WO2007090575A1 - Skin model with dendritic cells - Google Patents

Abstract

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 also dendritic cells, processes for producing the dermis, epidermis and skin equivalents, and also use thereof for testing substances, in particular with respect to a potentially sensitizing activity, preferably on the skin.

Description

 "Skin Model with 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 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. 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.

For the testing of raw materials and products, human cell cultures have mostly been used as in vitro systems in recent years. A further development of cell culture technology is organ-like human tissue or bioartificial constructs and coculture systems. The results obtained can be transferred to humans even better than the results obtained in single-cell cultures.

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. By introducing skin punch biopsies into the collagen lattice, keratinocytes from the punch biopsies can grow on the surface of the dermis equivalent, forming a skin 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. In this case, 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. After formation of the dermis equivalent, keratinocytes can then be seeded, forming a skin equivalent.

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.

Moreover, 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. In addition to the fibroblasts to be cultivated, 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.

In the case of the known skin models, it is disadvantageous that they usually only consist of one or more epidermal layer (s) of keratinocytes. In cases where a stratified epidermis is obtained, 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. If 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. If, in the case of the skin equivalents described in the prior art, 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. As a result, 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. Subsequently, 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.

Finally, it remains to be noted that the skin models known from the prior art are suboptimal in terms of their skin similarity. In particular, the expression of the important dermal differentiation marker elastin is not observed in the known skin models - in contrast to natural dermis.

Allergic reactions are mediated by dendritic cells. The dendritic cells of the skin 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.

The isolation and, in particular, the cultivation of adult Langerhans cells is difficult. Prolonged cultivation is not possible as these cells die within 1-2 days in one culture. Therefore, no skin models can be generated as test systems for sensitizing substances by cocultivation with adult Langerhans cells.

Sensitizing substances (raw materials or active substances), if they come into contact with the skin or the mucous membrane, can cause an allergic reaction (contact allergy).

Known examples of 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.

Such substances enter the vital cell layers of the epidermis during sensitization. In most cases, these are small molecules that can not trigger immune reactions themselves. In the skin, these molecules combine with the skin's own proteins to form a hapten-protein complex that can now be recognized by the immune system. In the case of contact allergy, the protein-hapten complex is taken up by the dendritic cells (Langerhans cells) and processed. Small molecules that do not sensitize per se can become a sensitizing agent (hapten) when altered by the skin's own enzymes. In this case, the actual hapten, which then forms an immunogenic hapten-protein complex with proteins, is formed from a prohapten by the biochemical conversion.

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.

Animal-free tests on whether certain new substances can have a sensitizing effect have hitherto been carried out on two-dimensional cell cultures (monolayers) of different cell types (eg monocyte dendritic cells). The disadvantage here is that in such simple models the complex interactions that occur in the skin when in contact with a sensitizing Agent can not be observed. In addition, only water-soluble raw materials or active ingredients can be introduced and tested in such cell cultures.

There is now a need, without recourse to animal experiments, to provide a test system which overcomes the disadvantages of the prior art and allows prediction of the sensitizing potential of substances in contact with the skin or mucous membranes.

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.

Within the meaning of the present invention, skin model is to be understood as meaning full-body equivalents (also called whole-skin models) and also dermis and epidermis equivalents.

According to the invention, 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.

Preferred are those cells which can express or regulate the sensitizing marker CD86 (B7.2) either basally or due to stimulation.

Particularly preferred are those cells which can either basally or due to stimulation express or regulate both the marker CD86 and the marker CD54. In particular, 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.

According to another preferred embodiment of the present invention, 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. In particular, 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.

In contrast to Langerhans cells and their precursors, as well as cells derived from blood, these cell lines are available as cell cultures and can be divided as often as desired and further cultured. The quality of the resulting skin model is therefore much less dependent on the quality of the individual cell preparations.

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.

According to another preferred embodiment of the present invention, the tumors are selected from myeloid or lymphoid tumors.

Preferably, 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. In this case, the designation of the tumors published in the DSMZ "German Collection of Microorganisms and Cell Cultures" was used.

