EP1504087A2 - Peau bioartificielle - Google Patents

Peau bioartificielle

Info

Publication number
EP1504087A2
EP1504087A2 EP03732323A EP03732323A EP1504087A2 EP 1504087 A2 EP1504087 A2 EP 1504087A2 EP 03732323 A EP03732323 A EP 03732323A EP 03732323 A EP03732323 A EP 03732323A EP 1504087 A2 EP1504087 A2 EP 1504087A2
Authority
EP
European Patent Office
Prior art keywords
fibroblasts
protein
growth factor
skin
autologous
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP03732323A
Other languages
German (de)
English (en)
Inventor
Hans-Günther Machens
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Individual
Original Assignee
Individual
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Individual filed Critical Individual
Publication of EP1504087A2 publication Critical patent/EP1504087A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/33—Fibroblasts
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/36—Skin; Hair; Nails; Sebaceous glands; Cerumen; Epidermis; Epithelial cells; Keratinocytes; Langerhans cells; Ectodermal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
    • A61K38/1866—Vascular endothelial growth factor [VEGF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • 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
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • 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
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/091—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells melanocytes
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
    • 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
    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts

Definitions

  • the invention relates to a bioartificial skin, comprising a porous matrix on which isogenic or autologous fibroblasts and keratinocytes are located, which express at least one angiogenic protein and / or at least one protein stabilizing the epidermodermal junction, a process for their production and their use ,
  • Bioartificial skin replacement is known.
  • Machens et al. in “Cell Tissues Organs” 2000, 167: 88-94 describes a skin replacement based on a glycosaminoglycan / chondroitin-6-sulfate matrix.
  • synthetic or xenogeneic see, for example, Machens et al. In “Chirurg” 2000, 71: 152-158.
  • this includes vascularization and innervation of the dermis, on the other hand, the sufficient molecular stabilization of the epidermodermal junction, i.e. the connecting layer between the epidermis and dermis.
  • the object of the invention is to eliminate these deficiencies inherent in the prior art.
  • a bioartificial skin which has a porous matrix corresponding to human skin on which there are isogenic or autologous fibroblasts which contain at least one angiogenic protein (for example VEGF165), a neurotrophic protein (for example NGF) and at least one which is epidermo- Express dermal junction stabilizing protein (eg collagen 17).
  • angiogenic protein for example VEGF165
  • NGF neurotrophic protein
  • epidermo- Express dermal junction stabilizing protein eg collagen 17.
  • the isogenic or autologous keratinocytes to be transplanted are enriched with isogenic or autologous melanocytes to such an extent that a color of the bioartificial skin corresponding to the recipient region is achieved.
  • the advantage that can be achieved with this is better vascularization and innervation of the matrix (it is sufficient, for example, if an angiogenic and neurotrophic active protein is secreted over a period of 2 to 3 weeks) and a more stable epidermodermal junction (ie connection points between the dermis and epidermis) and a skin color adequate to the environment.
  • an allogeneic acellular dermis from corpse skin is particularly suitable as a matrix. Due to its cellular nature, this dermis is immunocompetent and there are no rejection reactions.
  • a dermis can, for example, under the trade name Alloderm from the company Life-Cell Corp., USA, (LifeCell; One millenium Way; Branchburg, NJ 08876; USA).
  • Isogenic cells e.g. Fibroblasts
  • Fibroblasts in the sense of the invention, are genetically identical cells which do not lead to immune compatibility after retransplantation into an isogenic organism.
  • Autologous cells e.g. Fibroblasts are cells in the body in which the donor and recipient are one and the same organism.
