WO2007090575A1 - Modèle de peau avec cellules dendritiques - Google Patents

Modèle de peau avec cellules dendritiques Download PDF

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Publication number
WO2007090575A1
WO2007090575A1 PCT/EP2007/000914 EP2007000914W WO2007090575A1 WO 2007090575 A1 WO2007090575 A1 WO 2007090575A1 EP 2007000914 W EP2007000914 W EP 2007000914W WO 2007090575 A1 WO2007090575 A1 WO 2007090575A1
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matrix
cells
skin model
collagen
human
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PCT/EP2007/000914
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German (de)
English (en)
Inventor
Dietmar Eschrich
Rüdiger Graf
Karsten Rüdiger MEWES
Martina Spiegel
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Phenion Gmbh & Co. Kg
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Publication of WO2007090575A1 publication Critical patent/WO2007090575A1/fr

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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
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/125—Stem cell factor [SCF], c-kit ligand [KL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/15—Transforming growth factor beta (TGF-β)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/22—Colony stimulating factors (G-CSF, GM-CSF)
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/23—Interleukins [IL]
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/20—Cytokines; Chemokines
    • C12N2501/25—Tumour necrosing factors [TNF]
    • 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
    • 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
    • C12N2503/06—Screening or testing on 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
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to a skin model, in particular a dermis equivalent, an epidermis equivalent and a whole skin equivalent comprising a collagen matrix and dendritic cells, methods for producing the dermis, epidermis or skin equivalent and their use for testing substances, in particular with respect to a potential sensitizing Effect, preferably on the skin.
  • Skin-specific full-skin models which are also referred to as in vitro skin equivalents, can be used in dermatology in particular as a test skin to test substances, for example potential drugs or cosmetics, or agents, such as light and heat, for their pharmacological or cosmetic effects, in particular irritants. , Toxicity and inflammatory effects, and to assess their tolerability.
  • substances for example potential drugs or cosmetics, or agents, such as light and heat
  • Such a system can also be used for a variety of immunological, histological and molecular biological issues. These include, for example, studies on wound healing and the penetration and absorption of substances.
  • the investigations or tests of substances on such full-skin models offer significant advantages over animal experiments and experiments with human subjects, since the results obtained are more reproducible and the investigations can be carried out more cheaply and faster.
  • EP 0 197 090 B1 discloses a process for forming a skin equivalent wherein a hydrated collagen lattice is prepared by mixing an acidic collagen solution with contractile cells, for example fibroblasts. After neutralization of the pH, collagen fibrils are precipitated in the collagen lattice. The contractile cells attach to the collagen lattice and cause it to contract, forming a dermis equivalent.
  • contractile cells attach to the collagen lattice and cause it to contract, forming a dermis equivalent.
  • EP 0 285 474 B1 discloses a dermal equivalent comprising a dermis equivalent obtained from collagen and fibroblasts and a multilayer epidermis equivalent.
  • the dermis equivalent is inoculated with a human or animal explant such as a hair follicle to obtain the epidermis equivalent.
  • EP 0 020 753 B1 describes a process for the formation of tissue, in particular skin tissue, whereby fibroblasts are likewise introduced into a hydrated collagen lattice and a tissue forms after contraction of the collagen lattice. Onto this tissue, pre-in vitro cultured ke ratinocytes or foreskin-isolated keratinocytes may be applied to form a skin substitute.
  • EP 0 418 035 B1 discloses a tissue equivalent comprising a hydrated collagen lattice contracted by a contractile agent such as fibroblasts and a collagen gel in contact with a permeable element.
  • a contractile agent such as fibroblasts
  • a collagen gel in contact with a permeable element.
  • the mixture of collagen and contractile agent is applied to the collagen gel, wherein the radial or lateral contraction of the collagen lattice is prevented by the contact between collagen gel and permeable element, for example a polycarbonate membrane, so that the lattice contracts only with respect to its thickness.
  • permeable element for example a polycarbonate membrane
  • U.S. Patent No. 4,963,489 describes a stromal tissue prepared in vitro wherein the stromal cells, such as fibroblasts, encase a backbone made of a biocompatible material, such as cellulose.
  • the system described can be used, inter alia, for the production of a three-dimensional skin culture system in which keratinocytes and melanocytes are applied to the dermis equivalent, that is to say the three-dimensional stromal carrier matrix.
  • U.S. Patent No. 5,755,814 describes a skin model system that can be used both as an in vitro test system and for therapeutic purposes.
  • the system comprises a three-dimensional crosslinked matrix of insoluble collagen with fibroblasts contained therein and stratified layers of differentiated epidermal cells, wherein an epidermal cell layer is in direct contact with the surface of the collagen matrix.
  • the matrix can be crosslinked both by thermal treatment with dehydration and by chemical means, for example carbodiimide.
  • U.S. Patent No. 5,882,248 describes a method for determining the effect of chemicals or agents on a human skin model system according to U.S. Patent No. 5,755,814.
  • the interaction between the skin model system and the substances to be tested is determined by the release of substances by cells of the skin model system as well as the effects on metabolism, proliferation, differentiation and reorganization of these cells.
  • WO 95/10600 describes a method with which an epidermis equivalent can be obtained.
  • This epidermis equivalent can be used for pharmaceutical and / or cosmetic tanning tests.
  • International Patent Application WO 01/092477 discloses a method for differentiation and / or propagation of isolated dermal fibroblasts, wherein the fibroblasts are cultured in a three-dimensional gel-like matrix and can multiply there.
  • this matrix contains a framework of human or animal collagen constituted of a collagen solution, ie tissue-typical matrix proteins.
  • the method is to obtain an in vitro organoid skin model composed of two tissue-typical layers, namely a dermis equivalent and an epidermis equivalent.
  • the organotypical skin model should correspond to the native skin both histologically and functionally.
  • the known skin models it is disadvantageous that they usually only consist of one or more epidermal layer (s) of keratinocytes.
  • tissue explants are frequently used which harbor the risk of contamination with pathogens, which can lead to a falsification of the results when the skin equivalent is later used as a test skin.
  • the skin models described have a dermal part, this often consists of cancellous, cross-linked material which, in addition to collagen, may also contain other non-skin-specific materials.
  • the dermal part consists only of collagen and fibroblasts, it is subjected to an undefined shrinkage process, which is due to a strong shrinkage of the collagen gel and a liquid leakage therefrom.
  • the skin equivalents described in the prior art are suitable only to a limited extent as a test skin of defined size and the results obtained can be transferred only to a limited extent to native human skin.
  • Conventional Hautäqivente with a dermal part of collagen and fibroblasts are also expensive to produce, since a collagen gel is used, which is difficult to process.
  • the gel solution must be in the cold, that is best on ice, stirred and also pipetted cold, since it remains so fluid.
  • the gel solution is heated, e.g. at 37 ° in the incubator. Only under these conditions and the correct pH value does it gel. The warmer the gel solution becomes, the harder it can be processed. If the gel is overheated, there is also a risk that the collagen will denature and the gel becomes unusable.
  • the skin models known from the prior art are suboptimal in terms of their skin similarity.
  • the expression of the important dermal differentiation marker elastin is not observed in the known skin models - in contrast to natural dermis.
  • dendritic cells are the Langerhans cells. They are the essential antigen-presenting cells of the Skin and take in the epidermis antigens, migrate to the regional lymph nodes and present the penetrated through skin and mucous membranes antigens to the T lymphocytes.
