EP1290144A2 - Infection model - Google Patents
Infection modelInfo
- Publication number
- EP1290144A2 EP1290144A2 EP01947324A EP01947324A EP1290144A2 EP 1290144 A2 EP1290144 A2 EP 1290144A2 EP 01947324 A EP01947324 A EP 01947324A EP 01947324 A EP01947324 A EP 01947324A EP 1290144 A2 EP1290144 A2 EP 1290144A2
- Authority
- EP
- European Patent Office
- Prior art keywords
- cells
- human
- vitro
- tissue
- animal
- 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- Withdrawn
Links
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Classifications
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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
- A61L27/3895—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 using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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- A61K31/33—Heterocyclic compounds
- A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
- A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
- A61K31/4196—1,2,4-Triazoles
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
- A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin
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- A—HUMAN NECESSITIES
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- 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
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- 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
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- C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
- C12N5/0655—Chondrocytes; Cartilage
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- 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
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- G—PHYSICS
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- 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
- G01N33/5082—Supracellular entities, e.g. tissue, organisms
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- A—HUMAN NECESSITIES
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- 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
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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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- 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
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2503/00—Use of cells in diagnostics
- C12N2503/04—Screening or testing on artificial tissues
- C12N2503/06—Screening or testing on artificial skin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/52—Fibronectin; Laminin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2533/00—Supports or coatings for cell culture, characterised by material
- C12N2533/50—Proteins
- C12N2533/54—Collagen; Gelatin
Definitions
- the invention relates to means and methods for the analysis and diagnosis of infections caused by pathogenic and / or parasitic microorganisms and / or; Diseases of the human or animal body, means and methods for analyzing and diagnosing degenerate human and animal cells, means and methods for analyzing and diagnosing genetically modified human and animal cells, and means and methods for examining and testing anti-infectives and pharmaceuticals against Tumors, in particular cytostatics, and three-dimensional animal in vitro organ and tissue models, in particular tissues susceptible to infection, such as the intestine, skin, cornea, trachea and mucous membranes.
- Macroorganisms for example humans, can be infected by a large number of microorganisms, which include both prokaryotic organisms, such as bacteria, and eukaryotic organisms, such as fungi and protozoa, and also viruses in the broader sense. This can lead to very different consequences for an affected macroorganism. For example, infection by microorganisms can lead to the development of an infectious disease. Other microorganisms live in or on the host organism, which means that they live at the expense of theirs Host organism without seriously becoming ill. On the other hand, some viruses, especially oncogenic viruses, are known to be able to neoplastic transform human or animal cells in vivo, some being associated with the formation of degenerate cells and tumor pathogenesis.
- the infection of a human or animal organism by a microorganism comprises several steps, such as the transfer of the microorganism to its host organism, the adherence (adhesion) of the microorganism to cells or tissues of the host organism, the penetration of the microorganism (invasion or penetration) into spe - specific cells or tissues of the host organism and the multiplication of the microorganism therein.
- Infection is determined by the infectious properties, for example the transferability, contagiosity, adherence, penetration and reproductive ability, and the pathogenic properties of the microorganism.
- the development and course of an infectious disease is also determined by the susceptibility and immunity of the infected host organism.
- the mechanisms of an infection are very different.
- the development and course of a tumor disease also depend on the susceptibility and immunity of the host organism concerned. It is known that viruses preferentially lead to the formation of neoplasias in immunocompetent organisms.
- Anti-infectives used against infectious diseases that damage or kill microorganisms in the body of the affected organism are aimed at inhibiting the cell wall synthesis of the microorganism, disturbing the permeability of its cytoplasmic membrane, blocking its protein synthesis and / or suppressing its nucleic acid synthesis without, however, harming the host organism itself.
- the actual causative agent, the oncogenic virus cannot currently be targeted, but the degenerated cell is destroyed or its growth is inhibited, for example by using cytostatic agents.
- the technical problem on which the present invention is based is therefore means and methods for analyzing and diagnosing infections caused by pathogenic or parasitic microorganisms in human or animal host organisms, means and methods for analyzing and diagnosing degenerate or genetically modified human or to develop animal cells and means and methods for examining and testing diagnostic and therapeutic agents, in particular anti-infectives and anti-tumor drugs, in particular cytostatics, which overcome the above-described disadvantages in the prior art and which are particularly suitable for To investigate the development of infection-causing interactions between pathogenic or parasitic microorganisms and their target organs or tissues and the cellular and / or viral mechanisms leading to tumor formation, and the development and testing of these mechanisms allow specific diagnostic and therapeutic agents, such as anti-infectives or cytostatics.
