heart valves, the so-called "homografts", and in the storage of human veins or other tissues.

For example, the use of cryopreserved vein allografts is an established procedure in bypass surgery (Brockbank KGM et al., Cryopreserved vein transplantation. J. Cardiac Surg. 7: 170-176, 1992; Gelbfish J. et al., Cryopreserved homologous saphenous vein: Early and late patency in coronary artery bypass surgical procedures. Ann. Thorac. Surg. 42:70, 1986; Fujitani RM et al., Cryoperserved saphenous vein allogenic homografts: An alternative conduit in lower extremity arterial reconstruction in infected fields. J. Vase. Surg. 15: 519-526, 1992) and is used in patients who do not have sufficient body material or whose vessels are not of a suitable quality. Such cryopreserved veins are often used as bypasses in infected body regions. The use of prosthetic material is prohibited here due to the high, material-related, incidence of the prosthesis infection.

However, cryopreserved veins show poor long-term progression (Bilfinger TV et al., Cryopreserved Veins in Myocardial Revascularization: Possible Mechanism for Their Increased Failure. Ann. Thorac. Surg. 63: 1063-69, 1997 and comment in Ann. Thorac. Surg. 64 : 1524-5, 1997. Marshin RS et al., Cryopreserved Saphenous Vein Allografts for Below Knee Lower Extremity Revascularization. Ann. Surg. 219: 664-72, 1994). The main reason for this is probably an immunologically induced degeneration of the vein wall (Carpenter JP, Tomaszewski JE, Immunosuppression for Human Saphenous Vein Allograft Bypass Surgery: a Prospective Randomized Trial. J. Vase. Surg. 26: 32-42, 1997. Carpenter JP, Tomaszewski JE, Human Saphenous Vein Allograft Bypass Grafts: Immune Response. J. Vase. Surg. 27: 492-9, 1998). In addition, thrombotic early occlusions of cryopreserved veins are often observed. Both processes have so far been described in many publications on damage to the donor endothelium during cryopreservation, which can lead to the complete absence thereof, or on a restricted functionality of the endothelium obtained (Brockbank KGM et al., Cryopreserved vein transplantation. J. Cardiac Surg. 7: 170-176, 1992; Brockbank KGM et al., Functional analysis of cyropreserved veins. J. Vase. Surg. 11: 94- 102, 1990. Laub GW et al., Cryopreserved allograft veins as alternative coronary conduits: early phase results. Ann. Thorac. Surg. 54: 826-31, 1992. Louagie YA et al., Viability of long term cryopreserved human saphenous veins J. Cardiovasc. Surg. 31: 92-100, 1990).

As a result, previously known and already patented cryopreservation techniques for allo- and xenografts aim to ensure the highest possible degree of preservation of the vascular and microvascular donor endothelium after the cleaning process. The degree of preservation of the donor endothelium of the cryopreserved tissue is given in the literature as 50% - 80% (Bambang LS et al., Effects of cryopreservation on the proliferation and anticoagulant activity of human saphenous vein endothelial cells. J. Thorac Cardiovasc. Surg. 110: 998-1004).

In recent times, however, the vascular and microvascular endothelium has been given a key position in the context of acute and chronic organ rejection. Endothelium-specific non-HLA antigens, which lead to an activation of CD4 T cells, enable the donor endothelium - in cooperation with other accessory molecules - to offer foreign antigens to the recipient's immune system. The release of non-HLA antigens by damaged endothelial cells leads to a chronic immune reaction and possibly to graft vasculopathy and chronic rejection (Rose ML, Role of endothelial cells in allograft rejeetion. Vase. Med. 2 (2): 105-14, 1997; Reul RM, Fang JC, Denton MD et al, CD 40 and CD 40 ligand (CD 154) are coexpressed in microvessels in vivo in human cardiac allograft rejeetion. Transpalantation 64 (12): 1765-74, 1997; Salom RN, Maguire JA, Hancock WW, Endothelial activation and cytokine expression in human acute cardiac allograft rejeetion. Pathology 30 (1): 24-29, 1998). Conversely, the selective removal of the donor endothelium causes the absence of acute rejection reactions in the animal experiment (rat) (Ann. Surg. 206: 757-764, 1987), where spontaneous reendothelialization can then occur. Bypasses pretreated in this way showed an improved service life in animal experiments.

Based on the idea that the immunological graft rejection originates from all cellular components of the graft, various methods for removing these cells have been developed (US Pat. No. 5,613,982). Various hydrolytic enzymes (e.g. proteases, lipases, nucleotidases, glycosidases etc.), but also physical-chemical measures (use of hypotonic media or detergents, vapor phase freezing processes, pH extremes etc.) were used (US Pat. 5,192,312; U.S. Pat. No. 5,632,778; U.S. Pat. No. 5,613,982, U.S. Pat. No. 5,843,182, WO 95/24873). However, such measures have the fundamental disadvantage that it can never be ruled out that these treatments may also have a detrimental effect on the strength of other components which are also crucial for the structural integrity of hollow organ walls, such as, for example, collagens, in particular type I collagen, proteoglycans or glycoproteins. This is all the more important since the most serious complications, which are based on a structural wall weakness of the cryopreserved veins, such as tears in the vein wall or wall sacking (aneurysms) of the veins, which lead to complications-prone reoperations that also occur long after the implantation in humans can be well known (Lehalle B et al., Early rupture and degeneration of cryopreserved arterial allografts. J. Vase. Surg. 25: 751-2, 1997. Couvelard A. et al., Human allograft failure. Hum. Pathol. 26 : 1313-20, 1995). If decellularized tissue serves as a matrix for recellularization measures, it becomes necessary to incubate the tissue to be transplanted with high concentrations of special adhesion factors or growth factors in order to enable cells to resettle in the wall (US Pat. No. 5,632,778 and 5,613,982 and U.S. Pat. No. 5,192,312, U.S. Pat. No. 5,843,182, WO 95/24873). Such preparations are expensive, mostly not approved for clinical use and it is still largely unknown to what extent unphysiologically high concentrations of these active substances influence the functional differentiation of tissues. This is all the more important since, as mentioned above, only complete differentiated endothelium is effective (Ku D et al., Human coronary vascular smooth muscle and endothelium-dependent responses after storage at -75 ° C. Cryobiology 29: 199-209, 1992).

It is known today that a certain percentage of viable donor cells still remains after conventional cryopreservation, which has been discussed as a starting point for immunological reactions (Yankah AC et al., Antigenicity and fate of cellular components of heart valve allografts. In: Yankah AC , Hetzer R, Yacoub MH, eds. Cardiac valve allografts 1962-1987. Current concepts on the use of aortic and pulmonary allografts for heart valve substitutes. Darmstadt: Steinkopff Verlag 1988). Other authors, on the other hand, consider the viability of the graft to be immunologically insignificant and even prove that the life of viable grafts is longer (O 'Brian MF et al., A comparison of aortic valve replacement with viable cryopreserved and valves, with note on chromosomal studies. J. Thorac. Cardiovasc. Surg. 94: 812-23, 1987. Angell WW et al., Long term function of viable frozen aortic homografts: a viable homograft valve bank. J. Thorac. Cardiovasc. Surg. 93: 815-22, 1987). In general, however, viable homograft is preferred in cryopreservation today. A mild rejection reaction was found in heart valve replacement with cryopreserved homografts in ABO-compatible transplants, and moderate acute rejection in ABO-incompatible ones. In both forms of rejection, T cell activation was detectable for 4-10 days. There was no clinical relevance (Fischlein T et al., Immunologie reaction and viability of cryopreserved homografts. Ann. Thorac. Surg. 60: 122-6, 1995).

Apart from these disadvantages, hardly any knowledge of the distribution of antithrombogenic or prothrombogenic activities or active structures in the wall of hollow organs has been taken into account in the relevant literature. While vascular endothelium (as covering tissue of the inner or luminal side of all blood vessels and blood vessel valves) is characterized, for example, by a large number of antiaggregatory, anticoagulant and profibrinolytic activities (Z. Kardiol. 82: Suppl. 5, 13-21, 1993; FASEB J . 2: 116-123, 1988), are cellular components of the deeper vascular wall all characterized by the expression of tissue factor that initiates an immediate coagulation reaction in contact with plasma factors (Thrombosis Res. 81: 1-41, 1996; J. Clin. Invest. 100: 2276-2285, 1997; FASEB J. 8: 385- 390, 1994; Arterioscler. Thromb. Vase. Biol. 17: 1-9, 1997). Shielding the prothrombogenic wall activity of hollow organs is of great physiological importance not only for blood vessels and blood vessel valves, but also for all other cryopreserved and non-cryopreserved natural or artificial hollow organs or vessels.

