WO2002061420A2 - Systeme de culture a interaction cellulaire in vitro pour l'essai et le developpement de medicaments - Google Patents

Systeme de culture a interaction cellulaire in vitro pour l'essai et le developpement de medicaments Download PDF

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WO2002061420A2
WO2002061420A2 PCT/DE2002/000353 DE0200353W WO02061420A2 WO 2002061420 A2 WO2002061420 A2 WO 2002061420A2 DE 0200353 W DE0200353 W DE 0200353W WO 02061420 A2 WO02061420 A2 WO 02061420A2
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cell
culture
cells
vitro
culture system
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PCT/DE2002/000353
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WO2002061420A3 (fr
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Thomas Häupl
Christian Kaps
Michael Sittinger
Heike Smolian
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Oligene Gmbh
Transtissue Technologies Gmbh
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • 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
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0656—Adult fibroblasts
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • 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"
    • C12N2502/1317—Chondrocytes
    • 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
    • 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
    • C12N2503/02—Drug screening
    • 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
    • C12N2503/04—Screening or testing on artificial tissues

Definitions

  • the invention relates to an in vitro cell interaction system for drug testing and development in the form of interactive cell cultures.
  • tissue engineering tissue engineering
  • the techniques of three-dimensional tissue engineering are used to build complex in vitro test systems that closely approximate the in vivo conditions in healthy and chronically diseased tissue using the example of the joint. They are used for studies on the interactions of known and developing drugs with the tissue to be examined. They are also used to identify new therapeutic targets.
  • inflammation primarily from the synovial tissue with active invasion of the inflamed synovial tissue (pannus tissue) into the cartilage is considered to be the basis of joint destruction (Gay, 1993).
  • Numerous immunologically specific and nonspecific cellular reactions have been described as well as inflammation mediators, corresponding receptors, cartilage-destroying enzymes and increasingly also intracellular control loops and activation routes.
  • Osteoarthritis is seen primarily as a result of wear and tear with joint destruction from the cartilage, in which synovitis is interpreted as a secondary induced process.
  • the differentiation between the two diseases is made on the basis of anamnestic and clinical assessment, systemic inflammation parameters and imaging tests. Histological methods are only used secondarily, and there are currently no reliable molecular biological parameters. As a result, there is currently no adequate way to check the therapeutic influence and effectiveness of the drugs used or physical measures at the molecular level.
  • the invention has for its object to develop test systems that come close to the conditions in healthy as in diseased tissue using the example of the joint.
  • the task was solved by providing an in vitro cell interaction system. It consists of a first cell culture with pelleted and precultivated cells and a second culture that is applied to the first culture and contains the cells that simulate a diseased or healthy tissue.
  • the first cell culture consists of pelleted and precultivated mesenchymal cells and the second culture consists of human, immortalized synovial fibroblast cell lines.
  • 2.1. In vitro pannus and in vitro interaction culture 2.1.1. Interactive cultivation technique and drug testing.
  • Cultures of the first cell or tissue type are produced by centrifuging the cells in a culture vessel as a multilayered cell cluster or by converting them into a three-dimensional arrangement through an admixed substrate such as agarose. These cultures are kept in culture for 2 to 3 weeks in order to mature and build up a corresponding tissue structure.
  • the second type of cell or tissue is applied to the first culture, for example by pipetting and / or centrifuging. The therapeutic influence can take place immediately or after an incubation period of days to weeks.
  • Either primary cartilage tissue or cells that can differentiate into cartilage are used to build up an in vitro pannus interaction tissue. You can
  • Tissues or cells of different species from individual individuals or also pooled, e.g. from pork or beef.
  • Primary cells can also be genetically modified or used by genetically mutated individuals