From these myeloid tumors, cell lines can be obtained which are particularly easy to cultivate. In particular, it is preferred that 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). ,

These cell lines are particularly suitable because they can express or regulate both the surface marker CD86 (B7.2) as the marker CD 54 (ICAM) basal or after stimulation. The numbers in brackets indicate the cell lines. On the homepage of the DSMZ (www.dsmz.de) the mentioned cell lines are described with their origins as well as their characteristics. The information given refers to the November 2005 catalog.

According to another preferred embodiment, 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. In this case, the designation of the tumors published in the DSMZ "German Collection of Microorganisms and Cell Cultures" was used.

According to a particularly preferred embodiment of the present invention, 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).

It is very particularly preferred for the cells to be used according to the invention to be 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).

These cell culture lines are particularly well characterized, in particular with regard to their reactions in sensitization studies.

According to a particularly preferred embodiment of the skin model, 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. 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), TGFβ (Transforming Growth Factor beta), in particular TGFβi, or TNF-α (Tumor Necrosis Factor alpha) and mixtures thereof, more preferably from IL-4 and GM-CSF and mixtures thereof.

According to a particularly preferred embodiment of the invention, 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.

In particular, the 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.

According to a particularly preferred embodiment of the invention, the collagen matrix contains fibroblasts and / or keratinocytes.

As described below, the seeding of different cells can simulate different test systems of the real skin.

If only keratinocytes are seeded, an epidermis model results, while the sowing of fibroblasts without keratinocytes leads to a dermis model.

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.

Due to the presence of keratinocytes and fibroblasts in the skin model, it is possible to provide a test system that replicates the in vivo situation particularly well and realistically.

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.

Advantageously, in contrast to the previously used two-dimensional test cultures (monolayers) in a three-dimensional 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. After application of the test substance to the model, 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.

Furthermore, 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. In the case of a substance that is activated as a prodrug by such a metabolization only as a sensitizing agent (hapten), detection in monolayer cultures is not possible. For example, it is possible that only after the metabolization stable hapten-protein complexes are formed, which only trigger the sensitization reaction.

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.

Advantageously, 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. In addition, 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.

In the context of the present invention, 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.

In the context of the present invention, 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.

According to the invention, "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.

Before filling the containers, z. As the wells of a microtiter plate with the collagen suspension, 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. First, 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.

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.

For this reason, care is usually taken in the freeze-drying of biological material to cause the fastest possible cooling.

Surprisingly, however, 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. According to the invention is 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.

Advantageously, 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. On the other hand, 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. Surprisingly, the Applicant has found that, despite its cytotoxic and apoptotic properties, glutaraldehyde is preferably suitable as a crosslinker for the process according to the invention. However, the crosslinking can also be carried out by physical methods such as UV irradiation and dehydrothermal crosslinking (DHT).

According to the invention, 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.

Usable for the purposes of the present invention is also the crosslinking with carbodiimides such. EDC (1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide) involving N-hydroxysuccinimide.

Also possible according to the invention are 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). Further crosslinking possibilities are listed, for example, in EP-A2-0898973, to which reference is hereby made in its entirety.

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.

According to the invention, moreover, it is provided in a preferred embodiment that 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. 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. As a result, 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.

For cultivating the fibroblast-containing matrix, 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.

For further stabilization of the matrix B, fibronectin and / or laminin, preferably human fibronectin and / or laminin, can be added to the matrix or into the culture medium. 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. Thus, by adding fibronectins and / or laminin to the fibroblast collagen matrix, the binding of the fibroblasts becomes both favors collagen as well as one another. 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. In the context of the present invention, 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.

In addition to the described method of cultivating a full-skin model obtained according to the invention, 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.

Detailed information on improving the barrier function can be found, for example, in US-A1 -20020168768, the disclosure of which is hereby incorporated by reference.

In order to improve fibroblast growth in the dermis equivalent or in the whole skin model, factors may be added to the culture medium which are released from the keratinocytes in the case of cocultivation of fibroblasts and keratinocytes. For example, 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) can be used.

The 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).