For example, it is known that the thrombomodulin activity of the remaining donor endothelium is reduced after the cryopreservation. This leads to a restriction of the anticoagulatory function of the donor endothelium (Bilfϊnger TV et al., Cryopreserved veins in myocardial revascularization: possible mechanism for their increased failure. Ann. Thorac. Surg. 63: 1063-9, 1997). In order to avoid the disadvantages described above, it was proposed early on to use tissue engineering methods to develop new or modified organs with improved antithrombogenic properties (Weinberg CB, Bell E. A blood vessel model constructed from collagen and eultured vascular cells. Science. 1986 ; 231: 397-400.). It was possible to draw on the experience gained from settling a stable endothelial cell layer on the inside of plastic prostheses (Zilla P, Fasol R, Deutsch M, Fischlein T, Minar E, Hammerle A, Krupicka O, Kadletz M. Endothelial cell seeding of polytetrafluoroethylene vascular grafts in humans: a preliminary report. J Vase Surg. 1987; 6: 535-41.). Zilla et al. were able to prove convincingly that a stable endothelial cell layer does indeed improve the antithombogenic properties of such prostheses. Lining blood vessels to be implanted with a vascular endothelium is now a declared goal of biotechnology and "tissue engineering".

When trying to colonize natural hollow organs with cells, it turned out that the pre-coating of the organs affected with serum, which had been taken from the recipient of the organs modified in this way, is an ideal matrix for colonization of cells (Lamm P et al. New autologous coronary bypass graft: First clinical experience with an autologous endothelialized cryopreserved allograft. J Thorac Cardiovasc Surg 117: 1217-9, 1999). This pre-coating is distinguished from a pre-coating with fibronectin with and without a proteoglycan (eg heparin sulfate) (US Pat. No. 5,192,312; US Pat. No. 5,632,778; US Pat. No. 5,613,982, US Pat. No. 5,843,182, WO 95 / 24873, Zilla P. et al., Endothelial cell seeding of polytetrafluoroethylene grafts in humans. J. Vase. Surg. 6: 535-541, 1987) by shortening the coating process and dispensing with a number - clinically - common in other processes substances not previously approved (including fibronectin).

The object of the present invention is therefore to provide a new, generally applicable method for the preservation and storage of organs and tissues, but in particular of hollow organs. Another task was the provision of organs and tissues which have a higher mechanical stability and better suitability for further processing by the methods of "tissue engineering" compared to the organs and tissues produced using conventional methods. In particular, the immunological tolerance and antithrombogenicity of the processed organs and tissues is significantly improved by the washing-out process according to the invention of undesired cell degradation products and cell debris. Such organs and tissues show a significantly reduced thrombogenicity and immunogenicity in comparison to the original starting organs and tissues and also in comparison to organs and tissues that were not washed out according to the invention.

The solution to this problem results from the patent claims, the following description and the figures. The present invention provides a method for the devitalization and preservation of organs and / or tissues, in which the organs and tissues are removed sterile and stored in a liquid until the "devitalized steady state" is reached, selected from the group consisting of: sterile water, a crystalloid liquid, a colloidal liquid, a lipid-containing liquid and a combination of the liquids mentioned. Subsequently, cell debris, cellular degradation products and soluble substances are washed out under pressure (depending on the tissue or organ to be perfused) but preferably with physiological, ie the natural organ and tissue-specific perfusion pressures, with a liquid selected from the group consisting of the above-mentioned liquid ,

The washout is preferably pulsating, i.e. under variable (depending on the preservation time) pressure rise and pressure drop curves. Tissue- and organ-specific pressure waves are thus used. Suitable organ or tissue-specific pressure waves are determined by determining those pressure increases and pressure drop values (pressure waves) that are necessary to achieve devitalization in the respective organ or tissue. The pressure increase (the rate of pressure increase), the pressure level and the pressure drop are determined for the respective organ or tissue and are optimal if the washout leads to the washout of cell debris while at the same time maintaining the extracellular matrix. The preservation of the extracellular matrix and the successful washing out of cell debris (cell debris, remains) can be checked histologically. Conditions are chosen which in no way prevent the formation of the so-called "collagen cross linking". This can also be checked by histological examinations.

Furthermore, the present invention provides a method for producing matrices for the partial and new construction of organs and / or tissues. This method comprises the steps of the devitalization and preservation of organs and / or tissues according to the method described above and the cell repopulation of the organs and tissues, for example reendothelialization. additionally a cultivation device is provided for use in one of the methods according to the invention.

The method according to the invention is for the production of modified endogenous organs and tissues for immediate clinical use of these organs and tissues, e.g. suitable in the case of arteries and veins, the immediate implantation of which, after going through the manufacturing process, without additional treatment (e.g. cryopreservation) of the vessels. The organs and tissues produced according to the invention have significantly higher biomechanical stabilities than the same organs and tissues according to conventional storage and preservation methods (e.g. cryopreservation). In addition, the invention provides methods which are suitable for further treatment of the crgane and tissue according to the invention by the methods of "tissue engineering" in a clinically absolutely harmless manner. In particular, the present invention provides a method for lining hollow organs with a vascular endothelium, which are obtained with the inventive method for devitalization and preservation.

In addition, the method according to the invention makes it possible to use organic and / or artificial surfaces which have been precoated with components of the extracellular matrix (e.g. collagens, glycosaminoglycans etc.), such as e.g. pre-treat the inner surface of artificial hearts or PTFE and Dacron prostheses so that they have a significantly reduced thrombogenicity and immunogenicity compared to the surfaces that have not been pretreated.

Definitions The term "organs" means parts of the body made up of cells and tissues that form a unit with certain tissues.

The term "tissue" used here means individual types of cell assemblies that have common functions and that build up the body. "Organs according to the invention" are organs of the above definition that have undergone the manufacturing process according to the invention and can only perform their functions in whole or in part by additional further treatment in the form of cell repopulation of the organs, preferably by reendothelialization. The cell repopulation is preferably carried out by methods of "tissue engineering".

"Tissues according to the invention" are tissues of the above definition which have undergone the manufacturing process according to the invention and can be used clinically both with and without further treatment in the form of cell repopulation of the organs, preferably reendothelialization, in particular by the methods of "tissue engineering".

Hollow organs are also understood as tissues, as defined above. Hollow organs are, for example, blood vessels, blood vessel valves, lymphatic vessels, lymphatic valve valves, heart valves, ureters, vas deferens and bronchi.

“Organic or artificial surfaces according to the invention” are surfaces which have been precoated with extracellular matrix or matrix components and have been further treated with the method according to the invention.

The term "crystalloid liquid" means any form of buffered or unbuffered saline solutions. Preferred salt solutions in the context of the invention are phosphate-buffered saline solutions or clinically approved electrolyte solutions (Ringer's solution).

The term "colloidal liquid" used here means solutions containing protein and / or sugar. Preferred colloidal liquids are medium 199 and board-cutting cardioplegic solution. The term "lipid-containing liquid" means any form of fat-containing solutions.

The term "dark" used here means without the influence of a natural or artificial light source.

The term "under pressure" means perfusion of tissues and organs with fluids according to the invention under application of pressure, depending on the tissue or organ to be perfused, but preferably under application of organ and tissue-specific pressure values (ie the generally known and typical for the tissues and organs concerned Blood pressure values (= the physiological pressure)). In the case of hollow vessels according to the invention, a transmural pressure gradient across the wall of approximately 20-100 mmHg is preferred. In the case of complex organs, e.g. A pressure gradient is applied to the liver via the natural blood flow and the organ environment. "Sterile" means not exposed to germs.

The term "variable flow" means that various flow rates can be generated in the hollow organs via the bioreactors described in the patent. As a result, for example, in the early phase of coating hollow organs with endothelial cells according to the invention, the expression of adhesion factors from the endothelial cells can be increased by increasing the flow. This in turn facilitates the firm anchoring of the applied endothelial cells.

The term "lyophilization" describes a known method that is used to preserve unstable, aging biological substances. The substances to be dried are quickly and gently frozen in a cold mixture (eg carbonic acid snow in methyl alcohol) and then under high vacuum (upper limit of the vacuum: 0.05 - 0.1 Torr) The ice sublimes and the escaping water vapor is supported by the pump, supported by suitable hygroscopic means (eg freezer condenser) removed so quickly that the substance to be dried remains frozen as a result of the evaporative cooling.

The term “critical point drying” describes a known method for drying biological samples in which water is exchanged for an intermediate medium (for example ethanol) and this intermediate medium is then exchanged again for CO _2. This takes advantage of the effect that certain ones have Conditions (namely above the so-called critical point) can no longer be differentiated between liquid and gaseous phases and it is thus possible to avoid the direct phase transition liquid => gaseous.

The term "antibiotics" used here means fungal or bacterial metabolites and their modification products with inhibitory or killing action against viruses, bacteria and fungi. The preferred antibiotics according to the invention include gentamicin.

The term "devitalization" means killing all cells and reducing the corresponding organs and tissues to the level of the extracellular matrix of the connective tissue. This condition is also referred to below as "achieving devitalization". The achievement of devitalization can be checked histologically.

The term "devital steady state" used here means the state of the organs or tissues after devitalization. Devitalization is further characterized by the fact that major changes in the extracellular matrix, organs or tissues, such as intermolecular "crosslinking" of the collagens, have largely already taken place and these changes are irreversible. The term "tissue engineering" means techniques that make it possible to isolate, cultivate and multiply various, sometimes organ-specific cells (eg reendothelialization of hollow organs such as arteries or veins). Ultimately, these techniques create new organs and tissues.