- the invention solves the technical problem on which it is based by providing in vitro methods for differentiating and / or multiplying isolated animal and human cells, in the course of which three-dimensional animal or human in vitro organ or tissue models are produced.
- in vitro methods for differentiating and / or multiplying isolated animal and human cells in the course of which three-dimensional animal or human in vitro organ or tissue models are produced.
- primary cells or other cells from tissues or organs susceptible to infections such as the intestine, skin, cornea, trachea or mucous membranes, but also degenerate cells to be checked, in particular the organs and tissues mentioned above, or genetically modified cells to be checked , in particular the organs and tissues mentioned above, are used.
- the present invention provides in vitro methods for cocultivating the differentiated and / or increased cells with pathogenic or parasitic microorganisms and in vitro methods for investigating the interaction of substances with the in vitro co - Cultured differentiated and / or propagated cells and microorganisms ready. These methods allow the analysis of infection processes and the provision of diagnostic and therapeutic agents useful in the field of infection medicine.
- the results obtained with the organ test systems according to the invention can be more meaningful than the results determined with animal experiments and can ensure better transferability to humans.
- the cells described above are cultivated according to the invention in a three-dimensional, gel-like, connective tissue-like biomatrix and can multiply there.
- this biomatrix contains a framework of collagen constituted by a collagen solution, i.e. tissue-typical matrix proteins.
- tissue-typical matrix proteins i.e. tissue-typical matrix proteins.
- other cell types preferably other primary cells, can be applied to this cell-containing collagen gel.
- the cells contained in the biomatrix and the other cell types applied to the biomatrix can undergo a differentiation into a multi-layered three-dimensional animal tissue or organ test model.
- the in vitro organ or tissue test models according to the invention can be inoculated with a parasitic or pathogenic microorganism and, together with this, under suitable ones Conditions are cultivated.
- the cocultivation of the animal in vitro tissue or organ test system according to the invention with a parasitic or pathogenic microorganism offers the possibility of studying both the infection process itself and the defense reaction of the corresponding organoid cell systems. For example, larger amounts of an infected cell material and the pathogen itself can be obtained.
- the material obtained can then be further analyzed using conventional histological, biochemical, molecular biological or immunological methods, for example to determine morphological changes in infected cells, the release of specific substances by the pathogen, such as toxins or proteins relevant to resistance, or the release of specific substances by infested cells
- pathogen such as toxins or proteins relevant to resistance
- specific substances by infested cells To study cells, such as interleukins, more closely as a defense reaction or to create transcription and / or expression profiles, on the basis of which, for example, virulence factors can be identified as targets for the development of anti-infectives.
- the above-mentioned procedure according to the invention preferably allows the screening and analysis of potential diagnostics, with the help of which, for example, the presence of symptoms of infection can be demonstrated.
- the invention therefore also relates to methods for identifying diagnostic agents or analyzing their specificity, with a potential or testable diagnostic agent for its being included in the co-cultivation of the single- or multi-layered in vitro tissue or organ test systems according to the invention with infectious agents Ability to recognize infections or infection processes is tested.
- the diagnostics to be tested can be added to the system according to the invention, it being possible to determine to what extent there is a correlation between the state of infection and the marking or detection by the diagnostics on the basis of morphological, physiological or other parameters.
- the methods according to the invention and the agents used in them can be used to screen for potential active substances and to investigate properties such as specificities and interactions of active substances with target cells.
- active substance means any substance, but also other agents, including physical parameters such as electromagnetic radiation, radioactivity, heat, sound or the like, which can influence or recognize biological cells or parts thereof, in particular cell organelles , Such active substances can be, in particular, of a chemical nature, for example diagnostic or therapeutic agents.
- diagnostic agents are understood to mean any substances which 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.
- therapeutic agents is understood to mean in particular those substances which can be used either prophylactically or accompanying the disease in order to avoid, alleviate or eliminate disease states.
- diseases also understood states such as unnatural states of mind, pregnancies, signs of aging, developmental disorders or the like.
- therapeutic agents are also understood to mean those substances which have only or also cosmetic effects.