  • the at least one angiogenic protein is PDGF-A (platelet derived growth factor A), PDGF-B (platelet derived growth factor B), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast growth factor), TGFbeta (tumor growth factor beta), angiopoetin 1 and angiopoetin selected.
  • isologic or autologous keratinocytes and melanocytes are also applied to the isologic or autologous fibroblasts in such an amount and in such a ratio that a natural skin tone is produced.
  • the invention also provides a process for preparing the bioartifizellen skin according to the invention prepared in which a) in a body by implantation of biologically inert material, the formation of isogenic or autologous fibroblasts is triggered, b) formed in step a) fibroblasts from the 'body are obtained, c) the fibroblasts obtained in step b) are genetically modified so that they have at least one angiogenic Express protein, a neurotrophic protein and at least one protein stabilizing the epidermo-dermal junction and d) the fibroblasts obtained in step c) onto a porous one
  • Matrix for example alloderm, can be applied.
  • the fibroblasts are on the matrix, e.g. Alloderm, confluent, i.e. grown together. ⁇
  • the fibroblasts obtained in step c) are PDGF-A (platelet derived growth factor A), PDGF-B (platelet derived growth factor B), VEGF (vascular endothelial growth factor), bFGF (basic fibroblast express growth factor), TGFbeta (tumor growth factor beta), angiopoetin 1 and angiopoetin selected angiogenic protein.
  • isologic or autologous keratinocytes and melanocytes are applied to the isologic or autologous fibroblasts in such an amount and in such a ratio that a natural skin tone is produced.
  • the invention further specifies the use of a bioartificial skin according to the invention or a bioartificial skin according to the invention produced by a method according to the invention for covering wounds, in particular burns.
  • a biologically inert or metabolically inert material is a material that does not participate in the body's metabolic processes, but can still trigger a cellular reaction (accumulation of fibroblasts). If such a material (eg silastic) is implanted, a connective tissue capsule that contains the desired fibroblasts will form around the implant within a few days (5-7). This capsule together with fibroblasts can be removed and, after trypsinizing the material, the fibroblasts can be isolated and cultivated. These fibroblasts can be isogenic.
  • the fibroblasts are obtained from the patient, in which one later wants to use these cells after genetic modification in the bioartificial skin.
  • the cells would be autologous, i.e. cells in which the donor and recipient are identical.
  • a skin biopsy for the cultivation of keratinocytes and melanocytes can be carried out on a human donor (who will also be the recipient of the bioartificial skin). The technique of isolating and cultivating these cells does not differ in principle from the process for obtaining the fibroblasts.
  • GMFB isogenic fibroblasts of the rat
  • NMFB non-genetically modified fibroblasts
  • the inventor has also been able to demonstrate that, after adenoviral gene transfer in fibroblasts, a series of angiogenetically active proteins can be stably expressed for a period of 2-3 weeks and, after retransplantation into an isogenic recipient organism, induce therapeutic angiogenesis there.
  • Fibroblast cultures obtained from autologous Inbred rat strains (female Lewis CRL rats, weight 200-215 grams; Charles River Laboratories)
  • Virus-producing cell line amphotrophic psi-CRIP packaging cell line which is derived from murine NIH-3T3 fibroblasts which express the viral gene products gag, pol and env (R. Mulligan, Whitehead Institute of Biomedical Research, Cambridge, Mass.).
  • a padcos46 RESeGFP 39155 bp is used as the cosmid vector fragment and equipped with the corresponding desired gene sequence (W. Linden aier, Society for Biotechnological Research / Baunschweig).
  • PBS phosphate buffered saline
  • Trypsin solution (Trypsin 1-300, ICN Biochemicals), consisting of 0.1% D-dextrose (w / v) and trypsin 0.1% (w / v) in PBS at a pH of 7.5
  • Aechylacher (Fisher Scientific, Fair Lawn, NJ / USA)
  • Ketamine (Ketanest 100 mg / ml; Fort Dodge Laboratories, Iowa / USA)
  • VEGF mRNA quantification are known per se (competitive RT-PCR, Northern blot).
  • Autologous rat fibroblasts are grown in 5 animals of the above-mentioned rat strain as later carrier cells for the expression of the desired gene.