The term "matrix" used here means the basic structure for the reconstruction or the change of organs and tissues by methods of "tissue engineering". Cells in tissue culture are propagated on these "matrices".

The term "repopularization" means vine colonization with organ- or tissue-specific cells, so-called "repopulation cells".

"Apoptosis" (Greek: the leaves fall in the wind) is a process that is also called programmed cell death and is used to devitalize tissues and organs. Apoptosis is the most common form of cell death in the body. Apoptosis plays a fundamental role in maintaining tissue and organ homeostasis. The death of individual cells is an essential prerequisite for the survival of the entire organism, because the formation, design and maintenance of tissues is not only controlled by cell multiplication and differentiation, but also requires an orderly removal of cells that have become redundant or damaged. Apoptosis is defined by a large number of morphological and biochemical changes. These changes include the shrinking of the cell and the condensation of chromatin, which attaches to the inside of the nuclear envelope and specifically into high molecular fragments of 50 and 300 kbp (kilobase pairs) and in many cases into smaller fragments of about 200 bp (base pairs) and one Multiple of them split. For the targeted breakdown of essential proteins, such as the cytoskeleton, specific protein-splitting enzymes such as proteases (caspases) are activated. The composition of the plasma membrane changes and the cell, in particular the nucleus, shrinks, while the cell organelles remain functional for a relatively long time. Finally, the cell disintegrates into membrane-bound constrictions ("apoptotic bodies") that are broken down by phagocytes or neighboring cells. It is important that the plasma membrane remains intact during apoptosis, so that no inflammatory reaction is triggered.

The term "synthetic material" as used herein means any organic and / or inorganic product suitable for such purposes. In particular, the synthetic material is said to increase the mechanical stability of the organs and tissues according to the invention.

The invention thus relates to a method for the devitalization and preservation of organs or tissues until devitalization is achieved, comprising the sterile removal and storage of the organ or tissue in a liquid, selected from the group consisting of: sterile water, crystalloid liquid, colloidal liquid, lipid-containing liquid or a combination of the liquids mentioned and the subsequent - preferably pulsating - washing out of cell debris, cellular degradation products and soluble substances under pressure, preferably under physiological pressure, with a liquid selected from the group consisting of: sterile water, crystalloid liquid, colloidal Liquid, lipid-containing liquid or a combination of the liquids mentioned, preferred crystalloid liquids being Bretschneider's cardioplegic solution or medium 199 (Seromed). Also preferred is a crystalloid liquid that contains an antibiotic additive. In a preferred embodiment, the organ is stored in the dark for at least 2 weeks. The method can also be carried out under the influence of light, but preferably under UV radiation. This leads to photo-oxidation of organs and tissues. However, better results are obtained when stored in the dark.

The removal of the organ or tissue from the dead (multi-organ donors) is particularly preferred. In a further, particularly preferred embodiment, multiple rinsing takes place in the same liquid in which the storage is also carried out before storage. Cell debris, cellular degradation products and soluble substances can be stored and washed out in the same liquid. It is necessary here that in the case of tissues a pressure gradient is generated across the tissue (for example in the case of hollow organs a transmural pressure gradient, ie pressure gradient across the wall of the hollow organ). In the case of organs, a pressure gradient is created between the natural, organ-specific blood and / or lymphatic vessels and the rest of the organ.

In a further embodiment, cell debris, cellular degradation products and soluble substances are washed out several times with organ- and tissue-specific pressure waves (depending on the organs and tissues to be treated). The organs and tissues are preferably stored and washed out in a sterile liquid. In a particularly preferred embodiment, the washing-out process for hollow organs takes place in the cultivation device according to the invention (Fig. 1).

In a further embodiment of the method according to the invention, the organs and tissues are stored for at least 6 weeks in order to enable a "devital steady state". Storage is particularly preferably carried out under sterile conditions. In the case of veins, the particularly preferred storage time according to the invention is 6 months, after which washing out with pulsating pressure is preferably carried out.

In a further embodiment, the organs and / or tissues are stored at a pH between 3 and 9, preferably between 6.9 and 7.8, particularly preferably between 7.0 and 7.5 and at a temperature of 0 to 55 ° C. preferably 0 to 37 ° C, but particularly preferably at 4 ° C. In a further embodiment, the organs and tissues are stored under reduced oxygen pressure, particularly preferably under anaerobic conditions.

In a further embodiment, the devitalization and / or storage according to the invention is carried out with gases which can be in the liquid form (such as liquid CO ₂ ) or in the gaseous form. The gas is preferably an inert gas.

The organs and / or tissues according to the invention can be dried after the devitalization and preservation according to the invention has been completed. This drying can be done by lyophilization or critical point drying after dewatering.

Organs and tissues that were produced with the method for devitalization and preservation according to the invention have a structurally modified basic structure (extracellular matrix) compared to the native, freshly removed organs (intermolecular "crosslinking" and side chain modifications, also as "collagen cross linking") designated). These organs and tissues ideally - even without a special pre-coating - offer the prerequisites for a partial or new build-up of the affected organs using methods of "tissue engineering". In addition, as in the case of the pretreated blood vessels, the organs and tissues can also be clinically implanted directly without any further measures.

The method according to the invention enables the devitalization and preservation of hollow organs, for example blood vessels, blood vessel valves, lymphatic vessels, lymphatic valve valves, heart valves, ureters, vas deferens, bronchi and organs such as urinary bladders, livers, kidneys and hearts. The method according to the invention achieves decisive advantages over previously conventional methods. The organs and tissues produced in this way have significantly higher biomechanical stabilities than the same organs and tissues according to conventional storage and preservation methods (eg cryopreservation). In blood vessels produced according to the invention, there is a significantly increased burst pressure value compared to the same hollow organs after cryopreservation (Fig. 5). It is also of exceptional importance that the organs and tissues pretreated in this way are not or only slightly antigenic or thrombogenic.

Preferred hollow organs according to the invention, if they are immediately re-implanted as arteries and veins, are completely deendothelialized and have a staining for keratan sulfate that goes beyond the cell boundaries, with the extracellular matrix of the affected hollow organs being otherwise largely retained. This applies in particular to the three-dimensional preservation of the conserved collagen structures. Due to the slow disintegration of the viable structures during the storage and conservation period, cells that normally survive cryopreservation also die (e.g. pericytes, "pericyte-like cells"). The antigens formed by these cells, which are still detectable after cryopreservation (in the case of pericytes, "pericyte like cells", for example, the tissue factor initiating coagulation ("tissue factor")) can no longer be detected after pretreatment with the new method.

In a further aspect of the invention, the devitalization of organs and tissues is increased or accelerated in the method according to the invention, for example with the aid of low-molecular substances which directly or indirectly induce apoptosis. In a preferred embodiment, a chemotherapeutic agent (eg methotrexate) is used to induce apoptosis and thus to intensify and accelerate devitalization. The intensification of the devitalization, for example by means of low molecular weight substances, can take place during the storage of the organ or tissue according to the invention or in a preceding step. Substances in the sense of the invention mean any substance that Apoptosis is induced directly or the interaction of signaling molecules that are involved in the induction of apoptosis is weakened or strengthened. In particular, the chemotherapeutic agents mentioned above may be mentioned here.

The invention further relates to organs and tissues, in particular hollow organs, which are produced by the method according to the invention. The hollow organs, and in particular the vessels, which are devitalized and preserved with the method according to the invention, offer ideal conditions for their use as organ matrices (so-called scaffolds) in "tissue engineering" and, in the case of blood vessels, have autologous patients on the inner surface Endothelial cells are lined, better long-term openness rates than uncoated hollow organs or vessels. The organs and tissues for producing the matrices according to the invention for the partial and new construction of organs and / or tissues are available ubiquitously. The matrices according to the invention can be produced without any effort, they are - compared to artificial surfaces - significantly less thrombogenic and far less susceptible to infections. The production can also be carried out by trained, not specially trained personnel of the highest quality. In the case of blood vessels, the blood vessels according to the invention produced in this way have the same surgical properties (such as, for example, sewability and stingability) as untreated endogenous blood vessels. For the first time, xenogenic blood vessels can also be implanted in humans without any further treatment after the preparation according to the invention. There is the possibility of industrial implementation of the manufacture according to the invention in the largest style without any great effort. However, the production of a hydrated matrix is particularly preferred.

In the case of complex organs, such as the liver and kidney, but also in the case of hollow organs, the method according to the invention provides ideal matrices for the reconstruction or modification of these organs and tissues by means of "tissue engineering". In a further embodiment, the invention relates to new culture media which are characterized in that conventional culture media such as basal media or full media are supplemented with autologous (ie body / patient's own) growth factors and / or with autologous (ie body / patient's own adhesion molecules) MEM Eagle, DMEM, Medium 199, MCDB 131, Ham's Medium, Iscore, RPMI (available e.g. from Life Technology, Germany or Geromed, Germany).

