WO2003029446A2

WO2003029446A2 - Methods for producing in-vitro hollow organs

Info

Publication number: WO2003029446A2
Application number: PCT/EP2002/010605
Authority: WO
Inventors: Birgit Schäfer; Stefan Carl; Christian Lorenz; Karl-Herbert SCHÄFER
Original assignee: Cytonet Gmgh & Co. Kg
Priority date: 2001-09-24
Filing date: 2002-09-20
Publication date: 2003-04-10
Also published as: WO2003029446A3; DE10146903C1

Abstract

The invention relates to methods for producing animal and human in-vitro tissue and/or animal and human in-vitro hollow organs, particularly in-vitro urethra, in-vitro ureter, in-vitro bladder, in-vitro spermatic duct, in-vitro uterine tube, in-vitro blood vessel, in-vitro small intestine, and in-vitro large intestine hollow organs. The invention also relates to the use of this in-vitro tissue and to in-vitro hollow organs for transplantation or for testing purposes, and to the production of a matrix, which is colonized by fibroblasts and provided for the surgical elimination and/or covering of fistulae.

Description

Process for the production of in vitro hollow organs

description

The present invention relates to methods for the production of tissues or hollow organs, in particular urethra, ureter, bladder, vas deferens, fallopian tubes, blood vessels, small intestine and large intestine, so-called connective tissue, which have an organ-typical tissue layer based on animal and human tissue samples and for transplantation in vivo or are suitable for in vitro test purposes.

In principle, three methods are available in surgery for restoring or improving the shapes or functions of human organs and / or tissues. The resection procedures, in which mostly diseased organ or tissue parts are surgically removed, are contrasted with implantation and transplantation procedures. In the case of an implantation, implants made of materials that are foreign to the body are introduced into the organism for a limited period of time or for life, to fulfill certain replacement functions, for example plastic implants for the replacement of tendons and vessels or metal or ceramic implants for the replacement of joints or bones.

In the case of a transplant, cells, tissues or organs are transferred to another individual or to another part of the body of the same individual. wear. An allogeneic (homologous) transplant is understood to mean a transplant in which the donor and recipient belong to the same species, but are immunologically different. A syngeneic transplant is understood to mean a transplant from an immunologically identical twin sibling. Autogenous (autologous) transplantation is the transfer of one's own cells or tissues to other parts of the body of the same individual.

The success of an allogeneic transplant depends largely on the type and extent of the immune response on the part of the recipient. The differences in the antigen pattern in the donor and recipient can elicit an immune response against the transplant in the recipient organism, which is induced by the genetically determined histoco-patibility antigens of the donor tissue. In this way, sensitized lymphocytes and / or antibodies can be formed which have a cytotoxic effect on the graft. Ultimately, the graft can be rejected. A rejection reaction can be peracute, resulting in an irreversible loss of the transplant that cannot be influenced by medication. A rejection reaction can also be chronic, with a functional loss of the graft that can hardly be influenced by medication for weeks or years.

A transplant can also lead to a graft versus host reaction (GVH), that is, a graft versus host reaction. After the transfer of, for example, non-autologous immune competent cells from bone marrow, lymph nodes or spleen mediate these cells in the recipient's organism cellular immune reactions. In the healthy recipient organism, these cells are normally broken down quickly. In recipients in whom the immune system is suppressed by radiation or immunosuppressive treatment, GVH can lead to graft versus host disease, an acute or chronic disease with enlarged liver and spleen, atrophy of the lymphoid organs, diarrhea and cachexia.

Another major problem in transplantation surgery is generally the lack of available donor tissues or organs. As a result, there are waiting lists for patients to be treated for some organ transplants. With heart transplants, for example, the waiting time is currently around 12 months. If a patient's condition deteriorates very quickly during the waiting period, the impaired organ function can often only be bridged mechanically by means of an implanted support system in order to eliminate the acute dangerous situation at least for a short time. The lack of suitable donor tissues or organs is particularly noticeable in accident surgery, where it is important to restore or replace the function of injured or damaged organs or tissues within a very short time.

Due to the lack of available donor tissue or organs, attempts have been made to target organs from primates (concordant species) or non-primates (disconcordant species). For example, appropriately pretreated pieces of skin or parts of blood vessels from pigs were successfully transferred to humans. On the other hand, whole organ transplants usually ended in catastrophic failures. The immunological causes for this cannot be explained easily. Probably preformed antibodies

(so-called natural antibodies) is the biggest problem with such a xenotransplantation. There are also other obstacles to xenotransplantation, for example the very high risk of pathogen transmission.

In some cases, attempts are also made to remedy organ defects or disorders by transplanting tissue from other organs of the same organism. For example, the surgical restoration of the so-called neurogenic bladder, which occurs in spina bifida patients or paraplegics, is performed by sewing intestinal segments into the bladder. However, it has been shown that the incorporation of intestinal mucosa in the bladder leads to increased infections, metabolic disorders such as vitamin deficiency, stone formation, increased mucus production and the development of malignancies.

As an alternative to previous transplants, in vitro organ or tissue systems have therefore been developed in recent years, which are produced using defined cells and / or defined tissues and under defined culture conditions (so-called tissue engineering). Generally leave such in vitro organ or tissue systems are not only used for implantation or transplantation purposes, but also as in vitro test systems, for example for their effects and / or chemical substances such as potential pharmaceuticals or agents such as light and heat or to examine side effects on tissues or cells. Such in vitro systems can also be used for a wide range of immunological, histological and molecular biological problems. The examinations or tests of substances or agents on such in vitro organ or tissue systems offer some advantages over experiments on animals or experiments with human subjects, in particular because the examinations are carried out more cheaply and more quickly and can deliver results that are in particular compared to animal experiments are better transferable to humans. Under certain circumstances, it is even possible to individually predict the side effects of the patient concerned. In addition, the testing of pharmaceuticals on animals is subject to strict guidelines, which result in a great deal of red tape. For example, animal testing permits must be obtained. Animal experiments are also increasingly rejected for ethical reasons. In vitro organ or tissue systems can therefore make a significant contribution to avoiding animal testing.

Various methods have been described in the prior art for the production of in vitro organs or in vitro tissues which are used for the tion or as a replacement for diseased or damaged native organs or tissues.

WO 99/00152 discloses a method for producing a bioartificial transplant, wherein all antigen-reactive cells are removed from an allogeneic or xenogeneic tissue by enzymatic or chemical treatment and the cell-free, undenatured material obtained in this way is populated with desired autologous cells is obtained, whereby an immediately ready-to-use graft, for example heart valves, cartilage, trachea, skin or cornea, is obtained.

U.S. Patent No. 4,963,489 describes a three-dimensional cell culture system that includes a backbone that is made of a biologically compatible non-living material, which may or may not be biodegradable. In this system, for example, fibroblasts, endothelial cells, bone marrow cells and other cells can be cultivated, and the growth of the cells can be improved by adding proteins such as collagen or glycoproteins. The three-dimensional tissue models produced with the help of this system are also to be used in transplantation.

From WO 95/33821 a process for the production of stroma tissue is known, in which stroma cells, such as chondrocytes, fibroblasts, umbilical cord cells and the like, within a three-dimensional network made of a biodegradable material, for example cotton, gelatin or collagen, or a non-biodegradable material, for example a polyester or a polycarbonate, wherein the interstitial areas are bridged by the stroma cells and the cultivation of the network is carried out in a culture medium.

US Pat. No. 5,755,814 describes a skin model system that can be used both as an in vitro test system and for therapeutic purposes. The system comprises a three-dimensional crosslinked matrix of insoluble collagen, the matrix being crosslinked either by thermal treatment with dehydration or also by chemical means, for example carbodiide. Fibroblasts are cultivated in the matrix and keratinocytes are cultivated on the matrix, a skin equivalent being obtained.

US Pat. No. 5,567,612 describes a matrix for implantation in humans containing cells of the urogenital tract, which comprises a biodegradable, biologically compatible polymer and parenchyma cells of the human urogenital tract. The matrix material can consist, for example, of polyglycolic acid, polylactic acid, polyanhydride, polyorthoester and combinations thereof.

US 5,944,754 describes a method for generating new tissue on the surface of a mammalian tissue or organ using a hydrogel composition containing living mammalian cells, a liquid cell hydrogel composition being applied as a thin layer directly to the surface to be treated. The river cell-hydrogel composition solidifies to form a matrix and the cells contained therein subsequently form a new tissue. Polysaccharides such as alginates, polyphosphazenes and polyacrylates can be used to form the hydrogel. A biologically compatible adhesive such as methacrylate or methyl-α-cyanoacrylate can be used to improve the adhesion of the cell hydrogel composition to the treated organ surface. The surface to be treated can be inside or outside the body.

US 5,762,966 discloses the use of a submucosal matrix derived from vertebrates as a graft for damaged urinary bladder areas. The use of intestinal tunica submucosa tissue from dogs as xenograft in pigs is described as an example.