- the methods according to the invention are also suitable for examining the mechanisms of tumor pathogenesis and / or for examining substances for their suitability as medicaments, for example against a specific tumor.
- an in vitro organ or tissue test system constructed from degenerate cells, in particular the organs or tissues mentioned above, can be used to obtain larger amounts of a degenerate cell material.
- the material obtained can then be further analyzed using conventional methods, for example histological, biochemical, molecular biological or immunological methods, in order, for example, to examine morphological changes in degenerate cells or the release of specific substances more precisely or to create transcription and / or expression profiles.
- an in vitro organ or tissue test system constructed from degenerate cells can be co-cultivated with oncogenic viruses.
- the propagation and / or The spread of oncogenic viruses in the cells of the in vitro test system in the presence and absence of specific substances that can inhibit specific functions of the virus are examined.
- the methods according to the invention can also be used to check genetically modified cells, in particular the above-mentioned tissues and organs.
- cells can be tested that have been genetically modified with a view to gene therapy to eliminate gene-related malfunctions or to restore normal gene functions in diseases of the above-mentioned organs.
- a preferred embodiment of the invention comprises the cultivation of animal or human cells in a three-dimensional gel-like biomatrix to multiply these cells and to produce a three-dimensional animal or human in vitro organ or tissue test system.
- the invention comprises the cultivation of human dermal fibroblasts in the biomatrix to produce a three-dimensional human in vitro skin equivalent consisting of dermis equivalent and epidermis equivalent.
- the term “culturing cells” means maintaining the vital functions of cells, for example fibroblasts, in a suitable environment, preferably in vitro, for example with the addition and removal of metabolic educts and products, in particular also an increase in the cells.
- dermal fibroblasts are understood to mean naturally occurring fibroblasts, particularly those found in the dermis, or genetically modified fibroblasts or their precursors. Fibroblasts are the precursors of dermal fibrocytes. The fibroblasts can be of animal or human origin.
- the biomatrix provided for the cultivation of the fibroblasts contains the fibroblasts to be cultivated and a collagen scaffold newly constituted from a, preferably fresh, collagen solution at a concentration of preferably 3.5 to 4.5 mg collagen per ml biomatrix.
- the collagen structure is obtained from a, preferably cell-free, acidic solution of collagen I, the protein concentration of the collagen solution preferably being 5 to 7 mg / ml.
- the pH of the collagen solution is preferably 3.8.
- the collagen solution is preferably at 4 ° C.
- This mixture is gelled by raising the temperature to room temperature or 37 °. After the gels have gelled, fibronectin is added to the gels.
- the function of fibronectin in vivo consists in binding to other macromolecules, for example collagen, and in attaching cells to neighboring cells.
- the subsequent cultivation of the fibroblasts in the collagen gel is preferably carried out in submerged culture.
- a “submerged culture” is understood to mean a method for culturing cells, the cells being covered with a nutrient solution.
- the biomatrix containing fibroblasts is therefore covered with a layer of cell culture medium and incubated at 37 ° C.
- keratinocytes are understood to mean cells of the epidermis that form keratinized squamous epithelium, or genetically modified keratinocytes or their precursors, which can be of animal or human origin.
- the keratinocytes sown on the collagen gel are preferably as possible Pre-cultivated, undifferentiated keratinocyte stem cells from human biopsy tissue, ie cytokeratin 19- or integrin ßl-positive basal stem cells.
- the keratinocytes are sown on the biomatrix in a cell culture medium, preferably in KBM medium (clonetics), which is 5% fetal
- KBM medium human epidermal growth factor
- hEGF human epidermal growth factor
- BPE bovine pituitary gland extract
- an “airlift culture” is understood to mean a culture in which the height of the nutrient medium level is matched exactly to the height of the biomatrix, while the keratinocytes or the cell layers formed by the keratinocytes lie above the nutrient medium level and not from the nutrient medium that is, the cultivation takes place at the boundary layer air-nutrient medium, whereby the cultures are supplied with nutrients from below.
- a skin-typical, dermis equivalent and epidermis equivalent develops existing in vitro full skin model which can be used advantageously for the test methods according to the invention.
- the invention therefore also relates to a skin-typical in vitro whole skin test model, in particular animal or human in vitro whole skin test model, which was produced by the method according to the invention and which comprises at least 2 to 4 proliferative, some differentiating and at least 4 to 5 horny cell layers, wherein the epidermis equivalent stratum basale, stratum spinosum, stratum granulosum and stratum corneu, and wherein between the dermis equivalent and the epidermis equivalent there is a functional basement membrane made of matrix proteins.