  • Influence in the further course can be taken up by the measuring methods described in chapter 2.2.
  • mesenchymal stem cells in the interactive pannus model opens up the possibility of establishing genetically and physiologically identical cartilage cultures, which leads to a minimization of individual differences in the response to certain therapies. Furthermore, using stem cells, not only the effects of different therapies on already developed cartilage can be examined, but rather also the effects of therapeutic agents on developing cartilage structures and tissues can be analyzed comparable to the embryonic development. This leads to the possibility of not only testing a substance with regard to its therapeutic potential, but above all making statements about the teratogenic properties of this substance. Further therapeutic options for stimulating the regenerative potential can be tested.
  • pannus This provides starting points for testing molecules that can be used in gene therapy in the model of rheumatic diseases.
  • pannus 2.1.3.1. Individual components 0 In arthritis, various cells of the synovial tissue are involved in the inflammatory process. These include local fibroblastoid cells of the synovial membrane, immigrated macrophagocytic cells, T cells, B cells, endothelial cells and granulocytes. The importance of each individual cell population for the chronification of the inflammatory process has not yet been clarified. 5 In the in vitro pannus model, the effects of individual cell components on cartilage culture can be examined.
  • Established cell lines of appropriate origin (monocytic, macrophagocytic, T-cellular, B-cellular, endothelial, fibroblastoid, etc.) or cells obtained and purified by patients can be used.
  • synovial cell component of the in vitro pannus model primary patient materials - synovial tissue - from healthy donors or from patients with osteoarthritis (OA) or rheumatoid arthritis (RA) serve as tissue culture, whereby primary cell isolations are used in the model.
  • OA osteoarthritis
  • RA rheumatoid arthritis
  • primary cell lines and immortalized (spontaneously / actively immortalized) cell lines or progenitor cell lines e.g. fibroblasts, macrophages, monocytes, granulocytes, B / T cells, dendritic cells, endothelial cells, HSE or K4IM (transfection with an SV40 T antigen expression vector), U937, Jurkat.infected cell lines
  • primary cell lines and immortalized (spontaneously / actively immortalized) cell lines or progenitor cell lines e.g. fibroblasts, macrophages, monocytes, granulocytes, B / T cells, dendritic cells, endothelial cells, HSE or K4IM (transfection with an SV40 T antigen expression vector), U937, Jurkat.infected cell lines) to be used as individual components or in a defined combination , 2.1.3.2.
  • the different cell populations of the synovial tissue can be combined step by step and their interaction with the primary
  • This culture model is based on a co-culture of different cell populations (eg chondrocytes and synovial cells), which are cultivated in a three-dimensional matrix that takes the specific cellular microenvironment into account.
  • cell populations eg chondrocytes and synovial cells
  • chondrocytes and synovial cells eg chondrocytes and synovial cells
  • essential pathogenetic cellular interactions in the process of matrix destruction in rheumatoid arthritis can be simulated and investigated under in vitro conditions (Schultz, 1997; Sittinger, US Patent 5932459; Schultz, Patent DE 19540487 AI).
  • 2.2. Analysis of the interaction culture The analysis of the interactive cell cultures aims at the physiological and pathogenetic interactions to which the cell components used in the system are mutually dependent.
  • the histomorphological changes to evaluate cell invasion are to be analyzed histochemically, immunohistochemically and by in situ hybridizations to determine gene products that support the invasion.
  • cytokine ELISA for the cytokines IL-lbeta, IL-lalpha, IL-6, TNF-alpha, IL-10, IL-4, IL-13, IL-8, IL-2, IL-5, IL-18, IL-16, IL-17 and IL-11.
  • apoptosis / necrosis Bostosis / necrosis
  • apoptosis / necrosis Bostosis / necrosis
  • TNF family interleukins, interferons, cyclooxygenase
  • inflammation TIMP, interleukins, Oncostatine
  • tissue destruction matrix metalloproteinases, Nitric oxide synthase (NOS)
  • tissue integrity cartilage matrix molecules, such as collagens, proteoglycans, hyaluronic acid metabolism, link protein , Cartilage oligomeric matrix protein
  • inorganic materials in the form of bone prostheses serve, among other things, to replace degenerated bone structures (Hofmann, 1994).
  • the interaction of inorganic materials and organically grown bone can be simulated in vitro in order to test new prosthesis materials in advance for their bone compatibility, their bio- and osteocompatibility in vitro.