No. 5,851,833 describes a method for isolating and cultivating mammalian urothelial cells, the cells being cultivated on a biologically compatible, biodegradable polymer matrix. The matrix containing the cultured cells can be used to restore or replace damaged urothelial tissue. The matrix itself consists of polymers such as polyglycolic acid or polylactic acid. If necessary, the attachment of the urothelial cells to the matrix can be improved by coating the polymer matrix with basal membrane compounds, agar, gelatin, collagen, fibronectin, etc. The cells used can proliferate on the matrix in vitro, and then together with the matrix into one Recipient organism are introduced. The cells can also be transferred to the receiver immediately after adhesion to the matrix.

WO 98/06445 describes a method for multiplying or restoring tissue using isolated bladder mucosa. One example shows that an allogeneic bladder mucosa, to which urothelial and muscle cells have been seeded / applied in vitro, can form new bladder tissue after histological incubation in vitro and transplantation in recipients does not distinguish functionally from native bladder tissue.

WO 98/10775 describes a tissue graft for promoting the restoration of damaged tissue structures of warm-blooded vertebrates associated with the nervous system, the graft comprising intestinal submucosal tissue of a warm-blooded vertebrate and / or a digest thereof and a growth factor. The growth factor is a vascular epithelium growth factor, nerve growth factor or fibroblast growth factor. The submucosa tissue treated with the growth factor can either be transplanted directly or injected in fluidized form after enzymatic digestion. The method is used, for example, to regenerate the spinal cord, neuroglia, dura mater, pia mater and arachnoid.

Gabella and Uvelius describe in Neurosciences, 83 (1998), 645-653, the transplantation of a large part of the ganglion pelvinum of adult rats in the bladder of the same animals. The general structure of all ganglion grafts remained essentially unchanged. Although the synapses degenerated at the time of the transplant, new synapses were formed later. Overall, it was shown that the pelvic nerve cells transplanted into the bladder tissue could survive and grow in the long term while maintaining their morphology.

The in vitro organs or in vitro tissues known in the prior art have numerous disadvantages. In many of these systems, for example, the fact that the matrix used for the production consists partly or completely of foreign materials is disadvantageous. When such materials are used, mechanical, structural, functional or compatibility problems often arise after the transplantation or implantation has taken place. For example, it has been shown that artificial bladder wall grafts, which were produced using permanent, that is to say biodegradable, synthetic materials, fail mechanically in the body after a certain time. In addition, such grafts often result in a very strong formation of urinary stones. Artificial bladder grafts made using biodegradable matrix materials, on the other hand, lead to the deposition of fibroblasts, scarring, contracture, i.e. permanent shortening, of the graft and a reduced reservoir volume over time. Problems have also been shown with matrices based on collagen. In some cases, bladder grafts containing collagen matrices have been able to replace the function of the native organ for a relatively long time, but in most cases they have been subjected to undefined shrinkage processes. At least with some collagen matrices, this seems to be related to their cancellous, cross-linked structure, which is atypical for living tissue.

Another disadvantage of many in vitro organs or in vitro tissues described in the prior art is that even after a prolonged residence time in the body they do not have or develop the tissue layer that corresponds to the morphological structure of the native tissue or organ. This means that these in vitro organ or tissue systems in the body are not a complete functional replacement for native organs or tissues.

In some of the known methods for producing in vitro organ or tissue systems, there is furthermore no guarantee that the cells used, provided that they are already largely differentiated cells and these cells are subjected to cultivation in vitro, after several cultivation passages also maintain their natural metabolism. This problem is particularly known in the cultivation of cartilage cells, which are often subject to dedifferentiation in the course of the cultivation. But it also affects other cell types. Many of the known three-dimensional structures for culturing cells can often not be specifically adapted to the particular organ or tissue defect. Another major disadvantage is that many of the artificial grafts are only suitable for restoring or replacing relatively small damaged or diseased areas of an organ, but not for replacing an entire organ. Furthermore, there is no guarantee that the artificial organ or tissue grafts will remain permanently and differentiated at the location of the defect and thus enable the regeneration of the corresponding organ or tissue or its function.

Last but not least, a considerable disadvantage of the in vitro organs or in vitro tissues known in the prior art is that they often take several weeks to produce. The relatively long production time is a major disadvantage, in particular, if a rapid restoration or a quick replacement of a damaged or diseased tissue or organ is required, for example after an accident or if the patient's condition deteriorates rapidly.

The technical problem on which the present invention is based is therefore to provide methods and means for producing human or animal in vitro tissues or in vitro organs which overcome the above-described disadvantages in the prior art and which are particularly suitable for this in vitro To produce organ or tissue systems in a very short time, the tissue layering of the tissues or organs produced in this way largely corresponds to the native human or animal tissues or organs, and the tissues or organs produced in this way are particularly suitable for use as a permanently functional graft or as a test system suitable.

The invention solves the problem on which it is based, in particular by providing a method for producing a single-layer or multilayer human or animal in vitro tissue or a complete or partial, human or animal in-vitro hollow organ, comprising, preferably in the stated Sequence:

a) treating a biodegradable, biodegradable or non-degradable plate-shaped matrix with a biologically compatible, biodegradable adhesive or conditioned medium,

b) the application of isolated tissue-type or organ-type or tissue-type or organ-non-typical mesenchymal and / or epithelial tissue pieces of a biopsy to the matrix for the growth of the primary cells, that is to say the establishment of an explant culture, and / or the sowing of at least one Type of an isolated differentiated or non-differentiated tissue- or organ-typical or tissue- or organ-untypical cell, previously in vitro was cultivated and propagated, in low cell density on the matrix,

c) the culturing of the explant cells and / or cells previously cultured in vitro and, if appropriate, the matrix containing neurogenic cells under suitable conditions, and

d) cutting the matrix into the desired shape and size and / or shaping the matrix into a hollow organ.

In a preferred embodiment of the invention, neurogenic cells which are isolated, tissue-typical or organ-typical or tissue-typical or tissue-untypical, are sown in low cell density on the matrix after step b) and before step c).

The method according to the invention is thus characterized in that first a biologically compatible, biodegradable, plate-shaped matrix is treated, in particular coated, sprayed or soaked, with a biologically compatible, biodegradable adhesive or conditioned medium. Subsequently, epithelial or mesenchymal pieces of tissue and / or cells which have previously been cultured and grown in vitro and which are typical of the tissue or organ to be produced are applied to the treated matrix. In a preferred embodiment, isolated neurogenic cells are then sown on the treated matrix. According to the invention, it is particularly provided in a special embodiment that neurogenic cells and muscle cells on the underside of the matrix cells and fibroblasts are applied while epithelial cells are applied to the top of the matrix. The cell-containing matrix is then briefly cultivated in vitro and brought into the desired shape after cultivation and immediately before use as a transplant. Either the cell-populated matrix plate is cut so that it conforms to the shape of a tissue defect to be treated, or it is shaped into a hollow organ, for example brought into a cylinder or bubble shape.

The method according to the invention for the production of in vitro tissues or in vitro organs, in particular in vitro hollow organs, has several decisive advantages over the methods described in the prior art, which result inter alia from the use according to the invention of an adhesive or conditioned medium. Surprisingly, it has been shown that treating the matrix according to the invention with an adhesive or a conditioned medium significantly shortens the manufacturing time for an in vitro tissue or in vitro organ compared to the methods described in the prior art.

Since the matrix is treated with the adhesive or conditioned medium before the cells are sown, the sown cells or the applied explant culture cells have a considerably improved adhesion to the matrix. Surprisingly, it has been shown that if the in vitro hollow organ according to the invention or in vitro Tissue is produced using isolated, previously in vitro cultured and propagated cells, because of the improved adhesion of the cells to the matrix compared to the methods described in the prior art, significantly fewer cells have to be applied to the matrix. This in turn means that the in vitro pre-cultivation of the cells to be subsequently sown on the matrix can be considerably shortened. The in vitro pre-cultivation primarily serves to provide sufficient cell material for sowing on the matrix. Since fewer cells are required for sowing in accordance with the method according to the invention, the pre-cultivation of the cells can thus be considerably shortened because a lower cell density must be achieved. Particularly when used as a transplant, the cultivation of the tissue- or organ-typical cells and possibly the matrix containing neurogenic cells can also be significantly shortened due to the improved adhesion of the cells to the matrix. For example, it is sufficient to cultivate the matrix with the cells only for a few hours, preferably overnight, until the cells sown / applied thereon form a (se i-) confluent cell monolayer. When using explant cultures, the period from taking the biopsy to the finished graft can be reduced to two weeks. The matrix containing cells can therefore be used for the transplantation after a very short cultivation, although the cells contained on it have not yet formed complete and functional tissue layers, but only (semi) confluent monolayers. Should the cell-containing matrix be used as a test system Det, it can of course also be cultivated until the organ-typical tissue layers of the desired target organ are completely formed.

When the cell-containing matrix is used for transplantation purposes, the complete formation of the individual tissue layers typical of the organ and thus the full functionality of the graft produced in vitro is only achieved in vivo according to the invention. This preferred procedure according to the invention therefore imitates the primary wound healing process by setting in vitro generated “cell islands”. Advantageously, the cells of the (semi-) confluent monolayer contained on the transplanted matrix become in vivo, that is to say in Organism into which the cell-containing matrix has been inserted as a graft is nourished by diffusion processes, whereas fully developed tissue layers could only be nourished in vivo if these tissues had been vascularized.