According to a preferred embodiment of the skin models according to the invention, in particular the epidermis and the full-skin equivalent, the cells are sown together with the keratinocytes in step e. In the manufacturing process, the isolation, culture and differentiation of the dendritic cells then take place in parallel with the growth of the keratinocytes. After harvesting the two cell types they can be mixed together. 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.

According to another preferred embodiment, the sowing of the dendritic cells into the skin model, in particular the epidermis equivalent or the whole 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.

Particularly preferably, after the model has been transferred to 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. It is particularly preferred to introduce the skin model, in particular the finished skin model, preferably on a suitable carrier, into a culture of dendritic cells overlaid with suitable cell nutrient medium, the lower part of the skin model, in the case of whole skin models in particular the dermis, over the side but in particular only over the soil, which is preferably in contact with the medium via a membrane, which comes into contact with the cells.

The dendritic cells can also be present in a gel instead of in a normal cell culture. Preferably, collagen, agarose or fibrin gels are used, more preferably

Collagen gels.

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. For example, 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.

Advantageously, 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.

In the context of the present invention, 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.

In particular, 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.

According to a preferred embodiment, 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.

In the context of the present invention, 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.

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. Here, 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.

In the context of the present invention, "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:

Preferably, 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. Subsequently, 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 μg / 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.

Complete differentiation of the keratinocyte layers is achieved, for example, by airlift culture in DMEM without hEGF and BPE (bovine pituitary extract). In the context of the present invention, 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 typical dermis equivalent and epidermic equivalent dermal skin model is developed.

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.

However, 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.

Due to the complexity of the skin model produced this can be used specifically for various issues of the chemical-pharmaceutical industry and the cosmetics industry. In particular, 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. Using methods of quantifying cell damage, particularly using a vital dye, such as a tetrazolium derivative, 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. In a particularly preferred embodiment, 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.

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.

In a particularly preferred embodiment of the invention, the effects of chemical substances or other agents on specific skin types are examined. In this case, 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.

In a further particularly preferred embodiment of the invention, 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. For example, 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.

According to a preferred embodiment, the skin equivalent produced according to the invention can be colonized with microorganisms, in particular pathogenic microorganisms. Particularly preferred is a colonization with pathogenic or parasitic, especially human pathogenic microorganisms. In the sense of the invention, 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. As pathogenic and / or parasitic bacteria Staphylococcus aureus are particularly preferred.

With such a settled skin equivalent, both the process of colonization, in particular the infection process, by the microorganism itself as well as the response of the skin to this colonization can be examined.

Furthermore, with such a skin equivalent it is possible to study the effect of substances applied before, during or after the colonization on the colonization itself or on the effects of the colonization on the skin equivalent.

According to a preferred embodiment, 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. cross-linking to produce a second, mechanically stabilized matrix B, e) nucleating and growing keratinocytes on the second matrix B, and finally f) further culturing the keratinocytes growing in and on the matrix B until complete formation of the epidermis equivalent; Before, during or after sowing of the keratinocytes in step e dendritic cells are cocultivated with the epidermis equivalent.

In this case, unlike the method according to the invention for the production of the whole-skin model, only 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. Examples of 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. 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

According to the invention, the medium may contain other factors, for example hormones, growth factors, adhesives, antibiotics, selection agents, enzymes and enzyme inhibitors and the like.

To improve keratinocyte growth in the epidermis equivalent, factors may be added to the culture medium which are released from the fibroblasts in the event of cocultivation of fibroblasts and keratinocytes. For example, 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) can be used.

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.

In order to examine the dendritic cells and, if necessary, to be able to detect a stimulation, ie contact with a sensitizing substance, the cells are preferably removed from the skin model.

For the analysis of the epidermis of full-skin models and epidermis equivalents, they 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.

For the analysis of 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.

Preferably, they may be specifically labeled and analyzed in a cell sorter, e.g. with the FACS method (fluorescence-activated cell sorting).

Furthermore, 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).

The cells can also be examined in the intact skin model. For this purpose, for example, 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.

In particular, 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.

The following endpoints, which are found after contact with sensitizing substances, are particularly preferably investigated: 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.