  • the animals are anesthetized for this by intraperitoneal injection e.g. a combination of 0.05 mg / gm ketamine (Ketanest 100 mg / ml; Fort Dodge Laboratories, Iowa / USA) and 0.0013 mg / gm xylazine (Rampun 20 mg / ml; Bayer Corporation, Kansas / USA).
  • the spontaneously breathing animals are shaved from the xyphoid to the groin region and placed on an operating table.
  • Body temperature is measured during each experiment using a digital rectal thermometer and kept constant at 36 - 37 degrees Celsius using a warming mat. After sterile washing of the surgical area, a cut is made with the scalpel from the xyphoid along the linea alba towards the caudal end, whereby only the skin and the subcutaneous fatty tissue are severed and the fascia of the rectus muscles are left, followed by careful care of the epigastric skin vessels to create an approximately 4 x 5 cm wound pocket laterally on both sides.
  • silastic is only one example of a suitable material that causes the formation of isogenic fibroblasts after implantation and has no other side effects
  • PharmElast SF Medical
  • This membrane has a thickness of 0.0127 mm, for example, is particularly soft, extremely flexible and is supplied sterile packed.
  • the film is implanted, it is washed sterile. After the film has been fixed in situ, the wound is closed by an intracutaneous 6-0 ethilon suture with countersinking the corner knots.
  • the animals are observed daily for 7 days with free access to water and food in order to be able to uncover problems in wound healing at an early stage. After this period, the animals are anesthetized as described above and the silastic membrane, which should stimulate local fibroblast production as a foreign body, together with the scar tissue formed around it, are removed. The animals are euthanized by means of an intraperitoneal overdose of the anesthetic mixture mentioned.
  • the surgically obtained fibroblast conglomerate is immediately stored in DMEM at 4 degrees Celsius and processed as quickly as possible.
  • the material is detached from the silica membrane under sterile conditions. All work with the fibroblast culture takes place in a laboratory with genetic engineering level 2 instead of with air extraction at every sterile workplace.
  • the material is now subjected to extensive washing in a total of 10 plastic containers, each with 10 ml PBS (phosphate-buffered table salt 0.9%). Each container can be closed sterile so that the fibrocyte conglomerate can be shaken vigorously in the PBS for 10 x 60 seconds. After washing twice, the tissue is removed again in order to remove any remaining connective tissue residues and portions of silica that have been demarcated under sterile cauldrons.
  • the material is then placed in a sterile 25 ml monovette and enzymatically treated with 5 ml trypsin and 5 ml EDTA for 5 minutes in order to detach the fibroblasts from the collagen dressing.
  • the cells are filtered through sterile gauze and then washed in DMEM with 20% FBS to neutralize the trypsin activity.
  • the suspension obtained in this way is centrifuged at 800 rpm for 5 minutes.
  • the fibroblasts settle on the bottom and are mixed with 10 ml DMEM.
  • fibroblasts When the fibroblasts almost conflate, another cell separation is performed.
  • the cells obtained in this way can then be re-sown for further cultivation of new cells or can also be preserved and frozen. This is done by cell separation and subsequent suspension in DMEM with 10% FBS and appropriate antibiotic addition. If CRIP cells are preserved, they are suspended with BCS medium.
  • BCS medium As a protective protective agent, the substance DMSO is added to the medium in a mixing ratio of 1:10. Between 1 x 10 6 cells should then be contained per ml of medium. Then 1-2 ml of medium / container are filled and frozen for 24 hours at - 20 degrees Celsius. The following day, the containers are deep-frozen at - 80 degrees Celsius, in order to remain in liquid nitrogen at - 196 degrees Celsius 24 hours later. This gradual freezing prevents the formation of ice crystals in the suspension with consequent cell damage.
  • the first step in gene transfer is the production of a recombinant virus that encodes the gene to be transferred.
  • a cDNA that encodes the protein of interest is spaceted by means of PCR (polymerase chain reaction).
  • the corresponding primers produce, for example, a BspHl locus at the translation starter codon and, for example, a BamHl locus at the translation stop codon.