The matrix used according to the invention has an advantageous effect both on the formation of the cell monolayers in vitro and on the formation of the complete organ-typical tissue layers in vivo. The matrix is preferably a cellular matrix which originates from a biological source or is produced from a biological material and which preferably consists of collagen or other substances, for example chitosan, and also a large number of other substances. zen, such as chondroitin sulfate, fibronectin and growth factors. This provides the cells contained on the matrix with nutrients and at the same time promotes the regeneration of many tissues after transplantation into the organism. After regeneration of the individual tissue layers of the in vitro tissue or organ, the matrix is either completely broken down or remains permanently in the organism.

The inclusion of neurogenic cells in the method according to the invention, which is provided in a preferred embodiment of the invention, ensures that vascularization of the transplanted cell-containing matrix takes place more rapidly in vivo, since the growth of cells from the wound edges is greatly accelerated. The neurogenic cells contained on the matrix ensure that after the tissue formation and wound healing processes in the body have been completed, an organ replacement that is also functional from a nervous point of view is obtained.

With the aid of the methods according to the invention, animal or human in vitro tissue or organ systems are thus obtained which can be transplanted after a short time, the complete formation of the organ-typical tissue layers taking place in vivo. It is thereby achieved that the transplanted in vitro hollow organs or in vitro tissues largely correspond in structure to the native organs or tissues and can therefore take over the function of the replaced native organs or tissues in vivo. Since the materials used do not contain immunogenic substances, Rejection reactions on the part of the recipient organism are minimized and the survival time of the grafts is significantly increased. The in vitro tissues or in vitro organs produced according to the invention can, after appropriate cultivation, also be used in vitro for test purposes, for example to investigate the effect of potential medicaments on individual cell types, individual tissue layers or a complete tissue or organ.

In connection with the present invention, the term “biodegradable, biodegradable or non-degradable plate-shaped matrix” means a flat, flat structure to which cells can adhere and multiply, the components of this structure in the body of a recipient not being immunological, toxic or cause other reactions and can be metabolized in the body of the recipient, the metabolizing products also not causing any immunological or toxic reactions and, moreover, in the case of the degradable matrix in the recipient's body, are readily absorbable The matrix consists mainly of collagen or other substances such as chitosan or a mixture thereof. In addition, the matrix used according to the invention preferably contains substances that promote growth and / or promote the multiplication of the cells adhering to it. The matrix used according to the invention can be of natural origin or has been produced synthetically his. It is preferably acellular. This means that although it can come, for example, from a biological source, for example a tissue, due to its processing, it does not contain any cell structures, but only a collagen scaffold that largely corresponds to the complex extracellular matrix, or a scaffold of other substances, such as a Chitosan framework. The matrix used according to the invention is level in spatial projection, that is to say it has the shape of a plate before and during the method according to the invention. However, it has such elasticity or flexibility that it can be shaped into a three-dimensional body, for example a cylinder, in the last step of the method according to the invention. The matrix used according to the invention is therefore also characterized by good moldability.

In a preferred embodiment of the invention, the biodegradable, biologically compatible matrix used is a collagen-containing matrix.

In a particularly preferred embodiment, the collagen-containing matrix is a VET-BIO-SIS T ™ matrix from Cook Deutschland GmbH, Mönchengladbach, Germany. This commercially available matrix is made from the pig's small intestine submucosa. It is already disinfected so that all bacteria and viral components are removed. It contains type I, III and V collagen as well as fibronectin, decorin, hyaluronic acids, chondroitin sulfate A, heparin sulfates and growth factors, especially TGFß and bFGF. The VET-BIO-SIS-T matrix is usually used as an operative patch to restore and strengthen tissue. The matrix supports wound healing and tissue reforming by producing a biologically compatible, absorbable support tissue that serves as a scaffold for tissue growth. The VET-Biosis T ™ matrix is particularly suitable for the production of animal in vitro tissues or in vitro hollow organs, but not for the production of human tissues or organs.

In another particularly preferred embodiment of the invention, the collagen-containing matrix used is the Stratasis ™ matrix from Cook Urological, Spencer, Indiana, USA. Like the VET-BIO-SIS-T ™ matrix, the Stratasis ™ matrix is also made from pork small intestine subsubmucosa. Statasis ™ matrix is particularly suitable for the production of human in vitro tissues or human in vitro hollow organs.

In a further particularly preferred embodiment of the invention, the collagen-containing matrix used is produced using recombinant human collagen type I and / or recombinant human collagen type III. Type I collagen is collagen found primarily in skin, bones and tendons, while the more hemostatic type III collagen occurs primarily in blood vessels. The use of a matrix consisting of recombinant human collagen for the production of an in vitro Organ or in vitro tissue offers several key advantages over the use of a matrix isolated from a natural source such as porcine small intestine submucosa. Since the collagen contained in such a matrix is of human origin, the potential for immune reactions is significantly reduced. In contrast to a matrix isolated from natural sources, a recombinant matrix containing human collagen is also free of pathogenic agents, such as bacterial germs, viruses, prions and the like.

In a further preferred embodiment of the invention, the biodegradable, biologically compatible matrix used is a chitosan-containing matrix. Chitosan is a mixture of substances that is obtained when chitin is broken down by alkaline means. Chitin is an amino sugar-containing polysaccharide that can be isolated from animal organisms, especially arthropods, but also from the cell wall of fungi. Chitin consists of chains of β-1,4-glycosidically linked N-acetyl-D-glucosamine (NAG) residues. Chitosan is chitin, which can be deacetylated and depolymerized to different degrees. Methods for the production of chitosan are described, for example, in Malette et al., Ann. Thor. Surg., 36 (1983), 55. Chitosan has gel and film-forming properties. Studies have shown that chitosan can reduce the exophytic growth of callus in the regeneration of bone tissue and promote the ingrowth of vascular smooth muscles in vascular grafts (Malette et al., In: Chi- tin in Nature and Technology (ed. Muzzarelli, Jeuniaux and Gooday), (1986), 435-442, Plenum Press, New York). It has also been shown that chitosan has no adverse effect on the healing process of urogenital wounds (Bartone and Adickes, J. Urol., 140 (1988), 1134-1137). In a preferred embodiment of the invention, chitosan, which is about 80% to about 90% deacety- lated and has a molecular weight of 600,000 to 800,000, is used in particular to produce a matrix.

In connection with the present invention, an “adhesive” is understood to mean a substance or a mixture of substances with which tissue or cells can be connected to one another or to a matrix, the adhesion, that is to say the adhesion of the cells or tissue to one another or to the matrix The adhesive used according to the invention is preferably biocompatible and biodegradable, ie it contains no constituents that cause a toxic, immunological or other reaction in the body. The constituents of the adhesive used according to the invention are metabolized in the body, the Metabolism products also do not cause immunological or toxic reactions.

In a particularly preferred embodiment of the present invention, a fibrin adhesive is used as the adhesive. A fibrin glue is characterized in that it contains fibrinogen and thrombin components. Preferably lie these two main components of the fibrin adhesive initially appear separately and only come into contact with the matrix when the tissues and / or cells are actually bonded. When tissue and / or cells are bonded to a matrix using a fibrin adhesive consisting of two components, the matrix is preferably first treated with the fibrinogen component, for example a solution containing fibrinogen, by soaking the matrix in this fibrinogen solution, for example or the fibrinogen solution is sprayed onto the matrix. The thrombin component is preferably only later applied to the matrix together with the tissue-typical cells and / or neurogenic cells to be sown.

When a fibrin adhesive is used, processes take place which essentially correspond to the last phase of blood clotting, for example in the case of wound closure. When the cells are bonded to the matrix, just as with blood coagulation, fibrinogen is converted by thrombin to form monomeric fibrin by splitting off the fibrinopeptides A and B. For its part, monomeric fibrin spontaneously forms aggregated fibrin through end-to-end and side-to-side attachment. In the presence of calcium chloride, which is contained, for example, in commercially available fibrin adhesives, the aggregated fibrin is then converted into polymeric fibrin, covalent bonds being formed between adjacent γ and α chains of the fibrin monomers. After the cell-containing matrix has been introduced into a body as a graft, the polymeric fibrin formed by the fibrin adhesive promotes as in normal wound healing For example, the infusion of fibroblasts into the wound area, with fibrin acting as a guardrail. This process is a multifactorial process in which thrombin, fibrin and factor XIII have a stimulating effect on fibroblast proliferation. The gradual proteolytic degradation of the fibrin network then begins in the body.

In a particularly preferred embodiment of the invention, the fibrin adhesive is a commercially available fibrin adhesive for biological wound care, for example Tissucol-fibrin adhesive. In addition to thrombin and fibrinogen, Tissucol ^® fibrin glue contains calcium chloride and small amounts of plasminogen, which is converted to plasmin in vivo by calcium pure and other tissue factors and thus triggers fibrinolysis. The fibrin formed by the fibrin glue is thus gradually broken down in the body.

The invention also relates to the use of the adhesive defined above, in particular fibrin adhesive, for the production of human or animal single or multilayer in vitro tissues or complete or partial hollow organs.