In the full-skin or epidermis equivalents, in particular in the full-thickness model, it is also possible to investigate whether migration of the dendritic cells out of the epidermis takes place into the dermis or the matrix or into the surrounding medium (migration assay) and, in particular, a maturation of these cells is observable is.

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. The following examples illustrate the invention without, however, limiting it to:

Example 1 :

Production of the matrix A:

In a 1000 ml beaker 700 ml of deionized water are added, which is acidified with 875 .mu.l of concentrated acetic acid (glacial acetic acid). The solution is stirred using a commercially available magnetic stirrer with stirring bar. Into the rotating water column, 7.0 g of bovine tendon collagen are stirred in at once. The mixture is then removed from the stirrer and left for 4 hours without stirring at room temperature. Every 30 minutes, the pH of the mixture is controlled. The pH should be in the range between pH 3.5 and 4. Also every 30 minutes, the suspension is stirred for 1 minute at high speed. Care must be taken that the temperature of the suspension does not increase. Finally, the suspension is stirred for 3 minutes at high speed and thereby homogenized. To avoid heating by the homogenization process, the suspension is kept at room temperature. This gives a milky-turbid collagen suspension. In order to remove possibly existing air bubbles from the suspension, it can be centrifuged at 400 rpm (= 24.73 g) for 1 minute at 20 ^° C. (Hettich Universal 16 R, rotor diameter 138 mm). The bubbles then escape without foaming.

With a dispenser, 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.

Based on a temperature of the gel solution of 20 ° C, the freezing rate is 18 ⁰ C to 23 ° C per hour. The entire freeze-drying process takes about 20-27 hours.

Example 2:

Crosslinking of the matrix A to produce the matrix B

In order to obtain the largest possible and even surface of the skin models, the

Collagen matrices chemically fixed before sowing the fibroblasts.

The fixing agent used is glutaraldehyde (GA): C ₅ H ₈ O ₂ , MW = 100.12. 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, including washing and equilibration, may be carried out for a period of preferably 5 days but at least 4 days.

Example 3: Isolation of MoDC:

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).

Further isolation was carried out by the method of Berger et al. (Journal of Immunological Methods, 298, 2005, pp. 61-72). An alternative, equally applicable method is described in Nutt et al., Journal of Immunological Methods, 293, 2004, pp. 215-218. The identity of the monocytes is checked in a FACS analysis.

Cultivation of MoDC

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.

Example 4: Differentiation of the cells

After culturing in cytokine-supplemented medium (see Table 8), the surface marker CD86 (B7.2) can be detected by means of a specific antibody via flow cytometry (fluorescence activated cell sorting, FACS analysis). Example 5:

Production of a dermis equivalent

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.

From the wells of the microtiter plate containing the matrices equilibrated with fibroblast medium, the medium is aspirated so far that the matrices remain moist. In each case 1 ml of fibroblast medium, containing 1-6 × 10 ⁵ fibroblasts, is pipetted onto the surface of the matrices without injuring the surface. After sowing, 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.

Example 6:

Production of a full skin model

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.

From the wells of the microtiter plate containing the Matrices equilibrated with culture medium, the medium is aspirated so far that the surface of the matrices remains moist. In each case 1 ml of fibroblast medium, containing 1-6 × 10 ⁵ fibroblasts, is pipetted onto the surface of the matrices without injuring the surface. After sowing, 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.

After culturing the fibroblasts for 2-4 weeks, the keratinocytes are seeded on the dermis model. For this purpose, keratinocytes of a suitable passage in Pre-cultured cell culture flasks with keratinocyte 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 (keratinocyte medium) and centrifuged off. After determining the number of cells, the cell suspension is adjusted to a concentration of 1-6x10 ⁵ keratinocytes / ml. From the wells of the microtiter plate containing the dermis equivalents, 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. After completion of sowing, 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.

After the 3-7-day submerged culture, 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. After the unfinished full skin models are placed on the filter paper, 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). Depending on the desired degree of differentiation, the skin models are left in the Air-Liquid-Interface for 1-4 weeks. The culture medium is changed at regular intervals.