- This model is ideal as a test system for the investigation of potential or actual active substances. such as therapeutics, diagnostics or for investigations into the course of infection processes.
- Another particularly preferred embodiment of the invention comprises the cultivation of intestinal fibroblasts in the biomatrix for producing a three-dimensional human in vitro intestinal test system consisting of preferably Caco2 cells or also intestinal epithelial cells or other human cell lines.
- intestinal fibroblasts are understood to mean naturally occurring fibroblasts, in particular intestinal tissue, or genetically modified fibroblasts or their precursors.
- the intestinal fibroblasts can be of animal or human origin.
- intestinal epithelial cells are understood to mean naturally occurring epithelial cells, in particular in the intestinal tissue, or genetically modified epithelial cells or their precursors.
- the intestinal epithelial cells can be of animal or human origin.
- the collagen solution is mixed in a volume ratio of 1: 1, preferably at 4 ° C., with a solution also referred to as a gel solution, containing a, preferably 2-fold, cell culture medium, buffer, preferably He pes buffer and serum, preferably 10% serum, and preferably 1.5 x 10 5 / ml intestinal fibroblasts, especially pre-cultivated intestinal fibroblasts, mixed and mixed well.
- a gel solution with a concentration of x is present
- the collagen solution is preferably mixed in a volume ratio (xl): 1 with the gel solution, where x is the concentration factor. This mixture is gelled by raising the temperature to room temperature or 37 °.
- the subsequent cultivation of the intestinal fibroblasts in the collagen gel is preferably carried out in submerged culture.
- the biomatrix containing fibroblasts is incubated at 37 ° C.
- Intestinal epithelial cells are seeded onto the gel, preferably 1 to 3 days after incubation of the gels.
- the intestinal epithelial cells sown on the collagen gel are preferably pre-cultivated, undifferentiated intestinal epithelial cells.
- the intestinal epithelial cells are sown on the biomatrix in a cell culture medium, preferably in DMEM medium (Dulbecco's Modified Eagle Medium, Life Technologies, Cat. No. 41966 or 52100), the 10% FCS and glutamine (2 mM) and 1% non-essential amino acids (MEM, Life Technologies, Cat. No. 11140).
- a further advantageous embodiment of the invention comprises the cocultivation of a three-dimensional in vitro organ or tissue test system produced according to the invention with a pathogenic or parasitic microorganism.
- pathogenic or parasitic microorganisms are understood to mean both eukaryotic and prokaryotic microorganisms, such as bacteria, fungi, protozoa, vitoids, but also prions or viruses, which attack a macroorganism, in particular a human or animal organism, and which live in or on tissues of this organism and can lead to an infection of this organism, but need not necessarily lead to it.
- the term “cocultivation” means a, preferably In vitro simultaneous maintenance of the vital functions of animal cells and microorganisms in the same environment suitable for both, for example with the addition and removal of metabolic products and products, in particular also a simultaneous multiplication of the cells and the microorganisms.
- the human pathogenic fungus Candida albicans is cocultivated with the human in vitro skin test system produced in accordance with the invention in order to investigate the infection process of Candida on human skin tissue and with the human in vitro intestinal test system produced in accordance with the invention around the Infection process of Candida on human intestinal tissue to investigate.
- the results achieved with Candida in particular the detailed description of the infection process, can also be transferred to other pathogens.
- the present invention relates to the cocultivation of the human pathogenic microorganism Candida albicans with the human in vitro skin test system or the human in vitro intestinal test system in order to increase the first stage of the infection process, namely the adhesion of the pathogen to skin or intestinal cells investigate.
- the adhesion of the pathogen is examined using the virulent Candida strain Sc5314 and the avirulent Candida strain Can34, which have already been investigated in a mouse-macrophage model (Lo et al., Cell, 90 (1997), 939- 949).
- the in vitro skin test system or the in vitro intestinal test system is inoculated in submerged culture with about 10 3 pathogenic organisms in each case and cultivated with shaking.
- aliquots are removed and plated on Petri dishes with the appropriate nutrient media, for example YPD full medium (Difco). After an appropriate incubation period, the number of colonies on the Petri dishes is determined.