  • methods for anchoring the prosthesis in the bone can be carried out by examining the surface of the prosthesis with regard to the ingrowth of bone cells, the deposition of the bone matrix and the use of osteoinductive factors for stimulating the growth of bone cells.
  • the influence of the prosthetic material on these immune-competent cells can be considered for the induction of inflammatory mediators and bone degenerative factors are analyzed in more detail under defined conditions in vitro.
  • Lung fibrosis, hepatitis, pancreatitis, inflammation and destruction of the islets of Langerhans in type I diabetes mellitus, myositids or osteomyelitis can be simulated in vitro by building up interacting three-dimensional tissue cultures as described above.
  • Chlamydia are obligatory intracellular bacteria, which are associated with reactive arthritis, among others, and which, with the participation of macrophages and synovial cells, lead to pronounced tissue damage due to infection-related apoptosis, inflammation and immunological reaction (Beutler, 1994; Sieper, 1999).
  • synovial cells, macrophages or also cartilage cells can be infected with chlamydia under defined conditions and co-cultivated with cartilage cultures or synovial cells.
  • This interactive culture allows, for example, the recording of the morphological, physiological and genetic changes of infected cells and the creation of a cell type-related cytokine secretion pattern.
  • new medications can be examined with regard to their therapeutic effectiveness on intracellular bacteria or modulation of cytokine profiles triggered by chlamydia in the cellular context of a joint environment. Similar approaches can also be used for other infection-induced arthritis models e.g. with Borrelia burgdorferi as an extracellular or, optionally, intracellularly discussed pathogen or with parvovirus B19.
  • Lipopolysaccharides can be applied in isolation to the first or second tissue culture or their cells or the entire interaction culture. Furthermore, through targeted genetic engineering Influencing one or more of the cells used, for example by overexpressing TNF-alpha, can induce or intensify an inflammatory situation.
  • the in vitro cell interaction system consists of a first cell culture from pelleted and precultivated mesenchymal cells (preferably articular chondrocytes of the pig) and from a second cell culture which is centrifuged onto the first cell culture and from human, immorehased synovial fibroblast cell lines (preferably "HSE or K4IM , Haas, 1997 "). It is suitable for the investigation of the mode of action of biological and chemical substances, biomaterials, mechanical-physical influences and gene transfer systems for therapeutic purposes.
  • the in vitro cell interaction system is made using - mixed substrates (preferably agarose, alginate, fibrin, hyaluronic acid, resorbable and non-resorbable biomaterials) and / or a three-dimensional matrix of resorbable or non-resorbable biomaterials previously introduced into the culture vessel instead of pelleting. and / or centrifugation technology
  • - mixed substrates preferably agarose, alginate, fibrin, hyaluronic acid, resorbable and non-resorbable biomaterials
  • / or a three-dimensional matrix of resorbable or non-resorbable biomaterials previously introduced into the culture vessel instead of pelleting. and / or centrifugation technology
  • chondroid cell lines From native cartilage, chondroid cell lines, chondroid tumor cell lines, primary chondrocytes, chondrogen induced mesenchymal stem cells, chondrogen induced embryonic stem cells, chondrogen induced primary cells, chondrogen induced and immortalized primary cells instead of mesenchymal cells
  • fibroblastoid preferably human foreskin fibroblasts
  • macrophagocytic preferably Mono Mac 6
  • dendritic preferably U937
  • lymphocytic preferably Jurkat, EBV-transformed B cells
  • endothelial preferably HUVEC
  • a first cell culture with the properties of an organ system in vitro by using appropriate primary organ cells, derived cell lines or stem cells, which are differentiated into the corresponding organ cell instead of mesenchymal cells - from cell cultures with the Properties to induce infiltration, destruction and / or inflammation of the first cell culture (preferably by using primary tumor cells, tumor cell lines or cell lines or progenitor cell lines with fibroblastoid, macrophagocytic, granulocytic, dendritic, monocytic, lymphocytic human or endothelial combination instead of human or endothelial combination immortalized synovial fibroblast cell lines).