In a further particularly preferred embodiment of the present invention, the matrix is pretreated with conditioned medium. Conditioned medium is characterized in that it cultivates 24 hours with the cells to be transplanted before they are applied to the collagen membrane. was fourth. This cultivation enriches the medium with growth factors released by the cells. The pretreatment of the collagen membrane with conditioned medium improves the adherence, proliferation and survival of the cells on the membrane.

In a preferred embodiment of the invention it is provided that for the production of a single or multilayer human or animal in vitro tissue or a complete or partial, human or animal in vitro hollow organ at least one type, but preferably several types, of isolated differentiated or non-differentiated tissue- or organ-specific or tissue- and organ-unspecific cells, which were previously cultivated and propagated in vitro, are applied in low cell density to the matrix pretreated with an adhesive or conditioned medium.

In another preferred embodiment of the invention, epithelial and / or mesenchymal cells, which consist of tissue parts of a biopsy as so-called explant cultures, are used to produce a single- or multi-layered human or animal in vitro tissue or a complete or partial, human or animal in vitro hollow organ be obtained, applied to the matrix pretreated with an adhesive or conditioned medium. The explant cultures from mucosal or submucosal tissue parts are applied directly to the membrane. The tissue obtained as a biopsy is examined using a Scissors separated into a mucosal and a submucosal part. The respective tissue portions are chopped into 2 x 2 mm ² pieces and applied to the membrane. In the case of the collagen membrane, the mucosal tissue pieces are applied to the smooth side, the submucosal tissue pieces to the rough side. The cultures are then incubated for 10 to 14 days.

In a further preferred embodiment of the invention, epithelial and / or mesenchymal

Cells obtained from tissue parts of a biopsy as so-called explant cultures and isolated, previously cultivated and propagated in vitro

Cells combined applied to the pretreated matrix to produce an in vitro tissue or an in vitro hollow organ. For example, the membrane can coexist with epithelial cells, which are expanded by explant culture, and with

Muscle cells and fiboblasts that were previously grown in vitro are colonized.

The isolated cells and / or explant culture cells to be sown are generally selected so that they are compatible with the target organ or target tissue to be produced in vitro. The seeded cells and / or explant culture cells are preferably cells which are normally found in the target organ or target tissue to be produced. If the in vitro tissue or organ to be produced consists of several histologically and functionally distinguishable tissue layers and / or cell clusters, typical for each tissue layer and each cell cluster Cell types are sown or applied, the layering obtained according to the invention preferably being the same as the natural layering. For example, urothelial cells, muscle cells, fibroblasts and neurogenic cells are sown or applied to produce an in vitro urinary bladder. To produce an in vitro intestinal wall, for example, mucosa cells, muscle cells, fibroblasts and neurogenic cells are sown or applied.

The expression “sowing in low cell density” means that 0.01 cm × 10 ⁶ cells to 1 × 10 ⁷ cells, preferably 0.05 × 10 ⁶ cells to 2 × 10 ⁶ cells, are sown or applied per cm ² matrix surface. Preferably 0.1 to 0.5 x 10 ⁶ urothelial cells and muscle cells and 0.01 to 0.05 x 10 ^δ fibroblasts are seeded or applied per cm ² matrix surface.

In connection with the present invention, the term “cell” encompasses in particular isolated, naturally occurring or genetically modified human or animal cells or their precursors. Naturally occurring cells can be cells which have been changed non-pathologically, pathologically changed, not degenerate or "Pathologically altered cells" are cells whose normal cell functions, such as metabolism, stimulus response, motility or reduplication, are disturbed or damaged. The term “degenerate” encompasses all changes in a normal cell, for example cell polymorphism, anisocytosis, nuclear polyphosphy, polychromasia, disturbed Nuclear-plasma relation and aneuploidy, which can lead to disturbed differentiation or to undifferentiation and to a deregulated growth of the cell, and particularly affects cells of malignant tumors. In the invention, the term “genetically modified cells” is understood to mean all cells which have been manipulated with the aid of genetic engineering methods, with either foreign DNA being introduced into the cell or the cell's own DNA, for example by deletions, inversions and additions , was modified.

To produce a human or animal in vitro organ or tissue system, autologous, homologous or heterologous cells can also be used, based on the subsequent recipient organism. In preferred embodiments, the cells are homologous or allogeneic cells, which are obtained from a donor of the same type, so that immune reactions in the host organism are avoided or reduced after transplantation of the organ or tissue produced in vitro. In a particularly preferred embodiment, the cells are obtained from the organism into which the in vitro organ or tissue produced according to the invention is then to be placed. In a particularly advantageous embodiment of the invention, the cells used are human or animal stem cells.

The cells used according to the invention are cultivated in vitro and multiplied by the number of Increase cells, which can then be applied to produce an in vitro tissue or in vitro hollow organ on the matrix treated with an adhesive or conditioned medium. In connection with the present invention, the term “cultivating and multiplying cells in vitro” means that the vital functions of cells take place outside of the natural environment, that is to say outside the body, for example, in a suitable environment, for example with addition and removal Removal of metabolic products and products, the selected conditions guaranteeing the division of the cells and thus their multiplication.

According to the invention, the cells are cultivated and propagated in a full medium suitable for cell culture, for example DMEM medium, M199 medium, F12 medium, etc. The medium used for culturing and multiplying the cells can contain further additives, such as a buffer substance, for example Hepes buffer, serum, for example fetal calf serum (FCS), antibiotics, etc.

A cell culture medium containing components such as FCS, which have been obtained from natural, in particular animal sources, may possibly contain pathogenic agents, such as viruses, prions, bacterial germs and the like. If cell culture media of this type are used to cultivate and multiply the cells used according to the invention, there is therefore the potential risk that such pathogenic agents are transferred to the cells. In a particularly preferred embodiment of the invention, it is therefore provided that the cells used according to the invention are cultivated in a fully synthetic medium. In the context of the present invention, a “fully synthetic medium” is understood to mean a medium including all necessary additives, the components of which are produced exclusively using chemical synthesis processes and / or genetic engineering processes (DNA recombination processes) and not from animal starting materials, in particular not from Tissues, organs, body fluids or other parts of the body of a mammal, and in the genetic engineering of one or more components of the fully synthetic medium, the expression of these components is therefore preferably carried out in a prokaryotic host cell, for example a gram-negative or gram-positive host cell, or a eukaryotic non-animal Host cell, for example a fungal or plant host cell.

When the cells are cultivated and propagated in vitro, their ectodermal, mesodermal or endodermal characteristics are preserved. After differentiation of the cells, their specifically developed cell functions, such as specific metabolic performance, specific stimulus response capacity, mobility characteristics, etc., form again, that is to say that the cells have a differentiation status similar to the original state after cultivation, multiplication and subsequent differentiation. If necessary, the origin of the cells of the urinary tract can be varied, which means that, for example, bladder cells can be used for urethra or ureter or kidney pelvis. In a preferred embodiment, provision is also made to use muscle cells and fibroblasts from other organs, for example skin, to colonize the matrix.

In a particularly advantageous embodiment of the invention, it is provided that the cells to be sown come from a human or animal biopsy sample that was taken from the native target tissue or target organ in vivo. In the context of the present invention, the term “biopsy sample” means a tissue sample that a living organism can take as an untargeted biopsy sample, that is to say as a so-called blind puncture, by puncturing with a hollow needle, using special instruments such as pliers, punching instruments, and biopsy probes. Brushes, slings and similar instruments or surgically with a scalpel, or as a targeted biopsy under ultrasound or X-ray control or as part of an endoscopy or laparoscopy. Before further use, the bioptic material obtained is preferably subjected to histological, cytological, immunohistological, histochemical or genetic engineering controls and an additional test for viruses, bacteria, prions and other pathogenic agents.

In an advantageous embodiment of the invention it is provided that the biopsy sample was taken from a human or animal urethra. In a further advantageous embodiment It is intended that the biopsy sample comes from a human or animal bladder. In a further embodiment of the invention it is provided that the biopsy sample comes from a human or animal ureter or kidney pelvis. It is also provided according to the invention that the biopsy sample can come from a human or animal vas deferens or fallopian tubes. According to the invention, it is also provided that the biopsy sample comes from a human or animal blood vessel, human or animal small intestine or human or animal large intestine.

To obtain isolated cells, the biopsy sample obtained is preferably mechanically separated into individual tissue layers using scissors, a scalpel and / or tweezers and / or a similarly suitable instrument. The individual tissue layers are then mechanically shredded. According to the invention, it is provided in particular that the mechanically separated and comminuted tissue layers are digested enzymatically by using a protease mixture, for example the BZ1 mixture (Röche), which contains collagenase I, collagenase II, dispase and neutral proteases (caseinase) to obtain isolated cell types and to enrich them specifically. The enzymatic digestion degrades in particular the intercellular substance, which is an essential part of the connective and supporting tissues and consists of fibers and the light-microscopic homogeneous basic substance, which chemically mainly consists of mucopolysaccharides and proteins includes. It is provided according to the invention that the enzymatic cleavage of the separated and comminuted tissue layers carried out using proteases is stopped by adding an inhibitor, in particular by adding serum or other inhibitors such as soy bean trypsin inhibitor.

