WO2003029446A2 - Procede de fabrication d'organes creux in vitro - Google Patents

Procede de fabrication d'organes creux in vitro Download PDF

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Publication number
WO2003029446A2
WO2003029446A2 PCT/EP2002/010605 EP0210605W WO03029446A2 WO 2003029446 A2 WO2003029446 A2 WO 2003029446A2 EP 0210605 W EP0210605 W EP 0210605W WO 03029446 A2 WO03029446 A2 WO 03029446A2
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WIPO (PCT)
Prior art keywords
vitro
cells
matrix
animal
tissue
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PCT/EP2002/010605
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German (de)
English (en)
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WO2003029446A3 (fr
Inventor
Birgit Schäfer
Stefan Carl
Christian Lorenz
Karl-Herbert SCHÄFER
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Cytonet Gmgh & Co. Kg
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Publication of WO2003029446A2 publication Critical patent/WO2003029446A2/fr
Publication of WO2003029446A3 publication Critical patent/WO2003029446A3/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/383—Nerve cells, e.g. dendritic cells, Schwann cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

  • 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.
  • 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.
  • sensitized lymphocytes and / or antibodies can be formed which have a cytotoxic effect on the graft.
  • 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.
  • GVH graft versus host reaction
  • 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.
  • 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.
  • 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.
  • attempts are also made to remedy organ defects or disorders by transplanting tissue from other organs of the same organism.
  • 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.
  • 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.
  • 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).
  • 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.
  • in vitro systems can also be used for a wide range of immunological, histological and molecular biological problems.
  • 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.
  • 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.
  • 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.
  • a biodegradable material for example cotton, gelatin or collagen
  • non-biodegradable material for example a polyester or a polycarbonate
  • 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- ⁇ -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.
  • 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.
  • 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
  • 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:
  • 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
  • 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.
  • the sown cells or the applied explant culture cells have a considerably improved adhesion to the matrix.
  • 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.
  • the pre-cultivation of the cells can thus be considerably shortened because a lower cell density must be achieved.
  • 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.
  • 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.
  • 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”.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the biodegradable, biologically compatible matrix used is a collagen-containing matrix.
  • the collagen-containing matrix is a VET-BIO-SIS T TM matrix from Cook 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 TM 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.
  • the collagen-containing matrix used is the Stratasis TM matrix from Cook Urological, Spencer, Indiana, USA.
  • the Stratasis TM matrix is also made from pork small intestine subsubmucosa.
  • Statasis TM matrix is particularly suitable for the production of human in vitro tissues or human in vitro hollow organs.
  • 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.
  • 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 ⁇ -1,4-glycosidically linked N-acetyl-D-glucosamine (NAG) residues.
  • Chitosan is chitin, which can be deacetylated and depolymerized to different degrees.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • fibrin adhesive 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.
  • fibrinogen is converted by thrombin to form monomeric fibrin by splitting off the fibrinopeptides A and B.
  • monomeric fibrin spontaneously forms aggregated fibrin through end-to-end and side-to-side attachment.
  • 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 ⁇ and ⁇ chains of the fibrin monomers.
  • 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.
  • the fibrin adhesive is a commercially available fibrin adhesive for biological wound care, for example Tissucol-fibrin adhesive.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • epithelial and / or mesenchymal In a further preferred embodiment of the invention, epithelial and / or mesenchymal
  • the membrane can coexist with epithelial cells, which are expanded by explant culture, and with
  • 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.
  • 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.
  • urothelial cells, muscle cells, fibroblasts and neurogenic cells are sown or applied to produce an in vitro urinary bladder.
  • 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 ⁇ fibroblasts are seeded or applied per cm 2 matrix surface.
  • 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.
  • 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.
  • 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.
  • autologous, homologous or heterologous cells can also be used, based on the subsequent recipient organism.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a prokaryotic host cell for example a gram-negative or gram-positive host cell
  • a eukaryotic non-animal Host cell for example a fungal or plant host cell.
  • the cells 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the biopsy sample was taken from a human or animal urethra.
  • the biopsy sample comes from a human or animal bladder.
  • the biopsy sample comes from a human or animal ureter or kidney pelvis.
  • the biopsy sample can come from a human or animal vas deferens or fallopian tubes.
  • the biopsy sample comes from a human or animal blood vessel, human or animal small intestine or human or animal large intestine.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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%.
  • the precultivated and enriched cells of any cell type can be sown or applied to the matrix 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.
  • tissue layers which is to be used in particular as a transplant.
  • only muscle cells are sown on the underside of the matrix, with the remaining components being delivered by the recipient after implantation.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • neurogenic cells are applied to the matrix treated with an adhesive / conditioned medium ,
  • 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.
  • 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.
  • 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.
  • the effects of potential medications, pathogens, antigens or the like on the cultivated cells can also be investigated.
  • the cells are examined in the presence and absence of an agent and the observed effects are compared with one another.
  • the cells are cultivated on the matrix in order to produce an in vitro tissue in a cell reactor.
  • 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.
  • 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.
  • 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.
  • 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.
  • the cell-populated matrix plate is shaped into a suitable hollow organ.
  • 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.
  • 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.
  • the edges of the cell-populated matrix can be secured by a 3.0 or 4.0 suture or by small surgical clamps.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • active ingredients can be, in particular, of a chemical nature, for example diagnostic or therapeutic agents.
  • 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.
  • diseases are also understood to mean conditions such as unnatural states of mind, pregnancies, signs of aging, developmental disorders or the like.
  • 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.
  • 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.
  • a vital dye such as a tetrazolium derivative
  • 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.
  • 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.
  • 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.
  • primary cells and / or cell lines from patients with a specific 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.
  • 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 ,
  • 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 .
  • 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.
  • 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.
  • FIG. 1 shows muscle cells stained with B-TK26 one day after they have been applied to a collagen matrix.
  • Keratinocyte growth medium serum-free (GibcoBRL)
  • 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 ⁇ g / ml (0.5 ml to 500 ml BBS / DMEM; GibcoBRL 15750-037)
  • 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.
  • 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.
  • 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.
  • an explant culture is carried out on collagen-coated petri dishes with a diameter of 10 cm.
  • 200 ⁇ l thrombin are added to the mucosa pieces in 1.5 ml medium.
  • the petri dish is rinsed with fibrin-containing medium (200 ⁇ l. Fibrin in 1.5 ml keratinocyte medium) and then the supernatant is removed so that the petri dish is completely wetted.
  • fibrin-containing medium 200 ⁇ l. Fibrin in 1.5 ml keratinocyte medium
  • the cell or tissue suspension containing thrombin is applied in the middle of the Petri dish, where it polymerizes.
  • 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%.
  • 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 ⁇ l 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.
  • 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).
  • 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
  • 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.