  • the in vitro cell interaction system is added with the addition of pathogens (bacteria, viruses, protozoa, parasites, prions) or pathogenicity-imparting substances including endogenous inflammation and destruction mediators (preferably cytokines, arachidonic acid metabolites, autoantigens, hormones or matrix-degrading enzymes) Derivatives (preferably peptides, protein fragments or fusion proteins), foreign inflammation and destruction mediators (preferably lipopolysaccharides, lectins, foreign antigens of bacteria, viruses, protozoa, parasites, prions), chemical substances or dimerizing mediating substances - preferably for dimerizing receptors, such as quinazolines for EGFR (epidermal growth factor receptor) or Tomudex for Bax proteins.
  • pathogens bacteria, viruses, protozoa, parasites, prions
  • pathogenicity-imparting substances including endogenous inflammation and destruction mediators (preferably cytokines, arachidonic acid metabolites, autoantigens
  • the in vitro cell interaction system according to the invention is equally suitable in its use for identifying new therapeutic targets.
  • Example 1 Rheumatoid Arthritis (In Vitro Pannus) The in vitro pannus is characterized in that aggressive cells from the
  • the cartilage cell tissue is made from primary chondrocytes, obtained from porcine articular cartilage. During the cultivation in the form of pellet cultures, these cells are brought into a 3-D structure with the formation of new extracellular matrix.
  • synovial tissue cell cultures Cultivated synovial tissue cell cultures.
  • the synovial cells in suspension are centrifuged directly onto the chondrocyte pellet cultures.
  • This three-dimensional interactive cell culture system consisting of chondrocytes with synovial cells can be used with regard to the pathogenetically relevant cellular interactions based on the information in Chapter 2.2 compiled investigation methods are analyzed.
  • Example 2 Bones and implantable materials
  • three-dimensional artificial bone tissue is produced, attached to or around the prosthesis material and cultivated in vitro.
  • Increased cancellous cells, periosteal cells or undifferentiated mesenchymal stem cells from the bone marrow, blood or fat are used for the production of artificial bone tissue.
  • These cells are fixed in three-dimensional support structures with the help of fibrinogen / thrombin solution or collagen matrix and pre-cultivated under the influence of dexamethasone or osteogenic factors (e.g. BMP) for two weeks in vitro or directly cultured with the prosthesis material in vitro for four weeks.
  • Pre-cultured bone tissue is attached to the prosthesis material to be tested using fibrinogen / thrombin solution or collagen matrix and cocultivated for two weeks.
  • the bone-implant interactions are analyzed in accordance with Chapter 2.2. represented patterns with special consideration of bone-relevant gene families.
  • BMP Bone Morphogenetic Protein
  • EBV Epstein-Barr Virus
  • EGFR epidermal growth factor receptor
  • ELISA enzyme linked immunosorbent assay
  • DNA deoxyribonucleic acid
  • DNA deoxyribonucleic acid
  • HL-60 HL-60 a promyelocytic cell line derivative, see S.J. Collins, et al. Peripheral blood leukocytes were obtamed by leukopheresis from a 36-year-old Caucasian female with acute promyelocytic leukemia • HSE: SV40 large T antigen immorhased human synovial fibroblast cells from a normal donor (Haas C, Aicher WK, Dinkel A, Peter HH, Eibel H)
  • HUVEC human umbilical vein endothelial cells - umbilical vein endothelial cells
  • IAP family inhibitor of apoptosis proteins
  • IL Interleukin
  • K4IM SV40 large T antigen immortalized human synovial fibroblast lines from a patient with rheumatoid arthritis (Haas C, Aicher WK, Dinkel A, Peter HH, Eibel H: Characterization of SV40T antigen immortalized human synovial fibroblasts: maintained expression patterns of EGR -1, HLA-DR and some surface receptors)
  • MALDI-TOF matrix-assisted laser desorption / ionization time-of-flight
  • MMP matrix metalloproteinase
  • MonoMac 6 human monocyte cell line
  • RA Rheumatoid Arthritis • PCR: polymerase chain reaction - polymerase chain reaction
  • S V40 large T antigen Simian Virus 40 (S V40) belongs to a group of DNA tumor viruses, large T antigen is an early viral oncoprotein TIMP: tissue type inhibitor of metalloproteinases TNF: tumor necrosis factor • U937: s. Sundstrom and Nilsson 1974: from malignant cells obtained from the pleural effusion of a patient with histiocytic lymphoma, can be induced to terminal monocytic differentiation by supernatants from human mixed lymphocyte cultures, phorbol esters, vitamin D3, gamma interferon, tumor necrosis factor (TNF) and, retinoic acid. bibliography
  • Boss, J.H. et al. The nature of the bone-implant interface. The lessons learned from implant retrieval and analysis in man and experimental animal. Med.Prog.Technol. 20: 119-142, 1994