However, the actual separation of the individual cell types takes place according to the invention via in vitro pre-cultivation of the cell mixture obtained after the proteolytic cleavage. According to the invention, cell-specific media are used in particular, which are distinguished in that the growth of desired cells is promoted while the growth of undesired cell types is suppressed. Each proteolytic cell mixture of a previously mechanically separated and comminuted tissue layer is therefore cultivated according to the invention in different tissue-specific media, in order to specifically multiply and enrich specific cell types of this tissue layer. For example, it is provided that a smooth muscle cell serum (Promocell C-22060) low serum content is used to enrich muscle cells from bladder biopsies. Serum-free medium (Promocell C-23010) is preferably used to enrich fibroblasts from bladder biopsies. Serum-free keratinocyte medium (GibcoBRL 17005034) is preferably used to enrich mucosa cells from bladder biopsies. Other media for the selection and multiplication of the respective cell types, in particular media which are completely chemically defined, such as, for example, EpiLife (Cascade Biologics Inc.) can also apply. The cells are precultivated in vitro using conventional methods for cultivating human or animal cells. For example, the cells can be cultivated on microtiter plates or on the bottom of culture vessels such as bottles. Epithelial cells are preferably cultivated on collagen-coated bottles, and the culture of muscle cells, fibroblasts and neuronal cells in uncoated bottles. During the in vitro pre-cultivation of the individual cell types of a tissue layer, the cells of each cell type are preferably subjected to a passage into fresh medium two or three times, the medium being changed every two to three days, especially when the cell layer is one Has confluence of about 50 to 75%.

After two to three passages in vitro, the precultivated and enriched cells of any cell type can be sown or applied to the matrix according to the invention. According to the invention, cells are sown or applied to at least one side of the matrix in order to produce an in vitro hollow organ consisting of several tissue layers, which is to be used in particular as a transplant. For example, only muscle cells are sown on the underside of the matrix, with the remaining components being delivered by the recipient after implantation. In another embodiment, for example, the cells of at least one cell type on the top of the matrix (smooth-appearing side) and the cells of at least one cell type on the bottom of the matrix. seeded or applied at least to another or the same cell type. Preferably cells are sown or applied to the top of the matrix which form the cell layer delimiting the lumen in the native organ. The cells that form the cell layers in the native organ that face away from the lumen of the hollow organ are preferably sown or applied on the underside of the matrix (rough-appearing side). Of course, it is also possible to sow cells on only one side of the matrix, for example in order to produce a single-layer or multi-layer tissue which is to be used in particular for transplantation or for test purposes.

To produce, for example, an in vitro urinary bladder hollow organ, muscle cells and fibroblasts, which originate from a urinary bladder biopsy and have been isolated and enriched as described above, are sown / applied on the underside of the matrix. Accordingly, urothelial cells originating from a bladder biopsy are sown / applied to the top of the matrix used according to the invention.

According to the invention, the cells on the underside and on the top of the matrix have a density of 0.01 × 10 ⁶ to 0.1-0.5 × 10 ⁶ cells / cm ² matrix, preferably in a density of 0, 1 x 10 ⁶ to 0.5 x 10 ⁵ cells / cm ^{2 are} sown or applied. To produce an in vitro urinary bladder hollow organ according to the invention, for example, 0.5 × 10 ⁶ muscle cells and 0.05 × 10 ⁶ fibroblasts are removed per cm ² underside of the matrix. sows / applied, while 0.5 x 10 ⁶ urothelial cells are required per cm top of the matrix.

In a particularly preferred embodiment of the present invention, it is provided that, at the same time as the isolated and enriched cells, which were obtained from biopsy samples and cultured in vitro, or later, neurogenic cells are applied to the matrix treated with an adhesive / conditioned medium ,

In connection with the present invention, “neurogenic cells” are understood to mean nerve cells that come from larger nerve cell structures or nerve cell tissues such as a ganglion or a nerve plexus. It is provided according to the invention that the neurogenic cells used either come from the same native organ or from another organ to produce a special in vitro hollow organ. To produce an in vitro urinary bladder hollow organ, for example, neurogenic cells can be isolated from the native bladder tissue. However, the neurogenic cells can also be isolated from another organ, for example the Taeniae coli, that is to say the three strips of the longitudinal muscle layer of the colon. In contrast to the other cells sown / applied on the matrix, the neurogenic cells after their isolation are not cultivated in vitro using conventional methods, but are applied directly to the matrix immediately after isolation. According to the invention it is provided that the isolated neurogenic cells are sown / applied at a density of 1 x 10 ² cells / cm ² on the underside of the matrix.

After the cells have been deployed, the cell-containing matrix is cultivated under suitable conditions, so that an in vitro tissue or in vitro organ is obtained which can be used either for transplantation or for test purposes. Suitable conditions for culturing the cell-containing matrix include, inter alia, a suitable cell culture medium, preferably DMEM / F12 medium, a suitable temperature, preferably 37 ° C., and a suitable gas atmosphere which preferably contains 7% CO ₂ . If the cell-populated matrix is to be used later for transplantation, the matrix is cultivated until the tissue or organ-typical cells located on the bottom or top of the matrix have formed a monolayer, that is to say a complete single-cell layer. The formation of a cell monolayer is achieved, for example, when using cells which have been precultivated in vitro by culturing overnight. The matrix containing cell monolayers can then be transplanted. If the cell-containing matrix is used for test purposes, the cultivation can of course be carried out until all organ-typical tissue layers of the tissue or organ to be produced are completely formed. A culture of the cells in their respective cell-specific medium is sought under these conditions. For this purpose, the grafts are cultivated in specially made culture chambers. In an advantageous embodiment of the invention it is provided that the cells applied to the matrix are checked during and / or after the cultivation with regard to their function, their morphology and / or their differentiation status. The invention therefore also relates to screening and diagnostic methods carried out using such cells, the cells being cultivated and examined during and / or after the methods described above, for example for pharmacological, toxicological, physiological, morphological and / or molecular biological parameters. In the context of these investigations, the effects of potential medications, pathogens, antigens or the like on the cultivated cells can also be investigated. In a preferred embodiment, the cells are examined in the presence and absence of an agent and the observed effects are compared with one another.

In a particularly preferred embodiment of the invention, the cells are cultivated on the matrix in order to produce an in vitro tissue in a cell reactor. In connection with the invention, a cell reactor is understood to be a device for the automatic multiplication of cells and / or for the automatic production of in vitro tissues. The cell reactor is preferably a computer-based bioreactor of the Kerator type. A cell reactor consists of at least one cell growth chamber and a medium-containing chamber which is separated by a semipermeable membrane, for example se the collagen-containing matrix used according to the invention are separated, the membrane serving as a carrier for the cell layer to be produced. The advantage of producing an in vitro tissue in a cell reactor is, in particular, that the process runs automatically under computer control, whereby a very uniform growth of the individual cell layers is achieved and at the same time contamination with germs is avoided. The cell-covered membrane or matrix produced in the bioreactor can be used both for transplantation purposes and for test purposes.

In a preferred embodiment, the present invention therefore comprises the cultivation of human or animal cells for the production of single-layer or multilayer human or animal in vitro tissues or human or animal in vitro hollow organs, which can be used as a graft or partial or complete replacement to be used for a diseased or damaged tissue or organ in the body of a human or animal. According to the invention, it is provided in particular that the cell-containing matrix is brought into the form in which it is to be transplanted only after cultivation, that is to say after the cell monolayer has been formed, and immediately before transplantation. For example, if only small areas of a diseased or damaged tissue or hollow organ in the body of a human or animal are to be replaced, the line-populated matrix plate can be used by the doctor immediately after cultivation in the operating room can be adapted in size and shape to the defect by mechanical processing, for example with a scalpel or scissors. The cell-populated plate cut according to the defect is then sewn in and additionally fixed in the defect using a tissue adhesive, for example a fibrin adhesive.

If the diseased or damaged area of a hollow organ, for example a ureter, comprises larger sections, the cell-populated matrix plate is shaped into a suitable hollow organ. In connection with the present invention, a “hollow organ” is understood to mean an organ in which the tissue partially or completely encloses a cavity, the clear width or the diameter of the hollow organ being referred to as lumen. The formation of the cell-populated matrix plate into one The desired hollow organ therefore means that one or more row-populated matrix plates are / are brought into the three-dimensional body shape with the corresponding dimensions that the native organ has.

If, for example, a partial in vitro ureter is to be produced, the cell-coated matrix plate is cut to the appropriate size and then brought into a tube or cylinder shape. A tubular or cylindrical shape can, for example, be fixed by a surgical suture and additionally reinforced using a tissue adhesive, for example a fibrin adhesive. The cylindrical shape can additionally or exclusively with surgical Seam processes can be fixed. For example, the edges of the cell-populated matrix can be secured by a 3.0 or 4.0 suture or by small surgical clamps. If a fibrin adhesive is also used, the ends of the cell-populated matrix plate are brushed or sprayed with the adhesive and then briefly pressed together. If the section of a hollow organ to be replaced is very large, several line-populated matrices can optionally be connected to one another by surgical suturing and / or the use of a fibrin adhesive in order to produce a larger in vitro hollow organ.