The cultivation and multiplication of various, partially organ-specific cells is made possible by the method according to the invention and in particular by the culture media according to the invention. The organ-specific differentiation of the cells used is obtained or only produced by the culture media according to the invention. For example, when propagating liver cells (hepatocytes) using the culture media and methods according to the invention, the problems of multiplication and differentiation of these cells known from conventional techniques are meaningless. This means that the cells of the liver (hepatocytes) can be cultivated without any problems if culture media according to the invention supplemented with autologous growth factors are used. The same applies to the cells of the kidney. The cells obtained in this way are used to modify or rebuild organs and tissues, whereby the organs and tissues can also be pretreated (e.g. cryopreserved). In general, these organs and tissues serve as basic structures (scaffolds) for their modification by treatment with the above-mentioned cells obtained by the methods of "tissue engineering". In this way, three-dimensional constructs are ideally generated, which can take over the respective functions of the organ or tissue to be imitated in whole or in part.

Examples of hollow organs according to the invention, which immediately after the process for

Devitalization and preservation that can be reimplanted in humans without any further pretreatment are human blood vessels, i.e. arteries and veins.

Examples of organs according to the invention by the method of devitalization and preservation can be used as matrices for the reconstruction of organs include human blood vessels, livers, kidneys, ureters and bladder.

Particularly preferred vessels according to the invention are allo- or xenogenic vessels (arteries, veins, lymphatic vessels) with and without lining with autologous endothelial cells on the inner surface.

Furthermore, the invention relates to a method for producing matrices for the partial and new structure of organs, comprising the steps of devitalizing and preserving organs and / or tissues by the method according to the invention and, after reaching the "devital steady state" according to the invention, repopularizing these organs and / or tissue, for example by reendothelialization. The use of autologous cells, e.g. autologous endothelial cells for repopulation. The use of the new culture media containing autologous growth factors and / or adhesion molecules is particularly preferred.

In the case of hollow organs, which include allogeneic vessels, xenogeneic vessels and ureters count, the re-endothelialization takes place after the devitalization and preservation of these organs and tissues. For example, it is possible to use matrices of xenogenic origin for the construction of a new vessel and its endothelialization (e.g. bovine chest wall arteries which have been subjected to the method according to the invention).

The invention therefore also relates to those organs and tissues which have been produced by the method according to the invention, comprising devitalization and preservation and reendothelialization. The hollow organs according to the invention, which were reendothelialized before their implantation, preferably have a lining with autologous endothelial cells on the inner surface or the luminal surface. A particularly preferred embodiment of the present invention comprises vessels and their valves, which are lined with recipient autologous endothelial cells on the inner surface or the luminal surface.

Perfusion tests of endothelialized donor vessels according to the invention showed no differences in endothelial morphology and shear strength stability compared to completely intact, freshly obtained veins or arteries.

The endothelium of all blood vessels, vascular valves and cardiac cavities is not only characterized by the above-mentioned antithrombogenic features. In a healthy, intact state, it represents an immunologically important barrier against the mass of the defense cells in the blood (granulocytes, monocytes, lymphocytes), which slide by without direct contact with the endothelial layer.

The method according to the invention enables an absolutely confluent endothelial layer permanently anchored against the shear forces of the blood flowing past to be established on the luminal surface of a blood vessel or its flaps. This acts as a complex anti-thrombogenic and anti-inflammatory catalyst to prevent thromboembolization of the hollow organs. Organs that have been lined, modified or completely rebuilt with patient autologous cells not only do not trigger an immune response on their luminal surface, but experience has shown that they limit a possible immunological defense even in the area of deeper wall layers in such a way that there is no clinically relevant rejection the vessels come. In contrast to, for example, cryopreserved and autologous endothelialized blood vessels, for the first time the new method completely prevents the slight residual rejection reaction that is still present in these vessels and enables for the first time while simultaneously using the inventive method new culture media with autologous growth factors, clinically relevant re-colonization of complex organs such as the liver or kidney, which have so far failed due to the antigenicity of the basic substance of these complex organs.

Furthermore, the present invention relates to the clinical use of complex organs which have been produced in accordance with the method according to the invention.

In a particularly preferred embodiment, the cell repopulation, in particular reendothelialization, is carried out without any pre-coating of the donor vessels with adhesion factors or serum solely by directly sowing and settling the cells, which were produced using the new culture media with autologous growth factors, on the inner surface of the vessel. This is not possible with any coating process known to date.

The invention therefore also relates to methods for producing and using the new culture media. According to the invention, autologous growth factors and adhesion molecules are used for the first time as an additive to culture media and for the initial treatment of the organs and tissues to be repopularized. The use of culture media according to the invention enriched with autologous growth factors and / or adhesion molecules is particularly advantageous in order to achieve a pre-coating of the hollow organs with autologous adhesion molecules or with growth and adhesion factors before sowing the cells. These culture media according to the invention are preferably suitable for culturing cells from the vascular system of humans, in particular vascular endothelial cells. In biotechnology, especially for use in humans, the use of non-autologous or genetically engineered growth factors is often problematic clinically and forensically. This is impressively expressed by the fact that almost every culture medium currently on the market is not approved for use by humans, despite proven suitability for cell culture. This is caused by the media that are usually added to this media Components of human or animal origin, especially in the serum-free media. The resulting liability claims against the respective manufacturers prevent the clinical use of complex growth media in humans. The culture media according to the invention enable the cultivation of human cells to be problem-free.

Examples of basal media, i.e. basic chemically defined culture media for various cell types are: Minimal Essential Medium (MEM) for the cultivation of adherent mammalian cells (Dulbecco R, GF Plaque production by the Polyoma virus. Virology. 1959; 8: 396-397), Medium 199 for the cultivation of mice -Fibroblasts or RPMI medium for the cultivation of tumor cells. These media differ in their composition, among other things. of amino acids, vitamins, trace elements, organic salts and other organic substances that enable the growth of the cultivated cells.

The term basal media is used here synonymously with "basally chemically defined media". The term "basic chemically defined media" is used in tissue culture for culture media of known qualitative and quantitative chemical composition. In contrast, other media, here called full media, contain natural products such as Animal serum.

For the optimal cultivation of mammalian cells, basic chemically defined media are supplemented with various serums. Preferably with fetal calf serum (FCS) or newborn calf serum (NCS) and / or with other not precisely defined growth factors (e.g. endothelial cell growth supplement: ECGS).

A further partial aspect of the invention therefore relates to methods in which autologous growth factors obtained in various ways either alone or in combination with autologous serum or in combination with other non-autologous ones Growth factors are added to the corresponding culture media. It is irrelevant whether preferred chemically defined media (e.g. MCDB 131) or so-called full media (e.g. Gibco HE-SFM) are used. In any case, 1. the growth of the cells, especially the endothelial cells, is significantly accelerated (growth curves, see Fig. 2), 2. the differentiation of the cells is significantly increased, and 3. the lifespan of the cells is multiplied. Another, particularly noteworthy advantage is the absolute clinical harmlessness of the new culture media thus created from chemically defined media, which for the first time creates the prerequisite for the economic exploitation of almost all products previously produced using the methods of "tissue engineering". This should be a new milestone for the implementation of tissue and organs manufactured in vitro for safe use in humans.

Detailed description of the culture media according to the invention The new culture media serve to increase growth, remodeling processes and reducing dedifferentiation processes of vascular cells in cell culture and are characterized in that a basal chemically defined medium or a full medium autologous (i.e. body / patient's own) growth factors and / or autologous adhesion molecules can be added. In addition to the basal medium (= basal chemically defined medium) or complete medium, the culture medium according to the invention comprises 5-30%, preferably 5-20%, particularly preferably 10-15% autologous serum. Autologous serum means the patient's own serum (obtained from the patient) which contains the autologous growth factors and / or the autologous adhesion molecules and which is preferably not heat-inactivated.

In addition, recombinant growth factors can be added to the culture medium according to the invention. Examples of suitable recombinant growth factors are bFGF, VEGF, EGF, TGF, "scatter factor", PDGF or a combination of these growth factors. The autologous growth factors and adhesion molecules can be made from platelets and / or white blood cells. In a preferred embodiment, the autologous growth factors and adhesion molecules are obtained from enriched platelets. Furthermore, the autologous growth factors and adhesion molecules can be produced from coagulated autologous whole blood by centrifugation. The autologous whole blood obtained is preferably stored for at least 1 hour at 37 ° C or for 6 hours at 4 ° C (see Figure 6).

In a further embodiment, glycosaminoglycan can additionally be added to the culture medium according to the invention. Particularly preferred glycosaminoglycans are heparin, heparin sulfate, chondroitin, chondroitin sulfate, dermatin or dermatine sulfate.

In a particularly preferred embodiment, the culture medium according to the invention can additionally be supplemented with transferrin, hydrocortisone, insulin, selenium or albumin.

The culture medium according to the invention is suitable for the cultivation of vascular cells, in particular endothelial cells, pericytes, "pericyte-like cells" and smooth muscle cells. The culture medium is also suitable for growing non-vascular cells, in particular hepatocytes.