Example 7:

Production of a skin model with dendritic cells:

Cocultivation of the dendritic cells in the full skin model according to Example 6:

a) sowing together with the keratinocytes

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 TϊypanBlue. 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.

c) Cocultivation by media contact of a culture of dendritic cells with the skin model

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. In addition to 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).

On the surface of the skin model, which does not come into contact with the medium, substances can now be applied, which are to be tested for a sensitizing effect. Penetration through the skin model, if appropriate, modifies the substances analogously to the natural process before they show a detectable reaction in the skin model and in the culture of the dendritic cells.

In the previously described ways a) to c), the sowing of the MoDC, KG-1 and U937 cells on the skin models. The culture phase of the individual cell lines is carried out using the medium which is particularly suitable for this purpose (see Tables 5 to 8).

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. For this purpose, in each case the appropriate medium for cultivation in culture bottles (without or with cytokines, for example, Tables 3 to 7) is selected.

Analogously, the sowing of the dendritic cells into the dermis equivalent according to Example 5 and the epidermis equivalent prepared analogously to Example 5 with keratinocytes can also be carried out.

Example 8:

Production of an epidermis model with dendritic cells:

Cocultivation of the dendritic cells in the epidermis model according to Example 6:

d) sowing together with the keratinocytes The 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.

e) Seeding during the ALI phase

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.

f) Cocultivation by medium contact of a culture of dendritic cells with the epidermis model

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. In the culture vessel (in particular on the bottom of the culture vessel) there is a culture of KG-1 cells in addition to 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) which are in contact with the skin model via the medium.

On the surface of the epidermis model, which does not come into contact with the medium, substances can now be applied, which are to be tested for a sensitizing effect. 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.

In the previously described manners a) to c), 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. For this purpose, in each case the appropriate medium for cultivation in culture bottles (without or with cytokines, for example, Tables 3 to 7) is selected. Example 9:

Production of a dermis model with dendritic cells:

Cocultivation of the dendritic cells in the dermis model according to Example 6:

g) sowing together with the fibroblasts

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.

h) Sowing during fibroblast culture

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.

i) Cocultivation by medium contact of a culture of dendritic cells with the dermis model

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. In addition to 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.

On the surface of the dermis model, which does not come into contact with the medium, substances can now be applied, which are to be tested for a sensitizing effect. Penetration through the dermis model allows the substances to be modified, if appropriate, analogously to the natural process before they show a detectable reaction in the dermis model and in the culture of the dendritic cells.

In the previously described ways a) to c), 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. For this purpose, in each case 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:

Table 1: fibroblast medium:

Cultivation of THP-1 cells:

Table 3: Culture medium THP-1 I without cytokines:

Cultivation of KG-1 cells:

Table 5: Culture medium KG-1 I without cytokines:

Table 6: Culture medium KG-1 II with cytokines:

Table 7: Culture medium U937 I without cytokines:

Table 8: Culture medium of MoDC cells:

Component final concentration

RPMI 1640 incl. L-glutamine

Fetal CaIf Serum, heat-inactivated 10%

HEPES 1OmM

2-mercaptoethanol 50 μM

Penicillin 100 IU / ml

Streptomycin 100 μg / ml rhGM-CSF 200 IU / ml

IL-4 500 IU / ml

Table 9: Air Liquid Interface Medium:

Example 8:

Detection of elastin in the full skin model

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. As a secondary antibody, a goat anti-rabbit antibody with Alexa conjugate (Molecular Probes) is used in a 1:20 dilution. In order to block the high intrinsic fluorescence of the collagen matrix, 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.

Claims

claims:

A skin model comprising a collagen matrix and dendritic cells.

2. Skin model according to claim 1, characterized in that the collagen matrix is crosslinked.

3. skin model according to claim 1 or 2, characterized in that the dendritic cells are selected from cells or cell lines derived from tumors of the hematopoietic and / or immunological system.

4. Skin model according to claim 3, characterized in that the tumors are selected from myeloid or lymphoid tumors.

5. Skin model according to claim 4, characterized in that the myeloid tumors are selected from tumors called 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.