An in vitro hollow organ is also produced immediately after the cultivation of the matrix / matrices and before the transplantation. The in vitro hollow organ is preferably produced by the medical practitioner in the operating room.

The present invention therefore also relates to a partial or complete human or animal in vitro urethral hollow organ which has been produced by the method according to the invention and, if appropriate, a subsequent and / or preceding cultivation method of a conventional type, urothelial cells, muscle cells, fibroblasts and optionally neurogenic Cells were used. The in vitro urethral hollow organ can be used as a partial or complete replacement for a damaged or diseased animal or human urethra. An in vitro manufactured urethral hollow organ or an in vitro urethral replacement can for example, after a urethral injury, that is, an open or closed injury to the urethra as a result of violence in the area of the pelvis, perineum or other bladder injuries, in the case of urethral malformations, for example epispadia, hypospadias, partial or total obliteration or stenosis.

The present invention also relates to a partial or complete human or animal in vitro urinary bladder hollow organ which has been produced by the method according to the invention and a subsequent and / or preceding cultivation method of a conventional type, where appropriate, muscle cells, fibroblasts, urothelial cells and optionally neurogenic cells were used. The in vitro urinary bladder hollow organ can be used as a partial or complete replacement for a damaged or diseased animal or human bladder. An urinary bladder replacement manufactured in vitro can be used, for example, for bladder malformations, bladder dystrophy, for example bladder ecstrophy, bladder carcinoma or for secondary shrinkage blisters caused by neurogenic, hyperreflexible bladder dysfunction. When used for the surgical rehabilitation of a neurogenic bladder, the complications that can occur during bladder augmentation with the intestine or during auto-augmentation are avoided.

The present invention also relates to a partial or complete human or animal in vitro ureter hollow organ and a in vitro renal pelvis, which was produced by the method according to the invention and a subsequent and / or preceding cultivation method of a conventional type, using urothelial cells, muscle cells, fibroblasts and optionally neurogenic cells. The in vitro ureter hollow organ can be used as a partial or complete replacement for a diseased or damaged human or animal ureter. An in vitro urethral replacement can be used, for example, for kidney pelvic stenosis, subpelvine stenoses, ureter malformations such as the congenital megaureter, for obliteration or stenosis in the course of time, for example for stone problems or after surgery, for specific forms of ureter inflammation or traumatic lesions.

The present invention also relates to a partial or complete human or animal in vitro vas deferens hollow organ, which was produced according to the method according to the invention and an optional subsequent and / or preceding cultivation method of a conventional type, using epithelial cells, muscle cells, fibroblasts and optionally neurogenic cells become. The in vitro vas deferens can be used as a partial or complete replacement for a human or animal vas deferens. An in vitro produced vas deferens can be used, for example, for vas deferens, obstruction of the vas deferens due to inflammation or to restore continuity (Vasovasostomy) due to refertilization in the state after vasectomy.

The present ^'invention relates also to a partial or complete human or animal ULTRASONIC in vitro tubal hollow organ, which has been prepared by the process according to the invention and an optionally subsequent and / or preceding culturing methods of conventional type, where ciliated epithelial cells, muscle cells, fibroblasts and optionally neurogenic Cells were used. The in vitro fallopian tube can be used as a partial or complete fallopian tube replacement for a human or animal fallopian tube. A fallopian tube replacement made in vitro can be used, for example, after obliteration of the fallopian tube.

The present invention also relates to a partial or complete blood vessel hollow organ produced in vitro, which was produced by the method according to the invention and a subsequent and / or preceding cultivation method of a conventional type, where appropriate, using endothelial cells, muscle cells and fibroblasts. The in vitro blood vessel can be used as a partial or complete replacement for a human or animal blood vessel. A blood vessel replacement manufactured in vitro can be used, for example, to remove or circumvent flow obstacles in occlusive diseases, to eliminate pathological flow conditions, for example in varicosis, aneurysm, angioma and arteriovenous fistula, or to change the flow. direction can be used, for example, by creating a vascular surgical sub, etc.

The present invention also relates to a partial or complete human or animal in vitro small intestine hollow organ which has been produced by the method according to the invention and an optional subsequent and / or preceding cultivation method of a conventional type, using mucosa cells, muscle cells, fibroblasts and optionally neurogenic cells , The in vitro small intestine hollow organ can be used as a replacement for areas of a human or animal small intestine. A small bowel replacement manufactured in vitro can be used, for example, after complete or partial small bowel resection for small bowel tumors, inflammatory bowel diseases or other loss (intestinal infarction, volvulus, neurogenic damage). It is suitable to apply "pouches", such as those used for colon or small bowel surgery.

The present invention also relates to a partial or complete human or animal in vitro large intestine hollow organ which was produced by the method according to the invention and a subsequent and / or preceding cultivation method of a conventional type, using mucosa cells, muscle cells, fibroblasts and optionally neurogenic cells , The in vitro large intestine hollow organ can be used as a replacement for areas of a human or animal colon. An in Colon replacement produced in vitro can be used, for example, for complete or partial resection due to inflammatory bowel inflammation, colon cancer or for congenital innervation disorders (Hirschsprung's disease).

The present invention also relates to the production of a matrix populated with fibroblasts for the surgical removal / covering of fistulas which originate, for example, from the urogenital tract or intestine. The fistulas can be caused, for example, by inflammatory bowel diseases and by surgical interventions. To produce the matrix populated with fibroblasts, fibroblasts are either isolated from the skin or from the organs in question and cultured.

The present invention also relates to the production of a matrix populated with fibroblasts in the form of a loop for the operative lifting of organs, for example the urinary bladder, in women with weak pelvic floor.

Another particularly preferred embodiment of the invention comprises the use of the three-dimensional one- or multilayer human or animal in vitro organ or tissue systems produced according to the invention as test systems.

Due to the complexity of the in vitro organ or tissue systems produced according to the invention, in particular due to the use of primary cells, their arrangement in their natural state Layering and the inclusion of neurogenic cells, the in vitro systems can be used specifically for various questions in the chemical-pharmaceutical industry. This complexity of the in vitro systems according to the invention makes it possible to investigate the effect of a stimulus on individual cell populations and on the interaction of the cells. In particular, the use of neurogenic cells allows early statements to be made about the effects of active substances on nerve cells that could not previously be recorded. For example, the in vitro systems according to the invention are suitable for product testing, in particular with regard to effectiveness, mechanism of action, undesirable side effects, for example irritation, toxicity and inflammation effects, or tolerance of active ingredients. According to the invention, the term “active ingredient” means any substance, but also other agents, for example physical influencing variables such as electromagnetic radiation, radioactivity, heat, sound or the like, which can influence or recognize biological cells or parts thereof, in particular cell organelles. Such active ingredients can be, in particular, of a chemical nature, for example diagnostic or therapeutic agents. In connection with the present invention, diagnostic agents are understood to mean any substances that can specifically recognize the presence of conditions, processes or substances or their absence and, in particular, can provide conclusions about disease states. Diagnostics often have recognizing and marking functions. Such substances are used in particular under the term therapeutic agents. stands that can be used either prophylactically or accompanying the disease in order to avoid, alleviate or eliminate illnesses. In connection with the present invention, diseases are also understood to mean conditions such as unnatural states of mind, pregnancies, signs of aging, developmental disorders or the like. With the aid of the animal and human in vitro tissue or in vitro hollow organ test systems according to the invention, the effectiveness or the mechanism of action and / or the side effects of active substances can be analyzed much more precisely than in conventional test systems. The single-layer and multilayer in vitro tissue or hollow organ systems can also be used to screen potential active substances and to investigate properties such as specificities and the interactions of active substances with target cells.

These tests of active substances, therapeutic agents and diagnostic agents on the animal or human in vitro tissue or in vitro hollow organ test systems according to the invention can include conventional morphological or histological methods as well as conventional biochemical, toxicological, immunological and / or molecular biological methods. The effects of substances or agents on the cells of the three-dimensional in vitro organ or tissue systems according to the invention can be determined, for example, by the release of substances, for example cytokines or mediators, by cells, and by their effects on gene expression, the metabolism, the proliferation tion, the differentiation and reorganization of the cells of an in vitro organ or tissue test system. Using methods for quantifying cell damage, in particular using a vital dye such as a tetrazolium derivative, for example, cytotoxic effects on cells can be detected.

A preferred embodiment of the invention therefore includes methods for examining the pharmacological effects of active ingredients, diagnostics and therapeutic agents on human or animal tissue using the human or animal in vitro hollow organ or in vitro tissue systems produced according to the invention, the in vitro organ or tissue systems are treated with the active substance to be investigated and the results obtained in the presence and absence of the active substance to be investigated are compared with one another.

In a further particularly preferred embodiment of the invention, the human or animal in vitro organ or tissue systems produced according to the invention are used to examine tissue or organ-specific diseases and to develop new treatment options for these diseases. In an advantageous embodiment of the invention, the in vitro small intestine and / or in vitro large intestine systems according to the invention are used for the investigation of inflammatory bowel diseases such as Crohn's disease and ulcerative colitis. These diseases are characterized by relapsing, destructive inflammatory reactions of the intestinal mucosa. The invention provides for taking biopsy samples from patients with chronic inflammatory bowel disease and for producing tissue or a partial intestinal organ using the methods according to the invention. Potential active substances can then be examined on these in vitro tissues or in vitro organs. Yet another advantageous embodiment of the invention includes the use of the in vitro urinary bladder system produced according to the invention for the investigation of neurogenic, hyperreflexible and areflexible bladder disorders.