In addition, the culture medium can be used as a culture medium in the context of "tissue engineering". It is particularly suitable as a medium for "precoating" vascular prostheses, heart valves and bypasses in "tissue engineering". In a preferred embodiment, the culture medium according to the invention can be used as a preservation solution for tissue storage ("tissue banking"). In a further embodiment, the autologous growth factors can be obtained by mechanically destroying the body's own tissues. The autologous growth factors can preferably be obtained by chemical and / or biochemical destruction of the body's own tissues. The autologous growth factors can particularly preferably be obtained by apoptosis of the body's own tissue. Tissue destruction can also be carried out by ultrasound.

Preferred methods for obtaining autologous serum are described below:

Obtaining autologous serum:

Withdrawal of whole blood without anticoagulant substances (such as citrate) from the recipient of the tissue to be produced. Initiation of physiological coagulation through artificial surfaces (e.g. serum tubes) or coagulation activating substances (e.g. thrombin). Collection of the serum by centrifugation of the resulting blood cake at 400 g for 10 minutes. Then pipette off the supernatant (= serum).

Obtaining autologous serum enriched with autologous growth factors released from blood cells: Obtaining whole blood without anticoagulant substances by methods known to those skilled in the art. The initiation of coagulation (see above) also activates blood cells, in particular platelets and white blood cells. An initiation of coagulation and simultaneous activation of the blood cells can also be carried out by adding "activators" (eg HE / ml thrombin). This leads to the progressive release of growth factors (eg VEGF, PDGF, FGF). It is known, for example, that the Release of VEGF from the activated platelets reaches a maximum after 1 h at 37 ° C. In order to obtain as many of the growth factors as possible, the clotted blood is therefore kept for at least stored for 1 h at 37 ° C or at least 6 hours at 4 ° C. Subsequently, the above-mentioned centrifugation and pipetting off of the supernatant, which contains a large part of the growth factors, takes place in order to obtain the serum with the growth factors contained therein.

In a further process variant for the production of autologous serum with a higher proportion of autologous growth factors from blood cells, citrate blood is obtained and the solid components (blood cells) are centrifuged off. A portion of the supernatant (plasma) is pipetted off in accordance with the desired enrichment factor. After resuspension of the blood cells and recalcification, coagulation is initiated by artificial surfaces or by physiological activators (e.g. whole blood or thrombin). The growth factor-rich serum is obtained as described above.

The extraction of autologous growth factors from blood cells is described below:

Obtaining thrombocytic autologous growth factors:

Decrease in citrate blood. Platelet-rich plasma is obtained by careful centrifugation (315 g for 10 minutes) and pipetting off the supernatant. Release of the autologous growth factors by degranulation of the platelets after recalcification and activation with preferably whole blood (1 ml to 10 ml platelet-rich plasma). Enriched growth factors can be concentrated by prior concentration of the platelets e.g. to 2 million / ml and subsequent activation as described above.

White blood cells are obtained, for example, by centrifuging citrated blood, pipetting off the "buffy coat" and subsequent activation (for example with FMLP). In a further method form, concentrated white blood cells and platelets can be activated together. Serum rich in growth factors is obtained as above by centrifugation.

Obtaining autologous growth factors from blood cells and other tissues by mechanical disruption of the cells:

In a preferred embodiment, the cells are lysed by complement activation or apoptosis.

Obtaining concentrated autologous serum or concentrated growth factor-rich serum:

Withdrawal of water from the sera using a concentrator (e.g. dextranomer, polyacrylamide). Dextranomer and polyacrylamide concentrators are commercially available (Sephadex from Pharmacia, Bio-Gel P from Bio-Rad Laboratories). Alternatively, other concentrators such as silica gel, zeolites, dextramines, alginate gel, crosslinked agarose can be used.

If necessary, the mixture obtained can be dialyzed against physiological solutions (Hanks salts, Earle's salts, basal media).

In addition to the addition of autologous serum and autologous growth factors in basically chemically defined media, the additional addition of heparin, insulin, hydrocortisone, possibly transferrin and selenium has been shown to increase growth. Heparin in particular proved to be an important co-factor. Possible uses of the cultivation device according to the invention are described below:

The coating of hollow organs according to the invention can be carried out with all the equipment customary for such measures, but the cultivation device (bioreactor) according to the invention is particularly suitable (FIG. 1). The following advantages resulted from the use of this cultivation device:

A constant, arbitrary pressure gradient is generated across the vein wall. In addition, medium is transported over the vein wall, which is used to nourish the endothelial cells and, if necessary, other cells introduced into the vein wall. In addition, any antigens that are still present in the vessel wall are washed out into the external medium. In addition, the bioreactor according to the invention can be used for permanent perfusion, particularly of endothelialized hollow organs, if it appears necessary to particularly support certain differentiation states of cells. This leads to a significantly increased synthesis of the extracellular matrix, which particularly promotes the shear force stability of the applied endothelial layer of the affected hollow organs. This device is a simple, easy-to-use, inexpensive and safe aid for the endothelialization of hollow organs of all kinds.

Furthermore, the cultivation device according to the invention is also suitable for the following processes: For the re-cellularization of prosthetic and organic material, in particular for re-endothelialization with and without perfusion of the hollow organ.

• In modification (Fig. 3), for flushing out unwanted soluble and insoluble substances from prosthetic and organic material, especially from

Hollow organs. The flushing takes place by applying a transmural pressure gradient (pressure gradient over the wall of the hollow organ), which the Maintaining an osmotic and oncotic gradient between the tissue to be treated and the external medium, which is additionally desired for flushing out these substances, and by means of an additional exchange of the external medium.

The method according to the invention for the production of matrices for the partial and new construction of organs and tissues can be applied to all natural and artificial hollow organs and their components, for example natural blood vessels, blood vessel valves, lymphatic vessels, lymphatic valve valves, ureters and bladder, vas deferens, bronchi , with the heart and especially with heart valves.

In the production of so-called biological heart valves from xenogenic materials (for example from bovine pericardium), the affected starting materials are often fixed with so-called cross-linking agents (e.g. glutaraldehyde). This extends the possible duration of storage of the raw materials concerned. The starting materials are then mounted on so-called "stents" to achieve a biological form and biomechanical stability. These "stents" also serve as abutments for anchoring the surgical sutures during implantation. It is known that after implantation of such heart valves, there are chronic immunological processes which ultimately lead to degeneration of the affected heart valve. As a rule, the life of such heart valves after their implantation in humans is a maximum of 15 years, after which the heart valve must be replaced in a second operation with a significantly increased risk of surgery for the affected patient. The cause of these immunological processes are antigenic structures of the starting tissue used for the production of heart valves. When using the starting substances according to the invention, such antigenic structures are completely eliminated and the service life of biological heart valves is improved. Such heart valves according to the invention can now be implanted without any further treatment. However, they can also be further treated with any preservative substance or technique previously used in the manufacture of biological heart valves. It was found that, in the case of hollow organs, in particular veins, the mechanical stability of the coated and uncoated hollow organs according to the invention after 12 months of storage did not differ from that of freshly removed donor veins (burst pressure test and histological examinations of the extracellular matrix). However, the vessels of the invention showed a significantly increased mechanical stability compared to cryopreserved vessels (Fig. 5).

The method according to the invention can preferably be used for the production of matrices for the partial and new construction of organs and tissues in donor vessels (veins or arteries) and in xenografts. A particular advantage here is the possibility of treating these vessels with antiviral treatment before they are coated. This is possible because the vessel wall of the vessels manufactured according to the invention has a significantly higher mechanical stability than, for example, the wall of a cryopreserved vessel.

In a preferred embodiment, after washing out complex organs according to the invention, these organs are repopularized with cells in organ-specific three-dimensional rotary apparatus. Rotary apparatuses of this type are commercially available (Rotary Cell Culture System ™ from Synthecon, Ine, USA).

It is also possible to have vessels that are lined with autologous endothelial cells on the inner surface, the endothelial cells being obtained from other sources (e.g. peripheral blood, bone marrow, adipose tissue, genetically modified or produced endothelium, xenogeneic and, if appropriate, genetically modified xenogeneic endothelium) to use according to the invention. In addition, patient-autologous epithelium can be produced by genetic engineering, so that epithelium is obtained which imitates the patient-autologous epithelium in its surface properties or immunological properties.

In a further embodiment (described in detail in Examples 15-17), a precoating of artificial surfaces with cell populations is carried out, which are capable of forming extracellular matrix and then the surfaces pretreated in this way are transferred into the process according to the invention.

The further treatment of the surfaces produced in this way can be carried out using methods of "tissue engineering" or inorganic or organic chemistry (e.g. chemical coupling of antithrobogenic compounds). Further treatment with supporting substances such as e.g. Adhesion molecules take place.

In a further embodiment, the hollow organ according to the invention is additionally enclosed on the outer surface by a jacket made of a synthetic material. This synthetic jacket can consist of a resorbable material, for example synthetic polyglyconic acid. Hollow organs that are surrounded by a jacket made of synthetic material have the advantage that they are stabilized for several months.