  • Sensitizing substances raw materials or active substances
  • sensitizing substances are, for example, nickel, zinc and cobalt ions, chromate ions, fragrances and fragrances in perfumes, latex, preservatives (thiomersal, parabens, formalin), rosin, TNBF (2,4,6-trinitrobenzenesulfonic acid), DNCB (Dinitrochlorobenzene), TNCB (trinitrochlorobenzene), DNFB (2,4-dinitrodifluorobenzene), MBT (2-mercaptobenzothiazole), MCI / MI (methylchloroisothiazolinone / methylisothiazolinone), neomycin sulfate and other external antibiotics and rubbers (accelerators, antioxidants, vulcanizing agents, stabilizers) rubber production).
  • Other triggers are epoxy resins (resins, solvents and hardeners, usually only the smaller monomers and dimers, but not the polymers), drugs and plants.
  • hapten sensitizing agent
  • One way of determining the sensitizing potential of an agent is by using a murine lymph node assay ("mouse lymph node essay") to inject the test substance into the skin to stimulate the animals immune system. It is then determined whether the test substance triggers allergic reactions on repeated contact.
  • murine lymph node assay (“mouse lymph node essay")
  • the technical problem underlying the present invention is therefore to provide a dermal model which corresponds as far as possible to native human skin, a dermis equivalent, an epidermis equivalent and particularly preferably a human in vitro full skin model, and methods and means for their preparation, which overcomes the above-mentioned disadvantages of the prior art and can be used as a test skin, for example for the investigation of pharmacological and cosmetic effects and for skin compatibility testing, in particular for testing the allergenic or sensitizing potential of active substances.
  • the problem is solved by a skin model comprising a collagen matrix and dendritic cells.
  • the present invention relates to a skin model, in particular a dermis equivalent, an epidermis equivalent and a whole skin equivalent comprising a collagen matrix and dendritic cells, methods for producing the dermis, epidermis or skin equivalent and their use for testing substances, in particular with respect to a potential sensitizing Effect, preferably the effect on the skin.
  • skin model is to be understood as meaning full-body equivalents (also called whole-skin models) and also dermis and epidermis equivalents.
  • dendritic cells are to be understood as meaning those cells and their precursors which can express or regulate either the basal or stimulatory one of the surface markers CD86 (B7.2) or CD 54 (ICAM).
  • the stimulation may preferably be effected by factors known to the person skilled in the art, in particular chemokines and / or cytokines, or by the contact of the cells with a sensitizing agent.
  • dendritic cells are understood as meaning both induced or uninduced monocytic dendritic cells or induced or uninduced dendritic cells obtained from lymphoid, myeloid, histiocytic or monocytic cell lines.
  • the dendritic cells according to the invention are selected from cells or cell lines derived from tumors of the blood-forming and / or immunological system.
  • the cells or cell lines from tumors of the blood-forming and / or immunological system are preferably used.
  • they can be used after stimulation with suitable factors, in particular with chemokines or cytokines, since they preferably express or regulate the markers CD86 and / or CD54 after stimulation, and are obtainable from cell lines.
  • the skin models that are produced with the mentioned cell lines or contain them advantageously have a particularly high stability and reproducibility of the results.
  • the skin models derived from so-called immortal cell lines provide particularly well comparable values, especially in the case of several test series.
  • the tumors are selected from myeloid or lymphoid tumors.
  • the myeloid tumors are selected from human acute myeloid leukemia, human multiple myeloma, human acute myeloid (eosinophilic) leukemia, human multiple myeloma, human acute myeloid leukemia, myelomonocytic leukemia, human chronic myeloid leukemia in blast crisis, human multiple myeloma, human acute monocytic leukemia, human acute myelogenous leukemia, human histocytic lymphoma and human monocytic leukemia.
  • the designation of the tumors published in the DSMZ "German Collection of Microorganisms and Cell Cultures" was used.
  • cell lines can be obtained which are particularly easy to cultivate.
  • the cells to be used according to the invention are selected from myeloid tumors from cells of the human cell lines KG-1 (DSMZ no. ACC 14), U937 (DSMZ no. ACC 5) and THP-1 (DSMZ no. ACC 16).
  • the cells to be used according to the invention are selected from lymphoid tumors, from human plasma cell leukemia, human cell leukemia, human cell lymphoma, human B cell precursor leukemia, human B cell lymphoma, human chronic B cell leukemia, human T Cell acute lymphoblastic leukemia, human histiocytic lymphoma and human Hodgkin's lymphoma.
  • lymphoid tumors from human plasma cell leukemia, human cell leukemia, human cell lymphoma, human B cell precursor leukemia, human B cell lymphoma, human chronic B cell leukemia, human T Cell acute lymphoblastic leukemia, human histiocytic lymphoma and human Hodgkin's lymphoma.
  • the cells to be used according to the invention are selected from monocyte-derived dendritic cells (MoDC), cells from cell lines obtained from tumors selected from human acute myelogenous leukemia, human histocytic lymphoma, human monocytic leukemia, in particular the human cell lines KG ACC1 (DSMZ no. ACC 14), THP-1 (DSMZ no. ACC 16), Mono Mac 6 (DSMZ no. ACC 124) or Mono Mac 1 (DSMZ no. ACC 252) ) or MUTZ-3 (DSMZ no. ACC 295).
  • MoDC monocyte-derived dendritic cells
  • the cells to be used according to the invention are selected from monocyte-derived dendritic cells (MoDC) and in particular to the human cell lines KG-1 (DSMZ no. ACC 14), U937 (DSMZ no. ACC 5) and THP-1 ( DSMZ no. ACC 16).
  • MoDC monocyte-derived dendritic cells
  • the cells suitable according to the invention are treated before, during or after co-cultivation with the skin model with suitable factors, in particular chemokines or cytokines.
  • suitable factors in particular chemokines or cytokines.
  • the appropriate factors may be added to any of the appropriate culture media.
  • Preferred cytokines are selected from GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), SCF (Star Cell Factor), IL-4 (Interleukin 4), IL-13 (Interleukin 13), TGF ⁇ (Transforming Growth Factor beta), in particular TGF ⁇ i, or TNF- ⁇ (Tumor Necrosis Factor alpha) and mixtures thereof, more preferably from IL-4 and GM-CSF and mixtures thereof.
  • GM-CSF Gramulocyte Macrophage Colony Stimulating Factor
  • SCF Start Cell Factor
  • IL-4 Interleukin 4
  • IL-13 Interleukin 13
  • TGF ⁇ Transforming Growth Factor beta
  • TGF ⁇ i Tumor Necrosis Factor alpha
  • the collagen matrix is crosslinked.
  • the increased stability of the crosslinked collagen matrix (which, as described below, can be produced) in turn leads to an improved handling of the skin models and a higher reproducibility of the test results to be obtained.
  • crosslinking of the collagen matrix can be produced by crosslinking reagents, as described by way of example for the preparation process (step d), particularly preferably by glutaraldehyde.
  • the collagen matrix contains fibroblasts and / or keratinocytes.
  • the seeding of different cells can simulate different test systems of the real skin.
  • the skin model according to the invention particularly preferably contains both fibroblasts and keratinocytes, which are applied successively (in particular as described in more detail below) to the collagen matrix, which is preferably crosslinked. There, the cells are grown and, if necessary, converted to a finished skin model at the Air-Liquid interface.
  • the invention relates to a method for producing a skin model, which comprises a) recovering collagen sparingly soluble from collagen-containing tissue, b) converting the sparingly soluble collagen into a homogeneous collagen suspension by means of a mixing device, c) lyophilizing the collagen suspension and produces a first matrix A, d) subjecting the first matrix A to cross-linking and thus producing a second, mechanically stabilized matrix B, e) flocking and growing fibroblasts and / or keratinocytes (optionally one after the other) onto the second matrix B; f) further culturing the cells growing in and on the matrix B until complete formation of the skin model, before, during or after sowing the cells in step e) cocultivated dendritic cells with the skin model.