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Abstract

L'invention concerne un système de culture à interaction cellulaire <i>in vitro</i> pour l'essai et le développement de médicaments, qui est constitué d'une première culture cellulaire, à savoir une culture de cellules mésenchymales sous forme de culot et précultivée (de préférence des chondrocytes articulaires de porc), et d'une seconde culture cellulaire, culture qui est appliquée sur la première culture cellulaire par centrifugation et est constituée de lignées cellulaires de fibroblastes synoviaux humaines immortalisées (culture tissulaire tridimensionnelle-ingéniérie tissulaire). Ce système de culture peut être utilisé pour analyser le mode d'action de substances biologiques et chimiques, d'influences mécano-physiques et de systèmes de transfert géniques à des fins thérapeutiques qui se rapprochent extrêmement des conditions régnant <i>in vivo</i> dans des tissus sains et dans des tissus chroniquement malades, tels que ceux d'une articulation, et qui peuvent être utilisés pour l'identification de nouveaux points d'attaque thérapeutique.
PCT/DE2002/000353 2001-01-31 2002-01-28 Systeme de culture a interaction cellulaire in vitro pour l'essai et le developpement de medicaments WO2002061420A2 (fr)

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DE10105420A DE10105420B4 (de) 2001-01-31 2001-01-31 In vitro Zellinteraktionskultursystem für Medikamententestung und -entwicklung
DE10105420.3 2001-01-31

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1648389A2 (fr) * 2003-07-28 2006-04-26 The Board Of Trustees Of The University Of Illinois Genie biologique de structures articulaires contenant le cartilage et l'os
CN110484495A (zh) * 2019-08-23 2019-11-22 江南大学 一种从老年人体滑膜关节软骨分离培养细胞的方法

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US5932459A (en) * 1995-10-20 1999-08-03 Sittinger; Michael Artificial tissues, methods for the production and the use thereof

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US5932459A (en) * 1995-10-20 1999-08-03 Sittinger; Michael Artificial tissues, methods for the production and the use thereof
WO1998006825A1 (fr) * 1996-08-08 1998-02-19 KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG Tissu rhumatismal artificiel de type pannus et procede diagnostic pour mettre l'arthrite rhumatoide en evidence

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Title
SCHULTZ O ET AL: "DEVELOPMENT OF AN ARTEFICIAL PANNUS MODEL FOR DESTRUCTIVE JOINT DISEASES" ARTHRITIS AND RHEUMATISM, LIPPINCOTT, PHILADELPHIA, US, Bd. 39, Nr. 9, 1. September 1996 (1996-09-01), Seiten S-36, XP002050182 ISSN: 0004-3591 *
SCHULTZ O ET AL: "DEVELOPMENTS OF IN VITRO MODEL SYSTEMS FOR DESTRUCTIVE JOINT DISEASES" ARTHRITIS AND RHEUMATISM, LIPPINCOTT, PHILADELPHIA, US, Bd. 40, Nr. 8, 1. August 1997 (1997-08-01), Seiten 1420-1428, XP002050183 ISSN: 0004-3591 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1648389A2 (fr) * 2003-07-28 2006-04-26 The Board Of Trustees Of The University Of Illinois Genie biologique de structures articulaires contenant le cartilage et l'os
EP1648389A4 (fr) * 2003-07-28 2006-11-29 Univ Illinois Genie biologique de structures articulaires contenant le cartilage et l'os
CN110484495A (zh) * 2019-08-23 2019-11-22 江南大学 一种从老年人体滑膜关节软骨分离培养细胞的方法
CN110484495B (zh) * 2019-08-23 2021-03-09 江南大学 一种从老年人体滑膜关节软骨分离培养细胞的方法

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DE10105420A1 (de) 2002-08-14
WO2002061420A3 (fr) 2002-09-26

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