  • the product of the PCR can then be isolated by separating the gene product at the sites mentioned and inserting the gene obtained into the Ncol / BamHl loci of a viral vector called MFG. Successful transmission is then checked by DNA sequencing.
  • This vector comes, for example, from the murine Moioney leukemia virus and does not itself contain any viral genes other than those which are necessary for the transcription, packaging, reverse transcription, integration and expression of the viral vector with the modifying gene therein.
  • the vector In order to be able to produce virions that can genetically anchor this vector in target cells, the vector must be integrated into a special packaging cell line, which comes, for example, from murine 3T3 fibroblasts.
  • the transduction of the vector into the corresponding packaging cell line is facilitated by the addition of calcium phosphate, since this makes the cell membranes of the packaging cell lines temporarily porous and gives the viral vector easier access to the cell.
  • This packaging cell line (Psi-CRI) was specially produced to deliver the viral proteins pol, env and gag, which in turn can produce virions that code and transmit the vector with the modifying gene therein.
  • the packaging cell line itself cannot produce viruses that correspond to "wild type" replicants and are therefore virulent. Instead, she transcribes them.
  • PNS of the recombinant viral vector in RNA which is then integrated into the RNA of the virion.
  • the Psi-CRP packaging cell line then excretes the virion, ie the recombinant virus together with the modifying gene, into the cell medium.
  • a transfection of the target cells is effective with a quantity of 1.0 - 10.0 million virions / ml medium. Each transfected cell line is therefore checked for the highest titer production in order to then select it for gene transfer.
  • a padcos46 RESeGFP is used as a cosmid vector fragment for adenoviral gene transfer
  • the vector (39155 bp) used and equipped with the corresponding desired gene sequence. Furthermore, the vector can be constructed in such a way that gene expression takes place only with the simultaneous administration of another substance. This enables a time-controlled expression. Suitable vectors with so-called
  • the fibroblasts obtained are sown in brood chambers with a floor area of 75 cm 2 and with a medium of DMEM, mixed with 100 micrograms / ml streptomycin, 100 IU / ml penicillin, 3 microliters / l amphotericin and 5% FBS.
  • the fibroblasts are sown in a low density (5 x 10 5 cells) in order to achieve the greatest possible effectiveness of the transduction.
  • a medium change is carried out with medium from the Psi-CRIP cell line.
  • This medium contains 1.0-10.0 million virions / ml medium, which are freshly pipetted off from the medium of the packaging cell line (Psi-CRIP).
  • the medium is first filtered through a pore filter with a pore diameter of 0.45 micrometers in order to free it from cell debris and possible contaminants.
  • the substance polybrene is then added to the medium in a concentration of 8 micrograms / ml.
  • polybrene is a cationic polymer consisting of 1, 5-dimethyl-l, 5-diaxadecamethylene polymethobromide and exerts its transfection-supporting effect by adsorbing it on the virus particles and also on the surface of the target cell, so as to weaken the electrostatic repulsive forces of these two negatively charged substances.
  • the cells of the Psi-CRIP cell line must already be confluent and the medium changed on the day before the transduction in order to ensure the highest possible number of active viruses / ml medium.
  • the viruses have an average half-life of 6-8 hours at 37 degrees Celsius in the incubator. The lifespan of the viruses can be increased tenfold by lowering the incubator temperature to 32 degrees Celsius, thus considerably increasing the effectiveness of the transduction.
  • the virion When the recombinant viruses come together with target cells in the medium, the virion is bound to the cell surface of the fibroblasts by special receptors and releases the packaged RNA genome into the interior of the cell. This is reverse transcribed and the resulting DNA reaches the cell nucleus, where it is stably integrated into the genome of the target cell.
  • This integrated copy of the recombinant viral vector with the modifying gene is passed on to the daughter cells like any other autosomal gene.