  • the substances to be tested can be applied topically to the surface of the skin models. Therefore, formulations, e.g. Ointments, creams, gels and foams, and not just water-soluble raw / actives e.g. applied on top of the epidermis and thus tested.
  • a further advantage is the fact that the substances applied to the skin model according to the invention penetrate through the stratum corneum, ie the barrier layer of the skin.
  • the penetration rate depends significantly on the physico-chemical properties of the substances, e.g. Lipophilicity, molecular size, reactivity, which also have a decisive influence on the effect of a potentially sensitizing substance in vivo.
  • a distribution pattern of the substances is formed, from which the bioavailability of the substance can be derived. This effect can not be simulated on monolayer cultures.
  • the substance to be tested can be metabolized by tissue-specific enzymes or enzyme systems during the passage through the cell layers of the skin model.
  • tissue-specific enzymes or enzyme systems during the passage through the cell layers of the skin model.
  • detection in monolayer cultures is not possible.
  • a reverse mode of action the inactivation of a sensitizing substance by metabolisation in the skin, can also be detected by the model according to the invention, in contrast to two-dimensional cell culture test systems.
  • the collagen suspension obtained in step b) can be poured and pipetted without problem at room temperature, in contrast to the collagen gels used in the prior art.
  • the skin model according to the invention can be handled well in comparison to models from the prior art, since it is particularly robust.
  • the whole skin model obtainable according to the invention adheres well to the bottom of the culture vessels, so that it is not premature Replacement of the not yet fully developed skin model comes, whereby the model would be unusable.
  • the term "culturing” means maintaining, preferably in vitro, the life functions of cells, for example fibroblasts or keratinocytes, in a suitable environment, for example by adding and removing metabolic factors and products, in particular also one Multiplication of the cells.
  • fibroblasts are understood to mean naturally occurring fibroblasts, in particular occurring in the dermis, genetically modified fibroblasts or fibroblasts resulting from spontaneous mutations or their precursors.
  • the fibroblasts may be of animal or human origin.
  • “sparingly soluble collagen” is understood to mean coarse-fiber collagen which shows no visible or only slight swelling or no or only slight gel formation, in particular no gel formation, in the aqueous medium over several hours, while sparingly soluble collagen is preferably obtained from the tendons of animals , in particular of mammals, preferably of horses, pigs or cattle, very particularly preferably from the Achilles tendons or the skin of cattle, in particular from the Achilles tendons of cattle won.
  • the conversion into a homogeneous collagen suspension can be carried out with or in any mixing apparatus suitable from a professional point of view, preferably in a static mixer or with an Ultra Turrax.
  • the collagen suspension is now placed in containers whose dimensions correspond to those of the desired matrices, z. B. in the wells of a microtiter plate.
  • the vessels Before filling the containers, z.
  • the vessels can be coated with various agents to improve the adhesion of the lyophilized collagen matrix to the vessel wall in the following.
  • Suitable agents are, for example, collagen, gelatin, polylysine, fibrin or fibrinogen / thrombin or fibronectin.
  • the inner vessel walls are wetted with solutions or suspensions of the agents, the vessels are dried, so that a layer of the agents forms on the inner vessel surface and then first fills the collagen suspension.
  • step c The lyophilization (freeze-drying) in step c) takes place in these containers. If freezing takes place at a low cooling rate, large ice crystals are obtained Effect concentration differences and segregation in the product. Slow, pronounced ice crystal growth produces particularly large ice crystals which, if a temperature gradient has been present during freezing, are oriented in the direction of the gradient. In this case, products are obtained which are crossed by a large-lumen column or chimney structure. However, in these large-scale structures, the specific surface area is reduced, so that the reconstitution behavior can be adversely affected.
  • the Applicant has found that slow cooling of the collagen suspension during the lyophilization process has beneficial effects on the nature of the matrices A and B.
  • a cooling rate of up to 50 ° C per hour, in particular 5 0 C to 40 0 C per hour, preferably from 10 0 C to 30 0 C per hour, more preferably 15 0 C to 25 0 C per hour, most preferably from 18 ° C to 23 0 C, and even more preferably from 20 0 C to 22 0 C.
  • Such preferably available lyophilisates have a pellicle on the product surface.
  • this cuticle forms pores on the surface of the lyophilized matrix which offer particularly good conditions for the subsequent colonization with fibroblasts since they promote slow migration of the fibroblasts into the matrix.
  • faster cooling rates during the lyophilization process often result in an open-pore matrix with "craters" in the surface that can not completely fill the fibroblasts with newly synthesized extracellular matrix in the given culture time. whereby the stratification and thus the differentiation of the skin model can be disturbed sensitively.
  • the crosslinking in step d) can be carried out with the aid of any physical or chemical crosslinking process which is suitable for the person skilled in the art.
  • glutaraldehyde is preferably suitable as a crosslinker for the process according to the invention.
  • the crosslinking can also be carried out by physical methods such as UV irradiation and dehydrothermal crosslinking (DHT).
  • bifunctional substances whose groups react with the amino groups of the lysine and hydroxylysine residues on different polypeptide chains of the collagen fibers are suitable as chemical crosslinking reagents. Furthermore, activation of the carboxyl groups of the free glutamine and aspartic acid residues, followed by reaction with the Amino groups of another polypeptide chain, lead to a crosslinking of the collagen fibers according to the invention.
  • a crosslinking between two collagen fibers can also be effected according to the invention by a reaction of the amino groups with diisocyanates or by the formation of acyl azides.
  • EDC 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide
  • crosslinking reactions with homobifunctional crosslinking reagents which react with amino groups, for example with p-benzoquinone, dimethyl adipimidate, dimethylpimelinidate, dimethyl suberimidate, 1,4-phenylenediisothiocyanate, polyoxyethylene bis (imidazolylcarbonyl), bis [polyoxyethylene bis (imidazolyl) carbonyl)] and suberic acid bis (N-hydroxysuccinimide ester); as well as the use of a biological crosslinking agent, in particular with enzymes, preferably transglutaminase, which can link peptide chains together, or lysyl oxidases (EC 1.4.3.13).
  • enzymes preferably transglutaminase, which can link peptide chains together, or lysyl oxidases (EC 1.4.3.13).
  • the extraction and cultivation of the fibroblasts and keratinocytes is carried out according to methods known to the experts, which can be adapted according to the desired properties of the skin model to be produced.
  • step f before, during or after the seeding of the keratinocytes in step f), in addition to the cells used according to the invention, other cell types and / or other cells of other tissue types of human as well as animal origin, eg.
  • Mammalian, and / or progenitor cells thereof can be seeded on the matrix, for example, melanocytes, macrophages, monocytes, leukocytes, plasma cells, neuronal cells, adipocytes, induced and non-induced progenitor cells of Langerhans cells, Langerhans cells, and other immune cells , Endothelial cells, cells from tumors of the skin or skin-associated cells, in particular sebocytes or sebaceous gland tissue or sebaceous glands, cells of the sweat gland or sweat gland tissue or sweat gland explants, hair follicle cells or hair follicle explants; as well as cells from tumors of other organs or from metastases.
  • melanocytes for example, melanocytes, macrophages, monocytes, leukocytes, plasma cells, neuronal cells, adipocytes, induced and non-induced progenitor cells of Langerhans cells, Langerhans cells, and other immune cells , Endo
  • the differentiated cells in particular the melanocytes, can in this case be obtained both from a natural source, preferably a mammal, in particular human or mouse, or in vitro from omni-, pluri- or multipotent precursor cells, preferably stem cells, particularly preferably those from Mammals, especially from mouse embryonic stem cells, are obtained. It can also stem different Origin, tissue-specific stem cells, embryonic and / or adult stem cells are incorporated into the skin model. With the aid of the method according to the invention, an organoid in vitro skin model is thus obtained, which is composed of two tissue-typical layers, namely a dermis equivalent and an epidermis equivalent. The organotypical skin model corresponds largely to the native skin both histologically and functionally.