- the adhesion of the pathogens to the in vitro organ test systems can be determined on the basis of the comparison between the determined number of colonies and the originally inoculated number of pathogens. With the help of this method it could be shown that the virulent strain has the ability to probably has skin as well as intestinal cells, whereas only slight adhesion could be detected in the avirulent stem.
- the present invention the co-cultivation of human pathogenic microorganism Candida albicans with the inventively produced human in vitro skin test system relates respectively to the inventively produced human in vitro intestinal test system to a further stage of the infection process, namely the penetration / Invasion of the pathogen into cells.
- the organoid tissue test systems are co-cultivated in the airlift process with the avirulent and virulent pathogen strains described above.
- the pathogen is preferably fixed at 10 3 / ml in 1% agar and agar pieces with a diameter of 4 mm are placed on the organoid tissue test systems for a maximum of 98 hours.
- the penetration of the pathogen into the organoid structures is examined after 16 hours, 24 hours, 72 hours, 86 hours and 98 hours by means of histological methods on thin sections, using the PAS staining method (Mc Manaus, Romeis, 17th edition, page 393) , Using the histological sections, the invasion process of the virulent Candida strain down to the deeper layers of the connective tissue-like matrix can be documented.
- the term “agent” is intended in particular to include chemical, biological or physical agents, such as light or heat, which can have a potential effect on living cells.
- the studies on the action of anti-infectives have also been carried out with Candida there are two classes of substances, namely azoles and polyenes, which are preferably used as antifungals against systemic infections. Both classes of substances have disadvantages.
- the polyenes have strong side effects and azoles develop more and more resistance (DiDomenico, Curr. Opin. Microbiol. , 2 (1999), 509-515; Georgopapadakou, Curr. Opin. Microbiol., 1 (1999), 547-557. For this reason, the targeted development of new antifungals is urgently required.
- the method for examining the adhesion of Candida to the in vitro intestinal test system and the in vitro skin test system according to the invention is modified in such a way that the cocultivation approach contains an antifungal, in particular amphothericin B or fluconazole. With the help of this method it could be shown that both antifungals had no influence on the adhesion of the pathogen, but on its growth rates.
- the method for examining the penetration / invasion of Candida on the in vitro intestinal test system and the in vitro skin test system is modified such that the cocultivation approach contains amphothericin B or fluconazole. With the help of this method it could be shown that the invasion of the virulent pathogen strain could only be prevented by a complete inhibition of growth, whereby new active substances were also tested according to the invention.
- a particularly preferred embodiment of the invention comprises the analysis of degenerate cells.
- the term “degenerate” encompasses all changes in a normal cell, for example cell polymorphism, anisocytosis, nuclear polymorphism, polychromasia, disturbed nuclear plasma relation and aneuploidy, which lead to an impaired differentiation or to a dedifferentiation and to a deregulated growth of the Cell and malignant tumors are particularly affected, and degenerate cells, particularly the organs or tissues mentioned above, are used to build an in vitro organ or tissue test system to recover large amounts of the degenerate cell material, using conventional methods , for example histological, biochemical, molecular biological or immunological methods, are further analyzed in order to investigate the release of specific substances and to create transcription and expression profiles.
- the in vitro organ or tissue test system built up from degenerate cells is used for d he effect of drugs and of substances potentially suitable as drugs, in particular with regard to their ability to inhibit cell division.
- patient-specific degenerate cells are used to establish an in vitro organ test system in order to investigate therapeutic options for the specific tumor disease of the patient.
- the checking of genetically modified cells in particular the above-mentioned tissues and organs.
- genetically modified cells encompasses all cells which have been manipulated with the aid of genetic engineering methods, whereby either foreign DNA has been introduced into the cell or the own DNA has been modified, for example by deletions, inversions and attachments.
- it is provided to test genetically modified cells in vitro with a view to gene therapy of patient-specific diseases, in particular for their functionality, an in vitro organ test system being established using such genetically modified cells.
- the invention also relates to a, preferably gel-like, biomatrix in which the aforementioned cultivation processes can be carried out, specifically a biomatrix with cells of a tissue type.
- a biomatrix with cells of a tissue type can, as described above, be used to produce an in vitro organ or tissue test system.
- a biomatrix is understood to mean a gel structure which contains collagen, cell culture medium, serum and buffer, for example Hepes buffer.