The organs and tissues according to the invention show a significantly lower thrombogenicity in comparison to the results published to date on the antithrombogenicity of connective tissue. Fig. 4a and b show a vein according to the invention, which was removed 16 hours after its implantation in a patient. It shows a completely smooth surface without any attachment of fibrin, thrombo- and leukocytes. The body's own arteries and veins, which were also used in this operation, were all covered with platelet and leukocyte deposits (FIG. 4c) and completely thrombosed.

The organs produced according to the invention can be used in the entire field of medicine and veterinary medicine, surgery, in particular in cardiac and vascular surgery be used. Uncoated or coated vessels according to the invention are particularly used as aortocoronary bypasses for coronary artery disease and as vascular grafts for vascular reconstructions of any kind. This relates, for example, to peripheral arterial occlusive disease, aneurysmal changes to vessels that require replacement of these vessels, and all repetitive operations on the heart and on the vessels. In particular, these vessels are the ideal conduit for use in infected areas of the body. Further indications for the use of such vessels are a large number of congenital malformations (all types of shunt operations are mentioned as examples). In addition, such vessels are suitable for basic scientific studies such as arteriosclerosis research or permeation testing of pharmaceuticals. Of particular advantage is the possibility of being able to implant uncoated vessels at any time without any pretreatment. This also enables clinical storage of such vessels, as is known, for example, in the case of artificial prostheses. The manufacturing process according to the invention thus provides for the first time in the history of medicine an organic alternative bypass that is available at all times for use on the heart.

The figures serve to explain the invention.

Figure 1 shows a cultivation device (bioreactor) for use in the method according to the invention. It consists entirely of biologically inert, autoclavable parts and this device can be used to build up any pressure gradient across the vein wall. Furthermore, the vein can be perfused with any pressure and flow using a pump.

The cultivation device comprises a culture vessel (1) filled with medium in which the hollow organ is located (for example a vein (2)). The lumen of the two vein ends is connected to the two outlets of the culture vessel by means of two adapters (3). An adapter is connected to a computer controlled peristaltic pump (7). The other adapter ends at a storage or discharge container (5) with a riser pipe (4). If the vessel (5) represents a discharge vessel, the line (6) must be connected to a storage vessel. The pressure gradient Δp (depending on the riser pipe (4) and pressure transmitter (8)) is set according to the desired pressure gradient across the vessel wall. If the pressure generated by the riser pipe (4) is sufficient, the apparatus can also be used without a pressure sensor (8). The peristaltic pump (7) can be used to carry out a computer-assisted change of medium in the inner lumen of the vein or a continuous / discontinuous perfusion of the vessel (2). Pressure line (9), access ports (10).

Figure 2 shows the growth behavior of cultivated human macrovascular endothelial cells from the vena Saphena magna under different cultivation conditions and daily 50% medium change. a) Cultivation of the cells in medium MCDB 131 with 20% pool serum. b) Cultivation of the cells in medium MCDB 131 with 20% autologous serum. c) Cultivation of the cells in medium MCDB131 with 20% pool serum + 10 ng / ml rbFGF. d) Culturing the cells in medium MCDB 131 with 20% according to the invention

Autologous serum rich in growth factors (from platelet-rich plasma: 2 million platelets / ml).

It can be seen that the medium (d) according to the invention offers by far the best culture conditions for human endothelial cells.

Figure 3 shows a modification of the cultivation device in Figure 1 for the pressure-dependent flushing of a hollow organ according to the invention. There is no medium recirculation (see Figure 1). The storage vessel I (11) contains the Liquid that is applied into the inner lumen of the hollow organ (2) under pressure (depending on the riser pipe (4) and pressure transmitter (9)) via the pump (7) and is collected in the discharge vessel I (5). The storage vessel II (12) contains the liquid which is used to wash around the hollow organ (2) with the aid of the pump (8) and is collected in the discharge vessel II (6) with the liquid filtered over the wall of the hollow organ. Pressure line (10), access ports (13), sterile filter (14).

Figure 4 shows a vein according to the invention after implantation in comparison to a body's own vein. Figures 4a and 4b show a vein according to the invention which was removed 16 hours after its implantation in a patient. When histologically evaluated, it shows a completely smooth surface without any attachment of fibrin, thrombo- and leukocytes. In comparison, Figure 4c shows the inner surface of a body's own vein, which was also used in this operation. It was completely thrombosed and showed histological evaluation of fibrin, thrombo- and leukocyte deposits.

Figure 5 shows the burst pressure values of a) freshly removed veins, b) cryopreserved veins immediately after thawing and c) veins according to the invention after 12 months of storage according to the invention. Figure 6 shows a flow chart for obtaining autologous growth factors and adhesion molecules.

The following examples illustrate the invention and are not to be interpreted as restrictive.

Example 1: Initiation of devitalization and preservation in blood vessels

Donor veins are removed sterile from the organ donor using conventional techniques. Due to their integrity, these vessels are still in the operating room examined. Any branches or side branches of the veins are ligated with surgical suture material (eg Ethibond 4/0) in the usual way. The tubes are rinsed several times with crystalloid solution (e.g. Bretschneider's cardioplegic solution or Medium 199 (Seromed)) and then placed in a tube of approx. 1 cm caliber (either a sterile plastic tube or a specially made glass tube can be used for this). The vessel is filled with medium 199 until it overflows and then stored at 4 ° C. in the dark. Optionally, the veins can also be filled with medium 199, closed at both ends with vessel clips, for example, and then stored in medium 199. This has the advantage that the vessel no longer collapses when it is removed. The storage should be at least 2 weeks, preferably 6 weeks, particularly preferably 6 months. Even after a storage time of more than 24 months, the vessel is still fully suitable for its use according to the invention. After a microbiological test with proven sterility and irrigation according to the invention, the vessel can be implanted immediately after removal from the storage vessel. If desired, the inner surface of the hollow organ can be smoothed mechanically before implantation. For this purpose, a commercially available balloon catheter (Fogarty catheter) can be pulled through the hollow organ. This procedure is recommended because unevenness due to storage cannot be excluded.

Example 2:

Preparation of donor veins according to the invention according to Example 1 with a storage time of 6 months. In this way, a devital "steady state" is achieved.

Example 3:

Process according to the invention according to Example 2 at a pH of 7.0 and a temperature of 18-22 ° C. Example 4: Patient-autologous endothelialization of veins modified according to the invention

Approximately 500 ml of whole blood without anticoagulant substances are withdrawn from the patients to be operated on, stored at 4 ° C for 24 hours and then the solid components are centrifuged off. The serum is frozen until further use.

In a preliminary operation, an approximately 5 cm long piece of vein is removed from the recipient of the vein to be coated under local anesthesia. The isolation and proliferation of isolated endothelial cells is carried out according to common cell culture techniques (Jaffe EA, Nachman RL, Becker CG, et al. Culture of human endothelial cells derived from umbilical veins. Identification by morphologic and immunologic criteria. J Clin Invest 52: 2745-56, 1973 ). For example, medium 199 (Seromed) supplemented with 20% autologous serum and 2 ng / ml recombinant bFGF (basic fibroblast growth factor) can be used as culture medium. After reaching a sufficient number of cells for the coating, the stored donor vein according to the invention to be used is removed from its storage container. The vein is either filled directly with the patient's autologous endothelial cell suspension without any further pretreatment (see below) or filled with patient's autologous serum and then incubated in the filled incubator at 37 ° C for 12 to 24 hours. For this purpose, the two ends of the vein are provided with a continuous adapter plug (which is integrated), which in turn is closed by a removable plug. Subsequently, the serum is drained by removing the stopper, the pre-coated vein is filled with a defined cell number (80,000-120,000 cells / cm ² graft surface) of patient-autologous endothelium and closed by reinserting the stopper. Now the vein is rotated for several hours in a rotary device [Kadletz M, Moser R, Preiss P, et al. In vitro lining of fibronectin coated PTFE grafts with cryopreserved saphenous vein endothelial cells. Thorac Cardiovasc Surg, 35 Spec No 2: 143-147, 11/1987]) rotated in the incubator at 37 ° C. This leads to uniform adhesion of the cells on the Graft inner surface. The coated vein is then removed from the rotary device and placed in the special cultivation device (see FIGS. 1 and 3).

Example 5: Patient-autologous endothelialization of veins modified according to the invention using patient-autologous growth factors and adhesion molecules:

The endothelialization of allografts pretreated according to the invention was carried out according to the method of Example 2. In contrast to example 2, however, autologous growth and adhesion factor-rich serum according to the invention was used for the precoating and culture medium substituted with serum according to the invention with autologous growth factor rich serum (MCDB 131 + 20% serum according to the invention).