  • the crosslinking of the matrix A ensures that the dermis equivalent growing on and in the matrix B is not subject to any or only a very slight shrinkage process in the course of the cultivation period.
  • skin equivalents are obtained with a defined diameter, uniform surface and a defined degree to the edge of the culture vessel.
  • the uniform size and uniformity of the whole skin model (whole skin model) used as a test surface ensures that when testing substances for pharmacological and / or cosmetic effects, the quality of the results is increased and the test results more reproducible.
  • the cross-linked matrix B intended for cultivating the fibroblasts thus contains the fibroblasts to be cultivated and a collagen scaffold newly constituted from a, preferably fresh, collagen suspension of human or animal origin with a concentration of about 5 to 50 mg collagen per ml matrix, corresponding to 0.5 % to 5% collagen. Preference is given to the range from 8 to 25, corresponding to 0.8% to 2.5% collagen, in particular from 8 to 12 mg collagen per ml matrix, corresponding to 0.8% to 1, 2% collagen.
  • the collagen scaffold is obtained from a, preferably cell-free, acidic suspension of sparingly soluble collagen, the protein concentration of the suspension preferably being 5 to 15 mg / ml, corresponding to 0.5 to 1, 5% by weight.
  • the pH of the collagen solution is 0.1 to 6.9, preferably 2.0 to 5.0, preferably 3.0 to 4.5 and especially 3.5 to 4.0.
  • the matrix B is treated with a solution containing a cell culture medium (preferably DMEM cell culture medium), buffer (for example Hepes buffer), serum (preferably fetal calf serum (FCS), normal calf serum (NCS), normal lamb serum (NLS), defined serum or serum replacement products) and preferably 1-6 x 10 5 / matrix fibroblasts, especially precultured fibroblasts.
  • a cell culture medium preferably DMEM cell culture medium
  • buffer for example Hepes buffer
  • serum preferably fetal calf serum (FCS), normal calf serum (NCS), normal lamb serum (NLS), defined serum or serum replacement products
  • FCS fetal calf serum
  • NCS normal calf serum
  • NLS normal lamb serum
  • defined serum or serum replacement products preferably 1-6 x 10 5 / matrix fibroblasts, especially precultured fibroblasts.
  • fibronectin and / or laminin preferably human fibronectin and / or laminin
  • Fibronectins are structural or adhesion proteins produced in fibroblasts, the function of which in vivo consists in binding to other macromolecules, for example collagen, and in attaching cells to neighboring cells.
  • Laminin is a protein of the basement membrane to which cells can adhere.
  • Both proteins can either be incorporated directly into the matrix during their preparation or after the crosslinking step the matrix can be added in dissolved form, for example dissolved in culture medium. This can be done before or in parallel to cell seeding.
  • Subsequent cultivation of the fibroblasts in the collagen matrix is preferably carried out in submerged culture.
  • a "submersible culture” or a “submerged culture” is understood to mean a process for cultivating cells, wherein the cells are covered with a nutrient solution.
  • the fibroblast-containing matrix B is thus preferably coated with cell culture medium and incubated in the temperature range from about 30 0 C to about 40 0 C.
  • the model may be modified by altering culturing conditions, e.g. be optimized by medium components so that it gets a better barrier function, which comes even closer to the in vivo situation. This is important z. B. for the implementation of penetration studies, but also for the production of products that improve the barrier function of the skin. In addition, there are medically relevant disorders of the barrier function, whether due to environmental factors (contact with detergents) or genetically. Skin models with optimized barrier function would be important here.
  • the improvement of the barrier function can be achieved by changing culture conditions (e.g., humidity, temperature), chemical components in the medium (ceramides, vitamin C), or genetically engineered keratinocytes.
  • the change of the barrier function can be measured by measuring the surface electrical capacitance.
  • the culture medium which are released from the keratinocytes in the case of cocultivation of fibroblasts and keratinocytes.
  • the conditioned culture supernatant i.e., the supernatant of cultured keratinocytes and / or co-cultures of keratinocytes with other cell types, such as endothelial cells, immune cells, or fibroblasts
  • the conditioned culture supernatant i.e., the supernatant of cultured keratinocytes and / or co-cultures of keratinocytes with other cell types, such as endothelial cells, immune cells, or fibroblasts
  • Cocultivation of dendritic cells according to the invention with the skin model according to the invention can be carried out in various ways: Cocultivation can preferably be carried out by sowing the dendritic cells before, during or after step e) of the production process for the skin models (preferably whole or full skin model, dermis equivalent, epidermis equivalent).
  • the cells are sown together with the keratinocytes in step e.
  • the isolation, culture and differentiation of the dendritic cells then take place in parallel with the growth of the keratinocytes.
  • the mixture of both cell types preferably takes place in a ratio of 10: 1 to 1: 1 (keratinocytes to dendritic cells.
  • the cell mixture can now be carried out together in keratinocyte medium on the second matrix, which in the case of the whole-skin model was preferably pre-cultured with fibroblasts. Thereafter, the submerged cultivation is preferably continued, more preferably for 2-7 days. Thereafter, the model is preferably transferred to the air-liquid interface for further differentiation of the epidermis. The further cultivation can be 7-21 days.
  • the dendritic cells in the skin occur predominantly in the epidermis. Therefore, it is advantageous to secrete the cells after (or simultaneously with) the seeding of the keratinocytes into the skin model, in particular the epidermis equivalent or the whole skin model.
  • the sowing of the dendritic cells into the skin model takes place after the seeding of the keratinocytes in keratinocyte medium onto the second matrix (B). Cultivation of the keratinocytes may then continue to be submerged.
  • the dendritic cells may take place both during the submerged culture phase, which may preferably be 3 to 7 days, and after the transfer into the air-liquid interface.
  • the cells are sown onto the second matrix (B). It is very particularly preferred if the sowing of the cells to be used according to the invention are seeded onto the model after 1 to 3 days after transfer of the model into the air-liquid interface.
  • Another way to co-cultivate the skin model and the dendritic cells is to prepare the skin model according to steps a to f and cultivate further on the air-liquid interface.
  • This finished skin model can now be contacted with a culture of dendritic cells via a suitable medium.
  • the dendritic cells can also be present in a gel instead of in a normal cell culture.
  • collagen, agarose or fibrin gels are used, more preferably
  • the cells are stirred into a not yet or not completely gelled gel solution.
  • the gel is poured into a mold and allowed to gel.
  • collagen gels gel by heating to about 37 ° C (e.g., in the incubator).
  • the gel containing the cells is preferably poured or placed in a co-culture insert on a membrane, which is preferably porous and / or made of polymeric material.
  • the skin model can now be placed directly on the gel.
  • the Kokulturinsert is used in a culture vessel with culture medium.
  • the cells in the gel have no direct contact with the cells in the skin model, but can exchange messengers and other substances.
  • the dendritic cells can be harvested easier in this method after completion of the experiment and processed for further analysis.
  • the skin model thus obtained may be used for screening and diagnostic procedures, in particular for investigating the effects of chemical substances, plant extracts and metals, for example, potential drugs or components of cosmetics, or other agents (physical quantities), such as light or heat, radioactivity, sound, Electromagnetic radiation, electric fields and also for the investigation of phototoxicity, ie the damaging effect of light of different wavelengths, on cell structures.
  • the dermis equivalent prepared according to the invention can also be used to investigate wound healing. It is also suitable for investigating the effect of gases, aerosols, smoke, dusts on cell structures or metabolism or gene expression.
  • the term "agent” or “agents” is understood to mean, in particular, physical agents acting on the skin or skin cells, such as light, heat, or the like.