  • the gene is stable and regularly expressed, so that the target cells now secrete large amounts of the desired protein.
  • adenoviral vectors only temporary gene expression can be expected, which will be eliminated from the cell genome again after a few cell generations.
  • the medium should be left with the target cells for 24 hours to complete the transduction process in all cells.
  • the virus-containing medium can also be preserved for later use for transduction of fibroblasts.
  • the medium is pipetted off and snap-frozen on dry ice until it takes on a yellowish color.
  • the medium is then stored at - 80 degrees Celsius. However, you have to expect a loss of 30 - 50% viruses through this process.
  • the growth of the genetically modified compared to the untreated fibroblasts is examined by sowing 5 ⁇ 10 5 cells in each case into Petri dishes with a diameter of 60 mm and counting the cells at intervals of 12 hours over a total of 4 days according to the above-mentioned method.
  • This allows conclusions to be drawn about the biological activity of the secreted protein, since the substances used, with the exception of sFLTl D1-D6, also have an autocrine mitogenic effect, i.e. the fibroblasts that secrete the protein themselves stimulate cell division.
  • the number of fibroblasts is then measured under the fluorescence microscope using a counting chamber and the vitality of the cells is determined by Evans-Blue and compared with the controls, which consist of untreated fibroblasts.
  • Protein expression will also be determined in vitro using ELISA techniques. It becomes essential higher protein production expected from the genetically modified fibroblasts.
  • the transfected fibroblasts are applied to a matrix which has previously been cut in vitro according to the size of the graft to be used. This results in a design that corresponds exactly to the patient's needs.
  • the dermis is initially rehydrated with its basement membrane upwards and covered with DMEM with 10% FBS and corresponding antibiotic addition for 15 minutes.
  • the modified fibroblasts are then applied to the dermis with medium (1 ⁇ 10 5 cells / cm 2 ) and preserved in the incubator for 24 hours.
  • the confluence of the fibroblasts is waited for and is complete after approx. 2-3 days. During this time, the medium is changed every 48 hours.
  • the keratinocytes and melanocytes taken simultaneously with the fibroblasts have since been separated and cultivated and can be sown on the matrix if the fibroblasts are confluent using the same technique as the fibroblasts (2 x 10 5 keratinocytes / cm 2 ).
  • the amount of melanocytes to be transplanted with the keratinocytes from suspension depends on the desired color of the recipient region. For a rather light type (skin type 11) 1 x 10 4 melanocytes / cm 2 dermis are calculated, while for a rather dark European type '(skin type IV) it is 3 x 10 4 melanocytes / cm 2 dermis.
  • the wound areas to be transplanted are surgically subtly debrided again under sterile conditions and then the graft is precisely placed on the wound area.
  • a liquid-permeable layer of cuticerin (Beiersdorff) is spread over the graft.
  • a layer of 1 cm thick sterile sponge is placed over it. This sponge contains DMEM with 10% FBS and a corresponding antibiotic additive for the primary nutrition of the upper graft layers).
  • the entire wound area is then covered with a further layer of cuticerin and fixed with tie-on pads. After 2 days, the entire bandage can be removed and the transplanted wound can be moistly bandaged with Cuticerin soaked in Lavasept. The entire region, like any other skin graft, must be immobilized by splints for 10 days.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Zoology (AREA)
  • Veterinary Medicine (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Dermatology (AREA)
  • Transplantation (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Wood Science & Technology (AREA)
  • Organic Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Virology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Botany (AREA)
  • Vascular Medicine (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Microbiology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Dispersion Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Psychology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