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Abstract

La présente invention concerne des procédés de fabrication de tissus animaux et humains in vitro et/ou d'organes creux animaux et humains in vitro, notamment d'urètres in vitro, d'uretères in vitro, de vessies in vitro, de canaux déférents in vitro, de trompes de Fallope in vitro, de vaisseaux sanguins in vitro, d'intestins grêles in vitro et de côlons in vitro. L'invention concerne également l'utilisation de ces tissus et organes creux in vitro pour la transplantation ou à des fins de test, ainsi que la fabrication d'une matrice colonisée par des fibroblastes, destinée à l'élimination et/ou au bouchage de fistules.
PCT/EP2002/010605 2001-09-24 2002-09-20 Procede de fabrication d'organes creux in vitro WO2003029446A2 (fr)

Applications Claiming Priority (2)

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DE10146903.9 2001-09-24
DE10146903A DE10146903C1 (de) 2001-09-24 2001-09-24 Verfahren zur Herstellung von in vitro-Hohlorganen

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WO2003029446A2 true WO2003029446A2 (fr) 2003-04-10
WO2003029446A3 WO2003029446A3 (fr) 2003-09-12

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005113747A2 (fr) 2004-05-21 2005-12-01 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Systemes de culture tissulaire et organique multicellulaires

Families Citing this family (1)

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Publication number Priority date Publication date Assignee Title
DE102004025080B4 (de) * 2003-06-23 2007-05-03 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Multizelluläre Testsysteme

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006445A1 (fr) * 1996-08-16 1998-02-19 Children's Medical Center Corporation Sous-muqueuse vesicale ensemencee par des cellules pour la reconstitution de son tissu
WO1998010775A1 (fr) * 1996-09-16 1998-03-19 Purdue Research Foundation Composition et procede de reparation de tissus neurologiques
WO1999000152A2 (fr) * 1997-06-27 1999-01-07 Augustinus Bader Transplant biosynthetique et son procede de production
WO2000066036A2 (fr) * 1999-04-30 2000-11-09 Massachusetts General Hospital Fabrication de tissus vascularises a l'aide de moules bidimensionnels microfabriques

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998006445A1 (fr) * 1996-08-16 1998-02-19 Children's Medical Center Corporation Sous-muqueuse vesicale ensemencee par des cellules pour la reconstitution de son tissu
WO1998010775A1 (fr) * 1996-09-16 1998-03-19 Purdue Research Foundation Composition et procede de reparation de tissus neurologiques
WO1999000152A2 (fr) * 1997-06-27 1999-01-07 Augustinus Bader Transplant biosynthetique et son procede de production
WO2000066036A2 (fr) * 1999-04-30 2000-11-09 Massachusetts General Hospital Fabrication de tissus vascularises a l'aide de moules bidimensionnels microfabriques

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
SCHAEFER BIRGIT M ET AL: "Autologous transplantation of urothelium into demucosalized gastrointestinal segments: Evidence for epithelialization and differentiation of in vitro expanded and transplanted urothelial cells." JOURNAL OF UROLOGY, Bd. 159, Nr. 1, Januar 1998 (1998-01), Seiten 284-290, XP008016529 ISSN: 0022-5347 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005113747A2 (fr) 2004-05-21 2005-12-01 Fraunhofer Gesellschaft zur Förderung der angewandten Forschung e.V. Systemes de culture tissulaire et organique multicellulaires
US9375514B2 (en) 2004-05-21 2016-06-28 Fraunhofer-Gesellschaft Zur Forderung Der Angewandten Forschung E.V. Multicellular tissue and organ culture systems

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WO2003029446A3 (fr) 2003-09-12

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