  • the invention therefore also relates to screening and diagnostic methods using the skin models produced according to the invention.
  • a preferred embodiment of the invention comprises the treatment of the skin models in the presence and absence of the substance to be investigated and / or the agent to be examined and the comparison of the observed effects on the cells or cell components of the dermis equivalent.
  • a further preferred embodiment of the invention comprises a method for investigating the penetration of substances using the skin models produced according to the invention, in particular and using a whole skin model according to the invention which consists of a dermis equivalent and an epidermis equivalent.
  • the skin model according to the invention preferably the whole skin model according to the invention, is suitable for the investigation of such substances for which there is a suspicion of a sensitizing or allergy-inducing effect.
  • the method according to the invention is used to produce a dermis equivalent.
  • the fibroblasts are cultivated in matrix B in such a way that a dermis equivalent is subsequently obtained. This is preferably done by a process for the preparation of a dermis equivalent, which is characterized in that
  • a. collagen that is difficult to dissolve from collagen-containing tissue b. the sparingly soluble collagen is transferred by means of a mixing device into a homogeneous collagen suspension, c. the collagen suspension is lyophilized to produce a first matrix A, i. subjecting the first matrix A to cross-linking and thus creating a second, mechanically stabilized matrix B, e.
  • the fibroblasts are seeded on the second matrix B and allowed to grow, and finally f. the fibroblasts growing in and on matrix B are further cultured until complete development of the dermis equivalent, before, during or after the seeding of the cells in step e. cocultivated dendritic cells with the dermis equivalent.
  • a "dermis equivalent” is understood to mean a connective tissue-like layer of collagen and fibroblasts that largely corresponds to the native dermis.
  • Particularly suitable for the production of a whole skin model is to use a method in which a) from collagen-containing tissue sparingly soluble collagen, b) converting the sparingly soluble collagen into a homogeneous collagen suspension by means of a mixing device, c) lyophilizing the collagen suspension and thus producing a first matrix A, d) subjecting the first matrix A to cross-linking and thus producing a second, mechanically stabilized matrix B, e) F) festoon and grow fibroblasts onto the second matrix B, f) nucleate and grow keratinocytes on the second matrix B, and finally g) further cultivate the cells growing in and on the matrix B until complete formation of the dermal and epidermal part of the whole skin model in which cocultivation with the skin model before, during or after seeding of the cells in step e or step f dendritic cells.
  • step f It is particularly preferred to carry out the sowing of the dendritic cells before, during or after the sowing of the keratinocytes in step f.
  • a particularly preferred embodiment of the invention also relates to an aforementioned method of culturing dermal fibroblasts and epidermal keratinocytes in a matrix to produce a dermis equivalent and epidermis equivalent equivalent whole skin equivalent.
  • one to three weeks, preferably 10 to 14 days, after the preparation and incubation of the fibroblast-containing collagen matrix keratinocytes are seeded on the matrix.
  • keratinocytes are understood to mean cells of the epidermis which form keratinized squamous epithelium, genetically modified keratinocytes or spontaneous mutant keratinocytes or their precursors which may be of animal or human origin If keratinocytes depend strongly on the proportion of basal stem cells in the keratinocytes used, the keratinocytes seeded on the matrix according to the method according to the invention may be largely undifferentiated keratinocyte stem cells from human biopsy tissue, but it is also possible to use cell lines or use certain differentiated cells Alternatively to normal skin keratinocytes, mucosal keratinocytes or intestinal epithelial cells may also be applied to the matrix:
  • these are pre-cultured cells, particularly preferably keratinocytes in the first or in the second cell passage, but it is also possible to use cells from higher passages.
  • the seeding of the keratinocytes onto the matrix is preferably carried out in a cell culture medium, more preferably in DMEM / F12 medium, the approximately 1 to 30% fetal calf serum, NCS, containing defined serum or serum replacement products and various additives in varying concentrations that promote cell proliferation and differentiation.
  • the matrix is preferably supplemented with DMEM medium containing in particular EGF from the mouse or comparable preparations from other animals, epidermal growth factor (hEGF) (eg in a concentration of 0.2 ⁇ g / l medium), and for example 0 , 8 mM CaCl 2 , and subjected to preferably 1 to 10 days, preferably 3 to 7 days Submers cultivation.
  • DMEM medium containing in particular EGF from the mouse or comparable preparations from other animals
  • epidermal growth factor (hEGF) eg in a concentration of 0.2 ⁇ g / l medium
  • 0 , 8 mM CaCl 2 epidermal growth factor
  • an "airlift culture” is understood to mean a culture in which the level of the nutrient medium level is precisely matched to the height of the matrix, while the keratinocytes or cell layers formed by the keratinocytes are above the nutrient medium level and are not covered by the nutrient medium Cultivation takes place at the boundary layer air-nutrient medium, whereby the supply of the cultures takes place from below, for example the skin models are lifted out of a microtiter plate and placed on filter papers resting on metal spacers in culture vessels Medium is filled in the culture vessels so high that it does not completely cover the filter paper, but there is a liquid collar around the base of the skin models (air-liquid interface)
  • the period of the airlift culture can be varied as desired by one skilled in the art It is typically about 1 to 4 weeks. During this period, a
  • the method according to the invention for the production of an in-vitro whole skin model can advantageously be modified such that before, during or after the seeding of keratinocytes further cell types, such as melanocytes, macrophages, monocytes, leukocytes, plasma cells, neuronal cells, adipocytes, induced and uninduced precursor cells Langerhans cells, Langerhans cells other immune cells, endothelial cells and cells from tumors of the skin or skin-associated cells can be seeded on the matrix and further cultured.
  • the cells mentioned can be of both human and animal origin.
  • the invention therefore also relates to a skin-specific in vitro full skin model (whole skin model), in particular a human in vitro full skin model, which was produced by the method according to the invention and an optionally subsequent and / or preceding cultivation method of conventional type and which in the epidermal portion at least one proliferative cell layer , some differentiating cell layers and at least one keratinized cell layer, wherein the epidermal equivalent comprises stratum basale, stratum spinosum, stratum granulosum and stratum corneum and wherein between the dermis equivalent and the epidermis equivalent a basement membrane consisting of the characteristic BM proteins such as laminin and collagen IV is included and moreover skin typical Proteins such as transglutaminase, involucrin, collagen IV, laminin, filaggrin, fibronectin, Ki-67, cytokeratin 10 and especially elastin can be expressed.
  • BM proteins such as laminin and collagen IV
  • the stress marker cytokeratin 6 is expressed only to a small extent, which indicates that the cells in the in vitro full-skin model according to the invention are in a relatively low-stress state. Strong CK6 expression is found in the skin, e.g. during wound healing or regeneration.
  • the proliferative behavior as well as the differentiation of the dermal and epidermal cells can be modified by the application of electrical or electromagnetic fields to the dermis model or full skin model during the cultivation phase.
  • the skin equivalent produced according to the invention is suitable for product testing, for example with regard to efficacy, undesired side effects, for example irritation, toxicity and inflammation effects or allergenic effects, or compatibility of substances.
  • These may be substances which are to find potential use as medicaments, in particular as dermatics, or substances which are part of cosmetics, or also consumer goods which come into contact with the skin, such as e.g. Detergent etc.
  • the skin equivalent produced according to the invention can also be used, for example, for studies on the absorption, transport and / or penetration of substances. In addition, it is also suitable for the examination of other agents (physical quantities), such as light or heat, radioactivity, sound, electromagnetic radiation, electric fields, for example for the investigation of phototoxicity, ie the damaging effect of light of different wavelengths, on cell structures.
  • the skin equivalent produced according to the invention can also be used to investigate wound healing. It is also suitable for investigating the effect of gases, aerosols, smoke, dusts on cell structures or metabolism or gene expression.