In a particularly advantageous embodiment of the invention, primary cells and / or cell lines from patients with a specific disease, for example a specific bowel disease, are used in order to use them to produce patient-specific in vitro organ or tissue systems, for example a patient-specific in vitro intestinal system establish and use it to examine and assess the effectiveness of certain therapies and / or medications.

A preferred embodiment of the invention comprises the use of the human or animal three-dimensional in vitro organ or tissue systems according to the invention for examining the mechanisms of tumor pathogenesis and for examining active substances for their suitability as medicaments, for example against a specific tumor. In an advantageous embodiment of the invention, degenerated cells, in particular the organs or tissues mentioned above, built in vitro organ or tissue test system used to obtain larger amounts of a degenerate cell material. The material obtained is then further analyzed using conventional methods, for example histological, biochemical, molecular biological or immunological methods, in order, for example, to examine morphological changes in degenerated cells or the release of specific substances in more detail or to create transcription and / or expression profiles , In a further advantageous embodiment of the invention, the effect of drugs or other substances, for example with regard to their ability to inhibit cell division, is studied on an in vitro organ or tissue test system constructed from degenerate cells.

Another particularly preferred embodiment of the invention comprises the use of the in vitro organ or tissue systems according to the invention for checking genetically modified cells, in particular the aforementioned tissues and organs. In an advantageous embodiment of the invention, cells are tested which have been genetically modified with a view to a gene therapy to eliminate gene-related malfunctions or to restore normal gene functions in diseases of the organs mentioned above.

Further advantageous embodiments result from the subclaims. The invention is explained in more detail with reference to FIG. 1 and examples.

FIG. 1 shows muscle cells stained with B-TK26 one day after they have been applied to a collagen matrix.

example 1

Production of an in vitro hollow urinary bladder

1. Materials

Media:

Smooth muscle cell medium - low serum (P-romocell C-

22060 (Supplement: C-39265))

Fibroblast cell medium - low serum (B-romocell C-

23010 (Supplement: C-39315)) Keratinocyte growth medium serum-free (GibcoBRL

17005034 (Supplement: 37000015))

F12 medium (GibcoBRL 21765-029)

DMEM (GibcoBRL 41966-029)

FCS (SIGMA F-7524)

BZ1 (Röche 1988417): 9 mg B-roteases / ml BBS EDTA / trypsin (lx) (Sigma T-3924)

Trypsin inhibitor from soybeans (Gibco 17075-011) BBS with added antibiotics Transport medium with added antibiotics (DMEM)

antibiotics:

Claforan: 0.15 mg / ml (1 ml to 500 ml BBS / DMEM; Glaxo Claforan 2.0) Amphotericin: 2.5 U / ml (5 ml to 500 ml BBS / DMEM; GibcoBRL 15290-018)

Gentamycin: 50 μg / ml (0.5 ml to 500 ml BBS / DMEM; GibcoBRL 15750-037)

2. Procedure

A. Biopsy preparation

On the first day, a biopsy sample is taken sterile, placed in a transport medium and transported to a laboratory in it. An approximately 1 to 2 cm ² piece for immunohistological and other possibly molecular biological examinations is removed from each biopsy and frozen on the day of removal. The rest of the biopsy is stored overnight at 4 ° C in the transport medium. A sterile control of the solution is created to check all media and buffers used.

On the second day, the biopsy is removed from the transport medium and a culture of the transport medium is created for sterile control.

The muscularis and mucosa layers of the biopsy sample are separated using scissors. The separated layers are then shredded using scissors so that the tissue pieces are smaller than 0.5 mm ² . The shredded tissue layers are then washed three times in BBS containing Claforan, Amphotericin and Gentamycin and then weighed.

The crushed muscularis layer is then digested enzymatically, with 0.4-0.5 g in 3 ml of B-rotease medium being incubated for 15 minutes at 37 ° C. (1: 5; 1 ml of BZ1: 4 ml of F12 medium). Then another 2 ml of 1: 5 diluted BZ1 are added and the digestive reaction turns 15 Minutes continued. The crushed mucosa layer is treated identically, although the BZl-B rotease mixture is diluted 1:10 in Fl2 medium. After the tissue layer has been cleaved with boteases, the reaction mixture is examined microscopically and made up to 10 ml with FCS or to 50 ml with soy trypsin inhibitor in order to stop the reaction. The digested muscularis layer is resuspended in medium. 25 ml of this is transferred to a new 50 ml falcon tube so that one tube is available for the culture of smooth muscle cells and one tube for the culture of fibroblasts. Both batches are left for about 5 minutes to settle the larger pieces of tissue. The cells in the supernatant are transferred to a new 50 ml falcon tube. The cells are then centrifuged at about 1000 rpm for 10 minutes. The pellet is then washed three times in 15 ml of the respective cell-specific medium.

The settled pellets of the digested muscularis layer are discarded while the pellet of the mucosal layer is washed three times with 15 ml of keratinocyte medium.

The muscle cells and fibroblasts are counted microscopically, taken up in 12 ml of cell-specific medium and seeded / applied in 6 wells of a microtiter plate with 24 wells, 1 ml being applied to each well. The urothelial cells are also counted, taken up in 6 ml of keratinocyte medium and 3 ml of cell suspension are distributed over two 9 cm ² collagen-coated petri dishes.

From the remaining pieces of mucosa, an explant culture is carried out on collagen-coated petri dishes with a diameter of 10 cm. For this purpose, 200 μl thrombin are added to the mucosa pieces in 1.5 ml medium. The petri dish is rinsed with fibrin-containing medium (200 μl. Fibrin in 1.5 ml keratinocyte medium) and then the supernatant is removed so that the petri dish is completely wetted. The cell or tissue suspension containing thrombin is applied in the middle of the Petri dish, where it polymerizes.

The next day, the supernatants are removed from all cell cultures and either transferred to a 24-well microtiter plate or a fresh petri dish with a 9 cm ² collagen-coated surface in order to collect additional cells. The medium is changed approximately every two to three days, the passage of the cells taking place at a confluence of approximately 50 to 75%. In the case of urothelial cells, these are transferred to a collagen-coated bottle with a coating area of 25 cm ² , in the case of smooth muscle cells and fibroblasts in two bottles with a cultivation area of 25 cm ² .

The fibroblasts and muscle cells are detached by adding 100 μl of EDTA / trypsin, preheated to 37 ° C, are added per well of a microtiter plate containing 24 wells and incubated at 37 ° C for 10 min. The detachment of the cells is checked microscopically. The reaction is stopped with 100 ul FCS or 900 ul soybean inhibitor. The supernatants from 6 wells are combined and the cells are centrifuged off. The cell pellet obtained is washed twice in the appropriate cell-specific medium and the cells are then diluted by a factor of 1: 3.

The urothelial cells are detached by inhibiting the cells with 3 ml of prewarmed 0.2% EDTA at 37 ° C. for 10 minutes. The EDTA is then pipetted off. The urothelial cells are then incubated for a second time with 1.5 ml of 0.1% trypsin / 0.2% EDTA for 5 to 10 minutes. The reaction is then stopped with 5 ml FCS or 10 ml soybean inhibitor. The cells are centrifuged, washed twice in 15 ml of keratinocyte medium and diluted by a factor of 1: 2.

Production of the cell-seeded matrix

The matrix is soaked in DMEM / F12 10% FCS. The membrane is then incubated in 3 ml of DMEM / F12, which was mixed 1: 2 with fibrinogen solution (fibrinogen solution of the fibrin adhesive from Tissucol). The cell pellets for the underside of the membrane, i.e. muscle cells, fibroblasts and neurogenic cells, are taken up in 1.5 ml DMEM / Fl2 medium. An equal volume of thrombin solution (i.e. second one) is added to this cell suspension Component of the fibrin adhesive) added and mixed. This 3 ml solution is distributed homogeneously on the underside of the fibrin-soaked matrix. This causes the cell suspension to polymerize. 0.5 x 10 ⁶ muscle cells, 0.05 x 10 ⁶ fibroblasts and 1 x 10 ² neurogenic cells are sown / applied per square centimeter underside of the membrane. After the polymerization, the membrane is turned over. The urothelial cells are isolated according to the standard protocol and likewise suspended in 3 ml of DMEM / F12 medium, which also contains the thrombin solution, and distributed on the top of the membrane. 0.5 x 10 ⁶ urothelial cells are required per cm ² top. After the upper side has been polymerized, the membrane is cultivated overnight in DMEM / F12 at 37 ° C. and 7% CO ₂ and then transplanted.