- the collagen solution which is used for the preparation of the biomatrix is a solution which contains a high proportion of undenatured, native collagen in an acidic, aqueous medium, preferably with a pH of 3.8, for example in acetic acid. preferably in 0.1% acetic acid solution.
- a high proportion of undenatured collagen means a proportion of the total collagen in solution of
- no lyophilized collagen is used.
- the collagen content of the solution is advantageously 3 mg collagen per ml solution to 8 mg collagen per ml solution, more preferably 5 mg collagen per ml solution to 7 mg collagen per ml solution, most preferably 6 mg collagen per ml solution.
- collagen is preferably used, the strength after isolation, for example from rats tails in 0.1 "% acetic acid was incubated for three to fourteen days at 4 ° C under stirring and wherein non-dissolved collagen parts were removed by centrifugation.
- Fetal calf serum (FCS) or human antologous serum is preferably used as the serum and, for example, Hepes buffer as the buffer.
- the pH of the solution of cell culture medium, buffer and serum is 7.5 to 8.5, for example 7.6 to 8.2, in particular 7.8.
- the biomatrix can contain further factors, for example growth factors, adhesive agents, antibiotics, selection agents and the like.
- the invention therefore also relates to methods for producing a biomatrix containing cells, wherein in a first step fresh collagen, for example from rat tails, is produced by collagen fibers isolated from collagen-containing tissue being collected in buffer solution, disinfected superficially in alcohol and then washed in buffer solution and then in an acidic solution with a pH of 0.1 to 6.9, preferably 2.0 to 5.0, particularly preferably 3.0 to 4.0, in particular 3.3, for example a 0.1% solution Acetic acid solution to be transferred.
- a first step fresh collagen, for example from rat tails, is produced by collagen fibers isolated from collagen-containing tissue being collected in buffer solution, disinfected superficially in alcohol and then washed in buffer solution and then in an acidic solution with a pH of 0.1 to 6.9, preferably 2.0 to 5.0, particularly preferably 3.0 to 4.0, in particular 3.3, for example a 0.1% solution Acetic acid solution to be transferred.
- the collagen in the solution is stirred at 2 to 10 ° C, in particular 4 ° C, for a few days, for example 3 to 14 days, the undissolved collagen components are centrifuged off and a collagen solution with a collagen content of 3 mg / ml to 8 mg / ml at 2 to 10 ° C, for example 4 ° C. It is of course possible to temporarily store the solution in a frozen state, for example at -10 ° C to -80 ° C, in particular -20 ° C.
- a solution also referred to as a gel solution, of preferably multi-fold (x-fold) concentrated cell culture medium, serum and buffer mixed with precultivated and centrifuged cells, preferably 1 ⁇ 10 5 to 2 ⁇ 10 5 cells per ml, preferably 1 , 5 x 10 5 cells per ml, can be used.
- This solution or suspension with a pH of 7.5 to 8.5, preferably 7.6 to 8.2, in particular 7.8, is then, for example in a ratio of 1: 2, with the aforementioned collagen solution at 2 to 10 ° C, especially 4 ° C, mixed.
- the mixing ratio (volume) of collagen solution to gel solution is preferably 1: 1, with a volume ratio of (x-1): 1 collagen solution to gel solution being preferred for x-fold concentrated gel solution.
- the gel solution is then pipetted into culture vessels and, after gelling, overlaid with medium at 37 ° C.
- the biomatrix is then cultivated for at least 2 days and then cells of other tissue types, for example also immune system cells, can be applied to it.
- FIG. 1 shows a longitudinal section of the in vitro intestinal system produced according to the invention.
- FIG. 2 shows a longitudinal section of the in vitro intestinal system produced according to the invention with adherent cells of the Candida albicans fungus (adhesion of a virulent strain).
- FIG. 3 shows a longitudinal section of the in vitro intestinal system produced according to the invention with penetrated cells of the Candida albicans fungus (invasion of a virulent strain).
- Tissue containing collagen such as rat tail tails
- rat tail tails is used to prepare a collagen solution. All work is carried out under sterile conditions with sterile materials.
- the rat tails are surface disinfected at -20 ° C with 70% alcohol.
- the skin of the rat tails is pulled off and the individual collagen fibers are pulled out. If other starting tissues are used, existing cells can be gently removed by mechanical, enzymatic or chemical treatment.