  • the effects of substances or agents on human skin can be determined, for example, by the release of substances, for example cytokines or mediators, by cells of the human or animal skin model system, as well as the effects on gene expression, metabolism, proliferation, differentiation and reorganization of these cells.
  • substances for example cytokines or mediators
  • cytotoxic effects on skin cells for example, can be detected.
  • the tests of substances or Agents on the human dermal equivalent according to the invention may comprise both histological methods and immunological and / or molecular biological methods.
  • a preferred embodiment of the invention therefore comprises methods for investigating the effect, in particular the pharmacological effects, of substances or agents on human skin using the human skin model produced according to the invention, in particular a whole-skin model.
  • an XTT-tetrazolium reduction test (EZ4U test) or Alamar Blue (resazurin reduction test, Fa. Promega) is carried out.
  • EZ4U is a non-toxic water soluble yellow tetrazolium salt that can be reduced from living cells to intensely colored formazans. The reduction requires intact mitochondria and the test can therefore be used to demonstrate the vitality of cells.
  • Vitality assays which can be used according to the invention and which also rest on the basis of tetrazolium compounds are, for example, As the MTT, MTS or the WST-1 test. These are used as test kits u. a. offered by the company Roche.
  • the detection of the vitality of the cells can also be carried out via the release of lactate dehydrogenase (LDH) from cells with damaged cell membrane. With trypan blue cells with damaged cell membrane can be selectively stained.
  • LDH lactate dehydrogenase
  • a further preferred embodiment of the invention comprises a method for investigating the penetration of substances, wherein both a dermis equivalent produced according to the invention and a skin equivalent produced according to the invention are treated with the substances to be investigated and the results obtained in both systems are compared with one another.
  • the effects of chemical substances or other agents on specific skin types are examined.
  • cells of defined skin types for example skin types with few pigments and / or skin types with many pigments, are used to establish skin equivalents according to the invention and these are tested with regard to the effect of substances or agents.
  • the skin equivalent produced according to the invention is used as a model system for studies of skin diseases and for the development of new treatment options for skin conditions.
  • cells from patients with a particular genetic or acquired skin disease can be used to establish patient-specific skin model systems and to examine and evaluate the efficacy of particular therapies and / or drugs.
  • the skin equivalent produced according to the invention can be colonized with microorganisms, in particular pathogenic microorganisms.
  • microorganisms are to be understood as meaning, in particular, fungi, bacteria and viruses.
  • the microorganisms are preferably selected from fungi or pathogenic and / or parasitic bacteria.
  • Particularly preferred fungi are the species of the genus Candida, Malassezia and Trichophyton, more preferably Candida albicans, Trichophyton mentagrophytes and Malassezia furfur.
  • pathogenic and / or parasitic bacteria Staphylococcus aureus are particularly preferred.
  • the method of the invention is used to produce an epidermis equivalent. This can be achieved by a) recovering collagen which is sparingly soluble from collagen-containing tissue, b) converting the sparingly soluble collagen into a homogeneous collagen suspension by means of a mixing device, c) lyophilizing the collagen suspension and thus producing a first matrix A, d) the first matrix A.
  • step e dendritic cells are cocultivated with the epidermis equivalent.
  • keratinocytes are seeded onto the matrix, which acts as a framework or support for the epidermis model, and cultured under such conditions that the keratinocytes first proliferate and then differentiate in that an epidermis consisting of all 4 layers is formed.
  • the matrix is preferably pretreated with extracellular matrix or basement membrane proteins to provide better adhesion of the keratinocytes to the matrix material and to provide the cells with the signals mediated by these proteins.
  • proteins suitable according to the invention are collagen IV or collagens of other types, fibronectin and laminin.
  • the epidermis model thus formed is another object of the present invention and like the full-skin model can also be used for versatile tests.
  • Preferred cell culture media are DMEM (Dulbecco's Modified Eagle Medium), RPMI 1640, M199 and Ham's F12 medium. However, any other cell culture medium that facilitates the cultivation of fibroblasts can also be used.
  • the serum used is preferably fetal calf serum (FCS), but also NCS and serum replacement products, and as buffer, for example, Hepes buffer.
  • FCS fetal calf serum
  • the pH of the solution of cell culture medium, buffer and serum in a preferred embodiment is 6.0 to 8.0, for example 6.5 to 7.5, in particular 7.0
  • the medium may contain other factors, for example hormones, growth factors, adhesives, antibiotics, selection agents, enzymes and enzyme inhibitors and the like.
  • factors may be added to the culture medium which are released from the fibroblasts in the event of cocultivation of fibroblasts and keratinocytes.
  • the conditioned culture supernatant i.e., the supernatant of cultured fibroblasts and / or cocultures of fibroblasts with other cell types, such as endothelial cells, immune cells, or keratinocytes
  • the conditioned culture supernatant i.e., the supernatant of cultured fibroblasts and / or cocultures of fibroblasts with other cell types, such as endothelial cells, immune cells, or keratinocytes
  • the skin model can be used by topically applying in particular substances to be tested to the surface of the HM.
  • the expression of surface markers in the dendritic cells can be used to evaluate the sensitizing or allergenic properties of the applied substances.
  • the cells are preferably removed from the skin model.
  • the epidermis of full-skin models and epidermis equivalents are preferably incubated with thermolysin until the epidermis separates from the dermis or matrix. Subsequently, the epidermis is dissociated by treatment with enzymes, preferably proteolytic enzymes, e.g. Trypsin until the tissue disintegrates into individual cells.
  • enzymes preferably proteolytic enzymes, e.g. Trypsin until the tissue disintegrates into individual cells.
  • the dermis equivalent or the dermis of the full-skin model it is preferred to dissolve the dermis by means of proteolytic enzymes, preferably with collagenases, which can be obtained from a wide variety of sources. After that, the cells can be easily isolated. The cells thus obtained can be detected and assayed by methods known to those skilled in the art.
  • proteolytic enzymes preferably with collagenases
  • they may be specifically labeled and analyzed in a cell sorter, e.g. with the FACS method (fluorescence-activated cell sorting).
  • a cell sorter e.g. with the FACS method (fluorescence-activated cell sorting).
  • gene expression analyzes can also be carried out with the cells by PCR methods known to the person skilled in the art (for example also reverse transcriptase PCR (RT-PCR) or real-time RT-PCR).
  • PCR methods known to the person skilled in the art (for example also reverse transcriptase PCR (RT-PCR) or real-time RT-PCR).
  • the cells can also be examined in the intact skin model.
  • the presence of the cells or their differentiation state can be detected, for example, by immunohistochemical methods, microscopic, in particular electron or light microscopic methods, or by in situ or fluorescence in situ hybridization methods.
  • end points of dendritic cells can be investigated which occur after activation by contact with sensitizing substances or regulate their expression, in particular strongly regulated.
  • CD86 B7.2
  • CD83 HB15
  • CD54 IAM-1
  • DC-SIGN molecules of MHC classes 1 and / or 2, HLA -DR
  • IL-1 beta and IL-8 gene expression CD86 (B7.2), CD83 (HB15), CD54 (ICAM-1), DC-SIGN, molecules of MHC classes 1 and / or 2, HLA -DR; IL-1 beta and IL-8 gene expression
  • Soluble substances for example chemo- or cytokines, in particular interleukins, which are secreted by contact with sensitizing substances can moreover also be detected in the surrounding culture medium by methods known to the person skilled in the art, for example suitable are ELISA, RIA (radioactive immunoassay) or EIA (enzyme immunoassay), HPLC, FPLC, GC-MS or MALDI-TOF.
  • a further subject of the present invention is a method for detaching the epidermis from a skin model, preferably an epidermis equivalent, particularly preferably a whole skin model, characterized in that the epidermis of the skin model is treated with proteolytic enzymes, preferably thermolysin.