Production of the autologous growth and adhesion factor-rich serum:

Approximately 500 ml of whole blood with anticoagulant substances (preferably citrate) are withdrawn from the patients to be operated on in the preoperative course. Obtaining platelet-rich plasma (plasma with enriched platelets (platelets)) by careful centrifugation (315 g for 10 minutes) and pipetting off the supernatant (platelet-rich plasma). Release of the autologous growth factors by degranulation of the platelets after recalcification and activation with whole blood (1 ml whole blood on 10 ml platelet-rich plasma). Preferred enriched growth factors can be obtained by previously concentrating the platelets to, for example, 2 million / ml and then activating them. This serum according to the invention can optionally be concentrated using commercially available concentrators by removing water (for example dextranomer, polyacrylamide). Dextranomer and polyacrylamide concentrators are commercially available (Sephadex from Pharmacia, Bio-Gel P from Bio-Rad Laboratories). alternative other concentrators such as silica gel, zeolites, dextramines, alginate gel, "crosslinked" agarose can be used.

If necessary, the mixture obtained can also be dialyzed against physiological solutions (Hanks salts, Earle's salts, basal media).

Example 6: Patient-autologous endothelialization of xenografts pretreated according to the invention:

The endothelialization of xenografts pretreated according to the invention is carried out in accordance with the method of Example 4 or 5.

Example 7: Patient autologous endothelialization from another vessel, e.g. an artery

The endothelialization of an artery is carried out in exactly the same way as the endothelialization of a vein described in Example 4 or 5.

Example 8: Epithelialization from another hollow organ, namely from a ureter.

The epithelialization of a ureter is carried out in accordance with the endothelialization described in Example 4, with the difference that urothelium is used instead of endothelium.

Example 9: Coating process, the endothelial cells being obtained from other sources (see above) Coating method as in example 4. The corresponding endothelial cells are isolated from peripheral blood, bone marrow and abdominal fat by known methods. This isolation of endothelial cells has a clear benefit for patients, since these methods are also available for those patients who do not have a sufficient vascular substrate for the production of autologous endothelium. In addition, these procedures are less invasive to the patient.

The following examples relate to the use of the media according to the invention (media supplemented with autologous growth factors and adhesion molecules) in cell culture for tissue engineering.

Example 10: Isolation and Cultivation of Human Macrovascular Venous and Arterial Endothelial Cells:

The isolation is carried out as described above according to the Jaffe et al. The cells are preferably cultivated in medium MCDB 131 with 20% autologous thrombocytic growth factor-rich serum (from 2 × 10 ⁶ platelets / ml) additionally substituted with heparin (50 μg / ml). Example 11: Isolation and cultivation of human smooth muscle cells from the media of the aorta

There are two methods for isolating smooth muscle cells from the media of the aorta: 1. Outgrowth of the smooth muscle cells from explant pieces of the media or

2. Enzymatic disintegration of media. Option 2 is preferred due to higher cell yields. Pieces of human aorta are surgically freed from the intima and adventitia. The media obtained must be free of remnants of the intima and adventitia. The media is mechanically comminuted into 5 mm pieces and then incubated with a protease mixture (0.05% elastase type HI, 0.225% collagenase, 1% human albumin in PBS). At least 10 ml of protease solution is used for one gram of tissue. It is incubated at 37 ° C until the tissue is completely digested (generally 3-5 h). After complete digestion, the cell suspension is filtered through a nylon mesh (50 μm), centrifuged (190 g, 10 min) and resuspended in autologous culture medium (MI 99 + 20% autologous growth factor-rich serum). The cells are sown at a density of 10 ⁴ cells / cm ² and incubated at 5% CO ₂ , 37 ° C in the incubator.

Example 12: Isolation and Cultivation of Human Keratinocytes

For the isolation of human keratinocytes, the skin resectates left over during surgery can be used. For the transport of the skin piece, a basal medium (e.g. DMEM) is supplemented with an antibiotic additive (e.g. Gentamicin 50 ng / ml) to reduce the natural skin flora and prevent secondary infection.

Any hair and necrotic tissue that may be present are first removed from the skin, then the fatty tissue and vessels of the subcutis are carefully separated. For dissociation, the cleaned skin is placed in a trypsin / EDTA solution (0.25% / 0.2%) for 18 hours at 4 ° C. The 18-hour enzyme action on the skin is evident from its jelly-like nature. To free the skin of residual trypsin / EDTA solution, it is washed with PBS. The dissociated tissue is then removed from the skin with forceps and suspended in nutrient medium. This cell suspension is filtered through a sterile gauze compress (or a nylon mesh 50 μm mesh size) to remove necrotic tissue debris. The cell suspension is centrifuged (190 g, 10 min) and in autologous nutrient medium resuspended. The cells are then sown in cell culture dishes. At 5% CO ₂ fumigation and 37 ° C, the primary culture is kept in the incubator for 24 hours. After the 24 hours in which the cells can adhere to the bottom of the bottle, the medium is changed. Medium change takes place every 3 days.

The culture medium MCDB 153 (Boyce ST, Urn RG. Calcium-regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J Invest Dermatol. 1983; 81: 33s-40s) is substituted with insulin (5 mg / 1), hydrocortisone (1.4 μM, 0.5 mg / 1), ethanolamine (0.1 mM), phosphoethanolamine (0.1 mM) with 10% autologous serum and 10% autologous thrombocytic growth factors ( from 2xl0 ⁶ platelets / ml) used.

Example 13: Isolation and cultivation of human dermal fibroblasts

After the keratinocytes have been isolated, the remaining skin is placed in a culture bottle with autologous nutrient medium (MI 99 with 10% autologous growth factor-rich serum). After a few days of incubation in the incubator (5% CO ₂ , 37 ° C), fibroblasts grow out of the skin. After enough fibroblasts have grown out of the skin, the remaining skin is removed. Medium change takes place every 3 days.

Example 14: Cultivation of human hepatocytes

Human hepatocytes are isolated by the Berry et al. (Berry MN et al., High-yield preparation of isolated hepatocytes from rat liver. In Laboratory Techniques in Biochemistry and Molecular Biology, vo. 21 (ed. RH Burdon and PH van Knippenberg), pp. 15-58. Elsevier: Amsterdam , New York, Oxford, 1991).

The cells are sown at a density of 1.6 x 10 cells / cm in culture bottles. William's E Medium (Gibco, Grand Island, NY, USA) substituted with 15% autologous growth factor and adhesion molecule-rich (growth factors from 2 x 10 ⁶ / ml thrombocytes and 7 x 10 ⁵ / ml leukocytes) serum, 25 mM HEPES, 5 μg / ml insulin, 0.5 μg / ml Hydrocortisone, 5 μg / ml transferrin, 100 U / ml penicillin, 100 μg / ml streptomycin used. A 50% medium change takes place every 24 hours.

Example 15: Coating of an artificial surface (here PTFE prosthesis. Diameter 4 mm) with fibroblasts and subsequent further treatment according to the invention.

For the isolation and cultivation of fibroblasts, see Example 13. The sterile PTFE prosthesis to be coated is placed in autologous serum, taking care that the inner lumen of the prosthesis is completely wetted with the serum. The prosthesis is then stored at 37 ° C for approx. 12 hours. The prosthesis is then removed and filled with a fibroblast cell suspension (100,000 cells / cm ² inner prosthesis surface). The prosthesis is now rotated for 6-10 hours in a rotating apparatus (see also Example 4) in order to ensure uniform adhesion of the cells. The coated prosthesis is then transferred to a bioreactor and cultivated there for 4 weeks until a layer of extracellular matrix formed by the fibroblasts and firmly attached to the inner lumen has a layer thickness of at least 10 μm. The coated prosthesis is then transferred according to the invention into a devital steady state. After the devitalized steady state has been reached according to the invention, the prosthesis can either be implanted immediately or processed further as part of tissue engineering.

Example 16: Coating an artificial surface (here a polyurethane prosthesis, diameter 4 mm) with subintimal cells and subsequent further treatment according to the invention.

The subintimal cells are isolated from vessels which have been proteolytically isolated from the endothelial cells using the method described above (see example 10). A subsequent 15-minute proteo-lyric disintegration of the remaining cells (= subintimal cells) on the inner surface of the vessels with the same protease solution (collagenase) removes them from their cell structure and rinses the vascular segment. These cells are cultivated as in Example 13. The further coating is carried out analogously to Example 15.

Example 17: Endothelialization of a coated artificial surface pretreated according to the invention (here PTFE prosthesis, 4 mm diameter).

A PTFE prosthesis prepared according to Example 15 is endothelialized according to Example 4 after the "devital steady state" has been reached.

Claims

1. A method for the devitalization and preservation of organs and / or tissues, comprising the following steps: a) sterile removal and storage of the organ or the tissue until devitalization is achieved in a liquid selected from the group consisting of: sterile water, one crystalloid liquid, a colloidal liquid, a lipid-containing liquid or a combination of the liquids mentioned, b) washing out cell debris, cellular degradation products and soluble substances under pressure with a liquid, selected from the group consisting of: sterile water, a crystalloid liquid, a colloidal Liquid, a liquid containing lipids and a combination of the liquids mentioned.

2. The method according to claim 1, wherein the sterile removal of the organ and / or tissue from the dead (multi-organ donors).

3. The method according to claim 1, wherein the storage takes place for at least 2 weeks, preferably 6 weeks, particularly preferably 6 months in the dark.