L'invention concerne une peau bioartificielle présentant une matrice poreuse sur laquelle se trouvent des fibroblastes isogènes ou autologues qui expriment au moins une protéine angiogénétique, une protéine neurotrophe et au moins une protéine stabilisant la jonction épidermo-dermale ainsi que la formation d'un épiderme conforme au coloris, un mélange de kératinocytes et mélanocytes isogènes ou autologènes, leur procédé de production et leur utilisation.
EP03732323A 2002-05-07 2003-05-07 Peau bioartificielle Withdrawn EP1504087A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10220472A DE10220472A1 (de) 2002-05-07 2002-05-07 Bioartifizielle Haut
DE10220472 2002-05-07
PCT/EP2003/004784 WO2003095630A2 (fr) 2002-05-07 2003-05-07 Peau bioartificielle

Publications (1)

Publication Number Publication Date
EP1504087A2 true EP1504087A2 (fr) 2005-02-09

Family

ID=29285189

Family Applications (1)

Application Number Title Priority Date Filing Date
EP03732323A Withdrawn EP1504087A2 (fr) 2002-05-07 2003-05-07 Peau bioartificielle

Country Status (4)

Country Link
EP (1) EP1504087A2 (fr)
AU (1) AU2003239844A1 (fr)
DE (1) DE10220472A1 (fr)
WO (1) WO2003095630A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
ES2263382B1 (es) 2005-05-16 2007-11-16 Fundacion Para La Investigacion Biomedica Del Hospital Gregorio Marañon Matriz artificial de gel de fibrina endotelizada superproductora de factores proangiogenicos.
CN101955908B (zh) * 2010-07-22 2013-01-23 中国医学科学院皮肤病研究所 用于检测物质致敏性和药物筛选的人工表皮体外模型

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5266480A (en) * 1986-04-18 1993-11-30 Advanced Tissue Sciences, Inc. Three-dimensional skin culture system
US20010048917A1 (en) * 1998-03-09 2001-12-06 Warren Hoeffler Skin equivalent and methods of forming and using same
CN1333818A (zh) * 1998-11-19 2002-01-30 奥加诺吉尼西斯公司 生物工程化的组织构建物、其制备方法及其应用

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO03095630A2 *

Also Published As

Publication number Publication date
DE10220472A1 (de) 2003-11-27
WO2003095630A3 (fr) 2004-03-04
AU2003239844A8 (en) 2003-11-11
WO2003095630A2 (fr) 2003-11-20
AU2003239844A1 (en) 2003-11-11

Similar Documents

Publication Publication Date Title
EP1242129B1 (fr) Produit de synthese biologique pour articulation
AU731768B2 (en) Composition and method for repairing neurological tissue
CN105517587A (zh) 伤口愈合与组织工程
DE69930155T2 (de) Spray verabreichung von zellen
JPH08509356A (ja) 細胞の移植システム及び方法
Ui et al. Cell lines from imaginal discs of Drosophila melanogaster
US20030091543A1 (en) Therapeutic cell preparation grafts and methods of use thereof
DE69531712T2 (de) Hybridgel, das eine biologisch aktive Substanz sekretiert
DE69725317T2 (de) Implantierbare agarose-kollagenkügelchen enthaltende zellen, die ein diffusionsfähiges biologisches produkt bilden und ihre verwendung
WO1995023216A1 (fr) Mode de preparation de fibroplasts clonogenes, methode de transfection genique de fibroplasts et les fibroplasts genotransfectes ainsi obtenus
DE69233725T2 (de) Verwendung von DNA oder RNA zur Herstellung eines Medikaments zur Behandlung von Tumoren
CN1671836B (zh) 用于基因治疗的原代培养脂肪细胞
EP1286708A1 (fr) Procede de production d'un transplant bio-artificiel
DE60027724T2 (de) Verbesserte keratinozytenkultur und deren verwendungen
DE69824017T2 (de) Vehikel zur gentherapie enthaltend dermales huellgewebe
EP1989293B1 (fr) Constructions de transplantation tissulaire prévascularisées destinées à la reconstruction d'un organe humain ou animal
Schwenter et al. Survival of encapsulated human primary fibroblasts and erythropoietin expression under xenogeneic conditions
WO2004042038A1 (fr) Procede de traitement de tissus malades, degeneres ou endommages faisant appel a un tissu tridimensionnel obtenu in vitro associe a des cellules tissulaires et/ou des facteurs exogenes
EP1504087A2 (fr) Peau bioartificielle
CN113577108B (zh) 一种多功能胶原支架、其制备方法与应用
DE60221519T2 (de) Verfahren und zusammensetzung für hauttransplantate
Sato et al. Artifical Esophagus
DE19651992A1 (de) Serum als Zusatz von Kulturmedium bei der Züchtung von stratifizierten Hautäquivalenten aus Stammzellen
EP1476542A1 (fr) Agent d'induction ou d'inhibition de l'angiogenese
DE10220474A1 (de) Mittel zur Induktion oder Inhibition einer Angiogenese

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20041201

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PT RO SE SI SK TR

AX Request for extension of the european patent

Extension state: AL LT LV MK

DAX Request for extension of the european patent (deleted)
STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION HAS BEEN WITHDRAWN

18W Application withdrawn

Effective date: 20070313