  • proteolytic enzymes preferably thermolysin.
  • the suspension is kept at room temperature. This gives a milky-turbid collagen suspension.
  • the suspension is pipetted into the wells of a 24-well cell culture plate.
  • the filled plate is then picked in the hand, tilted slightly and turned to all sides so that the gel solution wets the wall of the wells also above the matrix surface. This procedure improves the adhesion of the matrices in the wells.
  • the thus treated filled plates are placed directly in the lyophilizer and frozen in the device.
  • the collagen suspension filled in the cell culture plates with 24 wells is frozen in a lyophilizer with temperature-adjustable shelves and then dried under reduced pressure.
  • the freezing rate is 18 0 C to 23 ° C per hour.
  • the entire freeze-drying process takes about 20-27 hours.
  • Collagen matrices chemically fixed before sowing the fibroblasts.
  • Glutaraldehyde solution is carefully added to each matrix of 24 matrices A in a 24-well cell culture plate with a pipette. The solution is slowly run down the inner edge of the well so as not to damage the matrix surface. The glutaraldehyde solution takes several minutes to penetrate the interior of the collagen sponges. The increasing saturation manifests itself in a color change of the matrices from pure white to gray to pale yellow. Then the lid is placed on the cell culture plates and the edge sealed airtight with Parafilm to avoid evaporation of the solution. The treated plates are stored protected from light at room temperature for 24 hours.
  • Cross-linking of the matrices may be carried out for a period of preferably 5 days but at least 4 days.
  • Monocytic dendritic cells are obtained by culturing PBMCs (peripheral blood mononuclear cells).
  • the starting material for the isolation of monocytes is a lymphocyte concentrate (buffy coat), which is obtained after centrifuging off whole blood.
  • the monocyte cells of the peripheral blood can be isolated from the lymphocyte concentrate by means of density gradient centrifugation or CCE (countercurrent centrifugal elutriation).
  • the isolated monocytes are cultured in RPM1 1640 with 25 mM HEPES (composition: see table) at 37 ° C in a CO 2 incubator.
  • the differentiation to immature monocytic dendritic cells is carried out by adding recombinant human GM-CSF and recombinant human IL-4 to the culture medium. Culture duration 2-7 days.
  • the surface marker CD86 (B7.2) can be detected by means of a specific antibody via flow cytometry (fluorescence activated cell sorting, FACS analysis).
  • flow cytometry fluorescence activated cell sorting, FACS analysis.
  • fibroblasts of a suitable passage Prior to seeding on the cross-linked collagen matrices, fibroblasts of a suitable passage are pre-cultured in cell culture flasks with fibroblast medium. After reaching the desired cell density, the culture medium is aspirated. The cells are detached from the bottom of the vessel by adding a trypsin solution to the culture flasks, washed with culture medium and centrifuged off. After determining the number of cells, the cell suspension is adjusted to a concentration of 1-6x10 5 fibroblasts / ml.
  • the medium is aspirated so far that the matrices remain moist.
  • 1 ml of fibroblast medium containing 1-6 ⁇ 10 5 fibroblasts, is pipetted onto the surface of the matrices without injuring the surface.
  • the plate lid is placed and the plate placed horizontally in the incubator.
  • the cultivation of the fibroblasts on the matrix takes place at 37 ° and 5% v / v CO 2 .
  • the culture medium is changed at regular intervals. After 2-4 weeks the dermis model is completely developed.
  • fibroblasts of a suitable passage Prior to seeding on the cross-linked collagen matrices, fibroblasts of a suitable passage are pre-cultured in cell culture flasks with fibroblast medium. After reaching the desired cell density, the culture medium is aspirated. The cells are detached from the bottom of the vessel by adding a trypsin solution (or another solution suitable for detaching adherent cells) into the culture flasks, washed with fibroblast medium and centrifuged off. After determining the number of cells, the cell suspension is adjusted to a concentration of 1-6x10 5 fibroblasts / ml.
  • the medium is aspirated so far that the surface of the matrices remains moist.
  • 1 ml of fibroblast medium containing 1-6 ⁇ 10 5 fibroblasts, is pipetted onto the surface of the matrices without injuring the surface.
  • the plate lid is placed and the plate placed horizontally in the incubator.
  • the cultivation of the fibroblasts on the matrix takes place at 37 ° and 5% CO 2 .
  • the culture medium is changed at regular intervals.
  • the keratinocytes are seeded on the dermis model.
  • the medium is aspirated so far that the surface of dermis equivalents remains moist.
  • 1 ml of keratinocyte medium containing 1-6 ⁇ 10 5 keratinocytes is pipetted onto the surface of the dermis models without injuring the surface.
  • the plate lid is placed and the plate placed horizontally in the incubator (Submerskultur).
  • the cultivation of the fibroblasts and keratinocytes on the matrix takes place for 3-7 days at 37 ° and 5% CO 2 .
  • the culture medium is changed at regular intervals.
  • the keratinocyte medium is aspirated from the wells.
  • the unfinished skin models are taken out of the wells and placed on filter papers.
  • the filter papers lie on metal spacers in each case in a culture vessel.
  • the culture vessel is filled with culture medium (airlift medium, air-liquid interface medium, ALI medium) so far that the medium reaches the upper edge of the filter paper and around the base the skin models distributed.
  • the surface of the skin models is not covered by culture medium (airlift culture or air-liquid interface).
  • the skin models are left in the Air-Liquid-Interface for 1-4 weeks.
  • the culture medium is changed at regular intervals.
  • the THP-1 cell suspension is centrifuged, the pellet subsequently resuspended in keratinocyte medium.
  • the cell count is determined in a Neubauer counting chamber after incubation of the cells with T ⁇ ypanBlue.
  • the THP1 cells are mixed in a ratio of 1: 2 to 1:10 with the keratinocytes. With a seeding of 4 * 10 5 keratinocytes per skin model, this corresponds to a THP1 cell count of 40,000 - 400,000 per skin model.
  • the skin models are then cultured submerged in keratinocyte medium for 2-7 days. Thereafter, they are transferred to the ALI phase (air-liquid interface phase) and cultivated in ALI medium for 7-21 days. b) sowing during the ALI phase
  • the keratinocytes (4 * 10 5 cells per skin model) are cultured at first 2-7 days in submerged keratinocyte medium and subsequently transferred to the ALI phase. After 2 days (1-5 days), the THP-1 cells are seeded to the keratinocytes in a ratio of 1: 2 to 1:10. Further cultivation takes place in ALI medium for 7-21 days.
  • a full skin model according to Example 6 is produced.
  • the full-skin model is cultured on the air-liquid interface as described above on a co-culture insert on a pore-penetrated polymer membrane and contacted with the underlying medium.
  • suitable cell nutrient medium in particular according to Tables 3 or 4
  • a culture of THP-1 cells which are in contact with the skin model via the medium is located in the culture vessel (in particular on the bottom of the culture vessel).
  • the cells of the cell lines THP-1, KG-1 and U937 can be introduced into the skin model both in the naive as well as in the activated state.
  • the appropriate medium for cultivation in culture bottles (without or with cytokines, for example, Tables 3 to 7) is selected.
  • KG-1 cell suspension is centrifuged, the pellet subsequently resuspended in keratinocyte medium.
  • the cell count is determined in a Neubauer counting chamber after incubation of the cells with TrypanBlue.
  • the KG-1 cells are mixed in a ratio of 1: 2 to 1:10 with the keratinocytes. With a seeding of 4 * 10 5 keratinocytes per epidermis model, this corresponds to a KG-1 cell count of 40,000 - 400,000 per epidermis model.