4. The method of claim 1, wherein the storage is carried out under sterile conditions.

5. The method according to any one of claims 1 to 4, wherein the liquid in which the storage takes place is a crystalloid liquid.

6. The method of claim 5, wherein the crystalloid liquid is Medium 199 (Seromed).

7. The method of claim 5, wherein the crystalloid liquid is Bretschneider cardioplegic solution.

8. The method of claim 5, wherein the crystalloid liquid contains an antibiotic additive.

9. The method according to any one of claims 1 to 7, wherein the method prior to storage comprises multiple rinsing in the same liquid in which the storage takes place.

10. The method according to any one of claims 1 to 8, wherein the storage and washing out of the organs and / or tissue is carried out with the same liquid.

11. The method according to any one of claims 1 to 9, wherein the storage takes place at a pH of 3 to 9.

12. The method according to claim 10, wherein the storage takes place at a pH of 6.9 to 7.8.

13. The method according to claim 10, wherein the storage takes place at a pH of 7.0 to 7.5.

14. The method according to any one of claims 1 to 12, wherein the storage takes place at a temperature of 0 to 55 ° C.

15. The method according to claim 13, wherein the storage is carried out at a temperature of 0 to 37 ° C.

16. The method according to claim 13, wherein the storage takes place at a temperature of 4 ° C.

17. The method according to any one of claims 1 to 15, wherein the storage takes place under reduced oxygen pressure.

18. The method according to claim 16, wherein the storage takes place under anaerobic conditions.

19. The method according to any one of claims 1-15, wherein the storage takes place with gases (in the liquid or gaseous phase).

20. The method of claim 18, wherein the gas is a rare gas.

21. The method according to any one of claims 1 to 20, wherein the washing is done pulsating.

22. The method according to claim 21, wherein the washing out of cell debris, cellular degradation products and soluble substances is carried out several times.

23. The method according to claim 22, wherein at least two washes are carried out, preferably at intervals of 6 weeks.

24. The method according to claim 1, wherein the organs and tissues treated according to the invention are dried after devitalization and preservation.

25. The method according to claim 1, wherein the organs are hollow organs.

26. The method according to claim 1, wherein the washout takes place in a culture vessel, comprising a culture vessel (1) filled with a liquid, two adapters (3) which are connected to the two outlets of the culture vessel, a hose which is connected to a pump and another hose,. which ends at a storage or discharge vessel (5), the pressure gradient Δp being dependent on the riser pipe (4) and pressure transmitter (8).

27. The method according to claim 1, wherein the washing out takes place in a cultivation vessel according to FIG. 1.

28. The method according to claim 1, wherein the organs and tissues are selected from the group consisting of: blood vessels, blood vessel valves, lymphatic vessels, lymphatic valve valves, ureters, urinary bladders, vas deferens, bronchi, livers, kidneys, hearts and heart valves.

29. Organs or tissues obtainable by the method according to one of claims 1 to 27.

30. A method for producing matrices for the partial and new structure of organs or tissues comprising the following steps: a) devitalization and preservation of organs and / or tissues according to the method according to one of claims 1 to 27, b) cell repopulation of the organs and Tissue, preferably through

Re-endothelialization.

31. The method according to claim 29, wherein the organs are hollow organs.

32. The method according to claim 30, wherein the hollow organs are selected from the group consisting of: allogeneic vessels, xenogeneic vessels and ureters.

33. Organs obtainable by the method according to any one of claims 29 to 31.

34. The method of claim 29, wherein the repopulation cells are autologous cells, e.g. autologous endothelial cells.

35. The method according to claim 29, characterized in that the reendothelialization is carried out by sowing cells on the inner surface of the hollow organs.

36. The method according to claim 29, characterized in that prior to the sowing of the cells, the hollow organs are precoated with adhesion molecules.

37. The method according to claim 29, characterized in that before the cells are sown, the hollow organs are precoated with patient-autologous serum.

38. The method according to claim 30, characterized in that prior to sowing the cells, the hollow organs are precoated with serum rich in growth and adhesion factors.

39. The method according to claim 30, wherein the natural and artificial hollow organs are selected from the group consisting of: blood vessels, blood vessel valves, lymphatic vessels, lymphatic valve valves, ureters, urinary bladders, vas deferens, bronchi, hearts and heart valves.

40. Use of the tissues or organs, obtainable by the method according to one of claims 1 to 28 in medicine and veterinary medicine.

41. Use of the vessels, obtainable by the method according to one of claims 1 to 28 in cardiac and vascular surgery.

42. Use of the vessels, obtainable by the method according to one of claims 1 to 28 as an aortocoronal bypass or as a vascular graft for vascular reconstructions.

43. Use of the vessels, obtainable by the method according to any one of claims 1 to 28 for peripheral arterial occlusive disease, aneurysmatic changes in the vessels, repeat operations on the heart and on the vessels and in the event of congenital malformations of the vessels.

44. Tissue or organs obtainable by the method according to one of claims 1 to 28, characterized in that they are additionally enclosed by a jacket made of a synthetic material.

45. Tissue or organs, obtainable by the method according to one of claims 1 to 29, characterized in that they are additionally enclosed by a jacket made of a synthetic resorbable material.

46. Tissue or organs, obtainable by the method according to one of claims 1 to 28, characterized in that they are additionally enclosed by a jacket made of a synthetic resorbable material consisting of polyglyconic acid.

47. Cultivation device for use in a method according to claim 1, comprising a culture vessel (1) filled with a liquid, two adapters (3) connected to the two outlets of the culture vessel, a tube connected to a computer-controlled peristaltic pump is connected and a further hose which ends at a storage or discharge vessel (5), the pressure gradient Δp being dependent on the riser pipe (4) and pressure transmitter (8).

48. culture medium for increasing growth, remodeling processes and reducing dedifferentiation processes of vascular cells in cell culture, characterized in that a basal chemically defined medium or a complete medium autologous growth factors and / or

Adhesion molecules are added.

49. Culture medium according to claim 48, wherein the autologous growth factors and / or adhesion molecules in the form of autologous, not heat-inactivated serum is added.

50. Culture medium according to claim 48, wherein the culture medium comprises 5-30% autologous serum.

51. Culture medium according to claim 48, wherein the culture medium comprises 5-20% autologous serum.

52. Culture medium according to claim 48, wherein the culture medium comprises 10-15% autologous serum.

53. Culture medium according to claim 48 to 52, to which additional recombinant growth factors are added.

54. Culture medium according to claim 48, wherein the autologous growth factors and adhesion molecules are produced from platelets.

55. The culture medium according to claim 48, wherein the autologous growth factors and adhesion molecules are made from white blood cells.

56. Culture medium according to claim 48, wherein the autologous growth factors and adhesion molecules are produced from platelets and white blood cells.

57. Culture medium according to claim 48, characterized in that the autologous growth factors and adhesion molecules are made from coagulated autologous whole blood by centrifugation.

58. Culture medium according to claim 57, characterized in that the autologous whole blood obtained is stored for at least 1 hour at 37 ° C.

59. Culture medium according to claim 57, characterized in that the autologous whole blood obtained is stored at 4 ° C for at least 6 hours.

60. Culture medium according to claim 54 and 57, characterized in that the autologous growth factors and adhesion molecules from enriched

Platelets are obtained.

61. Culture medium according to claim 53, wherein the recombinant growth factor is bFGF, VEGF, EGF, TGF, scatter factor, PDGF or the combination of these growth factors.

62. Culture medium according to claim 48 to 61, wherein the medium additionally glycosaminoglycan is added.

63. Culture medium according to claim 62, in particular if the glycosaminoglycan is heparin, heparin sulfate, chondroitin, chondroitin sulfate, dermatin or dermatine sulfate.

64. Culture medium according to claim 48 to 63, wherein the medium additionally transferrin is added.

65. Culture medium according to claim 48 to 63, wherein the medium additionally hydrocortisone is added.

66. Culture medium according to claim 48 to 63, wherein the medium is additionally added insulin.

67. Culture medium according to claim 48 to 63, wherein the medium is additionally added albumin.

68. Culture medium according to claim 48 to 63, for the cultivation of vascular cells, in particular endothelial cells, pericytes, "pericyte-like cells" and smooth

Muscle cells.

69. Culture medium according to claim 47 to 63, for the cultivation of non-vascular cells, in particular of hepatocytes.

70. Culture medium according to claim 48 to 69, when used for "precoating" vascular prostheses, heart valves and bypasses in "tissue engineering".

71. Culture medium according to claim 48 to 70, when used as a culture medium in the context of "tissue engineering".

72. Culture medium according to claim 48 to 71, as a preservation solution in "tissue banking".

73. Culture medium according to claim 48 to 72, characterized in that the autologous growth factors are obtained by mechanical destruction of the body's own tissue.

74. Culture medium according to claim 48 to 73, characterized in that the autologous growth factors by chemical and / or biochemical

Destruction of the body's own tissues.

75. Claims 48 to 74, characterized in that the autologous growth factors are obtained by apoptosis of the body's own tissue.

76. Culture medium according to claim 74, characterized in that the tissue destruction is carried out by ultrasound.