- the collagen fibers are collected in phosphate buffered saline (PBS) (pH 7.2), surface disinfected in 70% alcohol for 10 min and then washed thoroughly with PBS.
- the weight of the fibers is determined and the fibers are transferred to a 0.1% acetic acid solution (final concentration about 8 to 12 mg / ml).
- PBS phosphate buffered saline
- fibronectin 5 ⁇ g / ml
- 1 ml of M199 medium is added to each well, the inserts being overlaid with the medium.
- the fibroblasts contained in the gel are subjected to this submerged cultivation at 37 ° C. for about 1 to 2 days, the medium being exchanged for fresh medium after every 12 hours.
- the medium in the wells of the microtiter plate and the gels is carefully sucked off. Then 'per well 500 ul KBM medium (clone tics) containing 5% FCS was added. The gels are coated with 50 ⁇ l fibronectin solution and incubated for 1 hour at 37 ° C. Then 100,000 keratinocytes per gel are sown in 50-100 ⁇ l of KBM medium containing 5% FCS and incubated at 37 ° C. for 1 to 2 hours.
- the gels are transferred to a 6-well plate and 1.5 to 2 ml of KBM medium with a CaCl 2 content of 1.88 mM without hEGF and BPE are added to each well, the level of the medium being exactly at the level of the gel is adjusted, while the keratinocytes or the layers formed by keratinocytes are not covered by the medium. Airlift cultivation will continue for at least 12 to 14 days.
- 7.5 ml of collagen solution are placed in a 50 l centrifuge tube and placed on ice.
- Pre-cultivated fibroblasts are harvested and counted.
- 1.2 x 10 6 fibroblasts are in 7.5 ml ice-cold gel solution taken up, well suspended and added to the collagen solution without air bubbles.
- Collagen solution and gel solution with fibroblasts are mixed well.
- 300 ⁇ l of the mixture are carefully poured into the recess of an insert.
- the inserts are in a 24-well microtiter plate.
- the mixture is gelled by incubation at 37 ° C. for two minutes. After the mixture has gelled, 1 ml of medium are placed on and next to each insert.
- the fibroblasts contained in the gel are subjected to this submerged cultivation at 37 ° C. for about 1 to 3 days, the medium being exchanged for fresh medium after every 48 hours.
- the medium in the wells of the microtiter plate and the gels is carefully sucked off. Then 200,000 epithelial cells are sown in 200 ⁇ l DMEM medium (compare above) containing 10% FCS per gel, about 600 ⁇ l medium are added to the inserts and cultured at 37 ° C. for 10 to 20 days. The medium is changed every 48 hours.
- Example 3 An intestinal equivalent produced in this way is shown in FIG. 1.
- Example 3 An intestinal equivalent produced in this way is shown in FIG. 1.
- the penetration of the pathogen into the organoid structures is examined after about 18 to 24 hours by means of histological methods on thin sections, the PAS staining method being used both in the in vitro skin system and in the in vitro intestinal system.
- the invasion process of the virulent Candida strain down to the deeper layers of the connective tissue-like matrix was documented using the histological sections (FIG. 3). It was shown that the virulent Candida strain spreads in a star shape from the infection site into the connective tissue, whereas the avirulent strain could not penetrate the epithelial cells and showed no adhesion.