  • the epidermis models are then cultured submerged in keratinocyte medium for 2-7 days. They are then transferred to the ALI phase (air-liquid interface phase) and cultivated in ALI medium for 7-21 days.
  • the keratinocytes (4 * 10 5 cells per epidermis model) are first cultured submerged in keratinocyte medium for 2-7 days and then transferred to the ALI phase. After 2 days (1-5 days), the KG-1 cells are seeded in a ratio of 1: 2 to 1:10 to the keratinocytes. Further cultivation takes place in ALI medium for 7-21 days.
  • An epidermis model according to Example 6 is produced.
  • the epidermis model is contacted with the underlying medium at the air-liquid interface as described above in a co-culture insert on a porous polymeric membrane.
  • the culture vessel in particular on the bottom of the culture vessel
  • suitable cell nutrient medium in particular according to Tables 5 and 6 or the cell medium according to Tables 3, 4, 7 or 8 suitable for the respective cells
  • the substances can now be applied, which are to be tested for a sensitizing effect.
  • the substances By penetrating through the epidermis model, the substances may be modified analogously to the natural process, before they have a detectable response in the epidermis model and in the culture of the dendritic cells.
  • the MoDC, THP-1 and U937 cells are also sown on the epidermis models.
  • the culture phase of the individual cell lines is carried out using the medium which is particularly suitable for this purpose (compare Tables 3, 4, 7 and 8, respectively).
  • the cells of the cell lines THP-1, KG-1 and U937 can be introduced into the epidermis model both in the naive as well as in the activated state.
  • the appropriate medium for cultivation in culture bottles (without or with cytokines, for example, Tables 3 to 7) is selected.
  • Example 9 the appropriate medium for cultivation in culture bottles (without or with cytokines, for example, Tables 3 to 7) is selected.
  • the 11937 cell suspension is centrifuged, the pellet subsequently resuspended in fibroblast medium.
  • the cell count is determined in a Neubauer counting chamber after incubation of the cells with TrypanBlue.
  • the U937 cells are mixed in the ratio 1: 2 to 1:10 with the fibroblasts. With 4 * 10 5 fibroblasts seeding per dermal model, this corresponds to a U937 cell count of 40,000 - 400,000 per dermal model.
  • the dermis models are then cultured submerged in fibroblast medium for 7-28 days.
  • the fibroblasts (4 * 10 5 cells per dermis model) are first cultured submerged in fibroblast medium for 2-7 days. After 2-7 days, the U937 cells are seeded to the fibroblasts in a ratio of 1: 2 to 1:10. Further cultivation takes place for 7-21 days fibroblast medium.
  • a dermis model according to Example 6 is produced.
  • the dermis model is cultured in a co-culture porous polymer membrane insert and contacted with the underlying medium.
  • suitable cell nutrient medium in particular according to Table 8 or the cell medium according to Tables 3 to 7 which is suitable for the respective cells
  • a culture of U937 cells which are in contact with the medium via the medium is located in the multi-well shell (in particular on the bottom of the multi-well shell) Dermis model stand.
  • the MoDC 1 THP-1 and KG-1 cells are also sown on the dermis models.
  • the culture phase of the individual cell lines is carried out using the medium which is particularly suitable for this purpose (see Tables 3 to 6, 8).
  • the cells of the cell lines THP-1, KG-1 and U937 can be introduced into the dermis model both in the naive as well as in the activated state.
  • the appropriate medium for cultivation in culture bottles (without or with cytokines, for example, Tables 3 to 7) is selected.
  • the composition of the different culture media can be found in the following tables:
  • Ready-differentiated full-skin models with dendritic cells according to Example 7 are taken directly from the air-liquid interface, frozen in the cryostat and cut (slice thickness 7-9 microns).
  • the sections are fixed for 10 minutes in -20 C cold acetone and 0 connect with TBS (TRIS-buffered saline) washed several times.
  • the anti-elastin antibody (rabbit, Novotec) is diluted 1:40 with TBS, dropped onto the sections and left for 60 minutes. The sections are then washed three times for 5 minutes each in TBS.
  • a goat anti-rabbit antibody with Alexa conjugate (Molecular Probes) is used in a 1:20 dilution.
  • the dilution of the secondary antibody is made in a 0.1% Evans Blue solution.
  • the secondary antibody is dropped on the sections and left on for 60 minutes. Thereafter, the sections are washed three times for 5 minutes each with TBS. Finally, the antibody-labeled sections are capped in DAKO Faramount embedding medium. As a negative control, sections are incubated without the first antibody.

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Abstract

La présente invention concerne un modèle de peau, en particulier un équivalent du derme, un équivalent de l'épiderme et un équivalent de la peau entière, comprenant une matrice de collagène ainsi que des cellules dendritiques, des procédés de préparation de ces équivalents de derme, d'épiderme ou de peau entière, ainsi que leur utilisation pour le test de substances, en particulier pour rechercher un effet potentiel de sensibilisation, de préférence sur la peau.
PCT/EP2007/000914 2006-02-10 2007-02-02 Modèle de peau avec cellules dendritiques WO2007090575A1 (fr)

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FR2957088A1 (fr) * 2010-03-05 2011-09-09 Immunosearch Methode d'evaluation du potentiel sensibilisant et/ou irritant d'un compose test
FR2957090A1 (fr) * 2010-03-05 2011-09-09 Immunosearch Methode d'evaluation du potentiel irritant d'un compose test
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CN106568911A (zh) * 2016-10-26 2017-04-19 天津科技大学 一种离体状态的模拟皮肤模型
US10073085B2 (en) 2011-03-29 2018-09-11 Osaka University Method for producing artificial skin model, and artificial skin model
EP4299719A1 (fr) 2022-06-28 2024-01-03 Univerza v Mariboru Modèle complexe in vitro de peau humaine, procédé de préparation et utilisation de celui-ci

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JP2013520981A (ja) * 2010-03-05 2013-06-10 イミュノサーチ 試験化合物の感作ポテンシャルを評価する方法
FR2957088A1 (fr) * 2010-03-05 2011-09-09 Immunosearch Methode d'evaluation du potentiel sensibilisant et/ou irritant d'un compose test
FR2957090A1 (fr) * 2010-03-05 2011-09-09 Immunosearch Methode d'evaluation du potentiel irritant d'un compose test
WO2011107611A3 (fr) * 2010-03-05 2011-11-10 Immunosearch Méthode d'évaluation du potentiel irritant d'un composé test
WO2011107679A3 (fr) * 2010-03-05 2011-11-17 Immunosearch Méthode d'évaluation du potentiel sensibilisant d'un composé test
WO2011107614A3 (fr) * 2010-03-05 2011-12-01 Immunosearch Méthode d'évaluation du potentiel sensibilisant et/ou irritant d'un composé test
FR2957089A1 (fr) * 2010-03-05 2011-09-09 Immunosearch Methode d'evaluation du potentiel sensibilisant d'un compose test
US9255295B2 (en) 2010-03-05 2016-02-09 Immunosearch Method for evaluating the sensitizing potential of a test compound
US10073085B2 (en) 2011-03-29 2018-09-11 Osaka University Method for producing artificial skin model, and artificial skin model
US20150250925A1 (en) * 2012-09-04 2015-09-10 Biomedical Technology Hybrid Co., Ltd. Artificial skin tissue, artificial skin model and manufacturing method therefor
CN106568911A (zh) * 2016-10-26 2017-04-19 天津科技大学 一种离体状态的模拟皮肤模型
EP4299719A1 (fr) 2022-06-28 2024-01-03 Univerza v Mariboru Modèle complexe in vitro de peau humaine, procédé de préparation et utilisation de celui-ci
LU502391B1 (en) 2022-06-28 2024-01-09 Univerza V Mariboru A complex in vitro model of human skin, a process for preparation and use thereof

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