Claims

Expectations

1. A method for producing a single or multi-layered human or animal in vitro tissue or a complete or partial, human or animal in vitro hollow organ, comprising:

a) treating a biologically compatible, biodegradable plate-shaped matrix with a biologically compatible, biodegradable adhesive or conditioned medium,

b) applying isolated tissue-type or organ-type or tissue-type or organ-untypical mesenchymal and / or epithelial tissue pieces of a biopsy to the matrix for the growth of the primary cells, that is to say the preparation of an explant culture, and / or the sowing of at least one type of an isolated one differentiated or non-differentiated tissue or organ-typical or tissue or organ-untypical cell, which was previously cultivated and grown in vitro, in low cell density on the matrix, c) culturing the explant cells and / or cells previously cultured in vitro and, if appropriate, matrix containing neurogenic cells under suitable conditions, and

2. The method according to claim 1, wherein after step b) and before step c) isolated, tissue- or organ-typical or tissue- or organ-typical, neurogenic cells are sown in low cell density on the matrix.

3. The method according to claim 1 or 2, wherein the biologically compatible matrix is an acellular collagen-containing matrix.

4. The method of claim 3, wherein the acellular collagen-containing matrix is made from the porcine small intestine submueosa.

5. The method of claim 3, wherein the acellular collagen-containing matrix of ^' recombinant human

Collagen is made.

6. The method according to claim 1 or 2, wherein the biologically compatible matrix is an acellular chitosan-containing matrix.

7. The method of claim 6, wherein the acellular chitosan-containing matrix is made from chitosan that deacetylates to about 80% to about 90% and has a molecular weight of 600,000 to 800,000.

8. The method according to any one of claims 1 to 7, wherein the adhesive used to treat the matrix is a fibrin adhesive for biological wound care, which comprises at least the two components fibrinogen and thrombin, or conditioned medium.

9. The method according to claim 8, wherein the matrix is first treated with the fibrinogen component of the fibrin adhesive and the thrombin component of the fibrin adhesive is then applied to the matrix together with the in vitro cultivated cells and / or neurogenic cells or the matrix is first treated with conditioned medium and the cultivated cells are subsequently applied in conditioned medium.

10. The method according to any one of claims 1 to 9, wherein the in vitro cultured tissue or organ-typical or tissue or organ-typical cells are non-pathologically modified, non-degenerate, pathologically modified, degenerate or genetically modified cells or a mixture is about it.

11. The method according to claim 10, wherein the in vitro cultured tissue or organ-typical or tissue or organ-untypical cells come from a human or animal biopsy sample.

12. The method of claim 11, wherein the biopsy sample is from a human or animal urethra.

13. The method of claim 11, wherein the biopsy sample is from a human or animal bladder. •

14. The method of claim 11, wherein the biopsy sample comes from a human or animal ureter or kidney pelvis.

15. The method of claim 11, wherein the biopsy sample is from a human or animal vas deferens.

16. The method according to claim 11, wherein the biopsy sample comes from a human or animal fallopian tube.

17. The method of claim 11, wherein the biopsy sample is from a human or animal blood vessel.

18. The method of claim 11, wherein the biopsy sample is from human or animal small intestine.

19. The method of claim 11, wherein the biopsy sample is from human or animal colon.

20. The method of claim 11, wherein the biopsy sample is from human or animal skin.

21. The method according to any one of claims 11 to 20, wherein the biopsy sample is mechanically separated into individual tissue layers with the aid of scissors, a scalpel, tweezers and / or a similar suitable instrument, and the individual tissue layers are mechanically comminuted.

22. The method according to any one of claims 11 to 21, wherein the mechanically separated and comminuted tissue layers are subjected to a separate enzymatic digestion using a protease mixture in order to obtain individual isolated cell types.

23. The method according to any one of claims 11 to 22, wherein the enzymatic digestion of the separated and crushed tissue layers is stopped by adding FCS or soybean trypsin inhibitor.

24. The method according to any one of claims 11 to 21, wherein the cell mixture obtained by enzymatic digestion or by explant cultivation of each tissue layer for the enrichment of cells of each cell type is subjected to a separate in vitro cultivation in a cell-specific medium.

25. The method according to claim 24, wherein the cell-specific medium is a medium, the components of which are produced by means of chemical synthesis processes and / or genetic engineering processes.

26. The method according to any one of claims 11 to 25, wherein the cells of each cell type during in vitro cultivation at a confluence of 50% to 75% of a passage must be subjected to fresh medium.

27. The method according to any one of claims 11 to 26, wherein the cells of each cell type are used after the second or third passage in vitro for sowing on the biological matrix.

28. The method according to claim 27, wherein the cultivated cells are sown by at least one cell type on the underside of the matrix.

29. The method of claim 27, wherein the cultured cells are sown by at least one other cell type on top of the matrix.

30. The method according to any one of claims 27 to 29, wherein the cultivated cells are sown on the underside and on the top of the matrix in a density of 0.01 x 10 ^δ to 1 x 10 ^δ cells / cm ² matrix.

31. The method according to any one of claims 1 to 30, wherein the neurogenic cells applied to the matrix are cells isolated from intestinal tannins or from native bladder tissue.

32. The method of claim 31, wherein the neurogenic cells are sown at a density of 1 x 10 ² cells / cm ² on the underside of the matrix.

33. The method according to any one of claims 1 to 32, wherein the matrix with the seeded cultivated cells and neurogenic cells under suitable cultivation conditions at least overnight over o which is cultivated for 10 to 14 days in the case of explant cultivation.

34. The method according to any one of claims 11 to 21, wherein the mechanically separated and comminuted tissue layers are subjected to an explant culture on a matrix to obtain individual isolated cell types.

35. The method according to any one of claims 1 to 34, wherein the production of the in vitro tissue takes place in a cell reactor.

36. The method according to any one of claims 1 to 35, wherein the cultured cells and neurogenic cells containing flat matrix is formed into a cylindrical hollow organ with a diameter corresponding to the native organ and the cylindrical shape of the matrix using a fibrin glue and / or surgical Suture material is fixed.

37. Partial or complete human or animal in vitro urethral hollow organ, produced according to one of claims 1 to 12 and 21 to 36.

38. Partial or complete human or animal in vitro urinary bladder hollow organ, produced according to one of claims 1 to 11, 13 and 21 to 36.

39. Partial or complete human or animal in vitro ureter / renal pelvis Hollow organ, produced according to one of claims 1 to 11, 14 and 21 to 36.

40. Partial or complete human or animal in vitro vas deferens hollow organ, produced according to one of claims 1 to 11, 15 and 21 to 36.

41. Partial or complete human or animal in vitro oviduct hollow organ, produced according to one of claims 1 to 11, 16 and 21 to 36.

42. Partial or complete human or animal in vitro blood vessel hollow organ, produced according to one of claims 1 to 11, 17 and 21 to 35.

43. Partial or complete human or animal in vitro small intestine hollow organ, produced according to one of claims 1 to 11, 18 and 21 to 35.

44. Partial or complete human or animal in vitro large intestine hollow organ, produced according to one of claims 1 to 11, 19 and 21 to 36.

45. Partial or complete human or animal in vitro connective tissue equivalent, produced according to one of claims 1 to 11 and 20 to 36.

46. Use of a partial or complete human or animal in vitro urethral Hollow organ according to claim 36 as a graft for the treatment of damaged or diseased urethral areas.

47. Use of a partial or complete human or animal in vitro bladder

Hollow organ according to claim 38 as a graft for the treatment of damaged or diseased urinary bladder areas.

48. Use of a partial or complete human or animal in vitro ureter /

Hollow kidney organ as claimed in claim 39 as a graft for the treatment of damaged or diseased areas of the ureter.

49. Use of a partial or complete human or animal in vitro vas deferens

Hollow organ according to claim 40 as a graft for the treatment of damaged or diseased vas deferens.

50. Use of a partial or complete human or animal in vitro fallopian tube

Hollow organ according to claim 41 as a graft for the treatment of damaged or diseased fallopian tube areas.

51. Use of a partial or complete human or animal in vitro blood vessel

Hollow organ according to claim 42 as a graft for the treatment of damaged or diseased blood vessel areas.

52. Use of a partial or complete human or animal in vitro small intestine hollow organ according to claim 43 as a graft for the treatment of damaged or diseased areas of the small intestine.

53. Use of a partial or complete human or animal in vitro large intestine hollow organ according to claim 44 as a transplant for the treatment of damaged or diseased areas of the large intestine.

54. Use of a partial or complete human or animal in vitro connective tissue equivalent according to claim 45 as a graft for the treatment of fistulas and for "lifting" organs in the form of a loop plastic.

55. Use of a single- or multi-layered human or animal in vitro tissue system, produced according to one of claims 1 to 36 or a partial or complete, human or animal in vitro hollow organ according to one of claims 37 to 45 as a test system for determining the effect of an active substance on human or animal cells, the active substance being brought into contact with the in vitro organ or tissue system and the interaction between the in vitro organ or tissue system and the active substance being determined.

56. Use of a single or multi-layer human or animal in vitro tissue system, produced according to one of the claims 1 to 36 or a partial or complete, human or animal in vitro hollow organ according to one of claims 37 to 45 as a test system for determining the effect of active substances on pathologically modified human or animal cells, the active substance with the pathologically modified cells containing in vitro Organ or tissue system is brought into contact and the interaction between the pathologically modified cells containing in vitro organ or tissue system and the active substance is determined.