- Example 5 Example 5:
- Example 6 Based on the comparison between the determined number of colonies of the samples without the addition of an antifungal agent and the number of colonies of samples with the addition of an antifungal agent, it was determined that the adhesion of the virulent Candida strain could only be prevented by adding amphotericin B and fluconazole and by inhibiting growth , Example 6:
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Applications Claiming Priority (5)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10026789 | 2000-05-31 | ||
DE10026789A DE10026789B4 (en) | 2000-05-31 | 2000-05-31 | Cartilage replacement and process for its manufacture |
DE10062626A DE10062626B4 (en) | 2000-05-31 | 2000-12-15 | infection models |
DE10062626 | 2000-12-15 | ||
PCT/EP2001/006072 WO2001092476A2 (en) | 2000-05-31 | 2001-05-29 | In vitro tissue test system |
Publications (1)
Publication Number | Publication Date |
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EP1290144A2 true EP1290144A2 (en) | 2003-03-12 |
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ID=26005891
Family Applications (1)
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EP01947324A Withdrawn EP1290144A2 (en) | 2000-05-31 | 2001-05-29 | Infection model |
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US (1) | US20040023907A1 (en) |
EP (1) | EP1290144A2 (en) |
JP (1) | JP2004500855A (en) |
AU (1) | AU6904101A (en) |
CA (1) | CA2410946A1 (en) |
WO (1) | WO2001092476A2 (en) |
Families Citing this family (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US7785883B2 (en) * | 2004-04-28 | 2010-08-31 | Vax Design Corp. | Automatable artificial immune system (AIS) |
DE102010023156B4 (en) * | 2010-06-02 | 2012-07-05 | Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. | In vitro test system for viral infections |
US10267756B2 (en) | 2014-07-23 | 2019-04-23 | National Institute For Materials Science | Dryness/wetness responsive sensor having first and second wires spaced 5 nm to less than 20 μm apart |
CN206391052U (en) * | 2016-04-20 | 2017-08-11 | 珐博进(中国)医药技术开发有限公司 | Prepare the device of collagen cornea |
JP7300986B2 (en) * | 2016-11-10 | 2023-06-30 | オルガノボ インコーポレイテッド | Artificial intestinal tissue and uses thereof |
US11856664B2 (en) * | 2017-02-14 | 2023-12-26 | National Institute For Materials Science | Method and system for preventing dew condensation and light scattering due to dew condensation |
CN114107172A (en) * | 2021-11-12 | 2022-03-01 | 上海市农业科学院 | Method for constructing non-infectious gastric mucosa injury cell model and application |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4485096A (en) * | 1982-02-26 | 1984-11-27 | Massachusetts Institute Of Technology | Tissue-equivalent and method for preparation thereof |
US5266480A (en) * | 1986-04-18 | 1993-11-30 | Advanced Tissue Sciences, Inc. | Three-dimensional skin culture system |
US5032508A (en) * | 1988-09-08 | 1991-07-16 | Marrow-Tech, Inc. | Three-dimensional cell and tissue culture system |
US5863531A (en) * | 1986-04-18 | 1999-01-26 | Advanced Tissue Sciences, Inc. | In vitro preparation of tubular tissue structures by stromal cell culture on a three-dimensional framework |
US4835102A (en) * | 1987-03-31 | 1989-05-30 | Eugene Bell | Tissue equivalent test systems |
US4996154A (en) * | 1989-05-04 | 1991-02-26 | Millipore Corporation | Method for growing cellular tissue |
IL95429A (en) * | 1989-09-15 | 1997-09-30 | Organogenesis | Living tissue equivalents comprising hydrated collagen lattice and a collagen gel and their production |
ATE161408T1 (en) * | 1990-04-24 | 1998-01-15 | Mark Eisenberg | COMPOSITE EQUIVALENT OF LIVING SKIN |
CA2119064A1 (en) * | 1993-03-17 | 1994-09-18 | Richard A. Berg | Dermal-epidermal in vitro test system |
US5695996A (en) * | 1994-09-23 | 1997-12-09 | The United States Of America As Represented By The Department Of Health And Human Services | Artificial organ culture system |
US6497875B1 (en) * | 1996-04-26 | 2002-12-24 | Case Western Reserve University | Multilayer skin or dermal equivalent having a layer containing mesenchymal stem cells |
FR2757635B1 (en) * | 1996-12-24 | 1999-02-05 | Oreal | METHOD FOR EVALUATING UV-A DAMAGE TO THE SKIN |
-
2001
- 2001-05-29 WO PCT/EP2001/006072 patent/WO2001092476A2/en not_active Application Discontinuation
- 2001-05-29 CA CA002410946A patent/CA2410946A1/en not_active Abandoned
- 2001-05-29 JP JP2002500670A patent/JP2004500855A/en active Pending
- 2001-05-29 AU AU69041/01A patent/AU6904101A/en not_active Abandoned
- 2001-05-29 EP EP01947324A patent/EP1290144A2/en not_active Withdrawn
- 2001-05-29 US US10/296,965 patent/US20040023907A1/en not_active Abandoned
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Title |
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See references of WO0192476A2 * |
Also Published As
Publication number | Publication date |
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WO2001092476A2 (en) | 2001-12-06 |
JP2004500855A (en) | 2004-01-15 |
US20040023907A1 (en) | 2004-02-05 |
AU6904101A (en) | 2001-12-11 |
CA2410946A1 (en) | 2002-11-29 |
WO2001092476A3 (en) | 2002-11-07 |
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