6. Skin model according to claim 5, characterized in that the cells from myeloid tumors are selected 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).

7. skin model according to claim 4, characterized in that the cells are selected from lymphoid tumors called human plasma cell leukemia, human T cell leukemia, human T 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 lymphoma.

8. Skin model according to claim 1, characterized in that the cells are selected from monocyte-derived dendritic cells (MoDC), cells from cell lines derived from tumors, preferably selected from tumors called human acute myelogenous leukemia, human histocytic lymphoma, human monocytic leukemia, in particular the human cell lines KG-1 (DSMZ no. ACC 14), U937 (DSMZ no. ACC 5), 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).

9. skin model according to claim 8, characterized in that monocyte derived dendritic CeIIs (MoDC) and in particular the human cell lines KG-1 (DSMZ no. ACC 14), U937 (DSMZ no. ACC 5) and THP-1 (DSMZ no. ACC 16).

10. Skin model according to one of the preceding claims, characterized in that the cells are treated before, during or after co-cultivation with the skin model with chemokines and / or cytokines.

11. skin model according to claim 10, characterized in that the cells are treated before, during or after the cocultivation with the skin model with cytokines selected from GM-CSF (Granulocyte Macrophage Colony Stimulating Factor), SCF (Star Cell Factor) , IL-4 (Interleukin 4), IL-13 (Interleukin 13), TGFβ (Transforming Growth Factor beta), in particular TGFβi, or TNF-α (Tumor Necrosis Factor alpha) and mixtures thereof, more preferably IL-4 and GM -CSF and mixtures thereof.

12. Skin model according to one of the preceding claims, characterized in that the collagen matrix contains fibroblasts and / or keratinocytes.

13. Skin model according to one of the preceding claims, characterized in that it is a full-skin equivalent, are contained in the fibroblasts in the matrix and keratinocytes on the matrix.

14. skin model according to one of claims 1 to 12, characterized in that it is a dermis equivalent, are included in the fibroblasts in the matrix.

15. skin model according to one of the preceding claims 1 to 12, characterized in that it is an epidermis equivalent, are contained in the keratinocytes on the matrix.

16. Skin model according to one of the preceding claims, characterized in that it is colonized with microorganisms, in particular pathogenic microorganisms.

17. skin model according to claim 16, characterized in that the microorganisms are selected from Staphylococcus aureus and species of the genus Candida, Malassezia and Trichophyton.

18. A method for producing a skin model, 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. lyophilizing the collagen suspension and thus producing a first matrix A, d. subjecting the first matrix A to cross-linking and thus creating a second, mechanically stabilized matrix B, e. Flock fibroblasts and / or keratinocytes (optionally one after the other) to the second matrix B and grow, f. culturing the cells growing in and on the matrix B until complete formation of the dermal and epidermal part of the whole skin model, before, during or after the seeding of the cells in step e. dendritic cells cocultivated with the skin model.

19. The method according to claim 18 for the preparation of a dermis equivalent, 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.

20. The method according to claim 18 for the preparation of an epidermis equivalent, 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. Keratinocytes are seeded on the second matrix B and allowed to grow and finally f. the keratinocytes growing in and on the matrix B are further cultured until the epidermis equivalent is fully formed, coculturing dendritic cells with the epidermis equivalent before, during or after the keratinocytes are seeding in step e.

21. The method according to claim 18 for the preparation of a whole skin model, 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. Seeding and growing fibroblasts onto the second matrix B, f. Keratinocytes are seeded on the second matrix B and allowed to grow and finally g. culturing the cells growing in and on the matrix B until complete formation of the dermal and epidermal portions of the whole skin model, co-culturing dendritic cells with the skin model before, during, or after seeding the cells in step e or step f.

22. A method for producing a whole skin model according to claim 21, characterized in that the dendritic cells are cocultured before, during or after seeding of the keratinocytes in step f with the skin model.

23. Method according to claim 18, characterized in that the sparingly soluble collagen is a coarse-fiber collagen which shows no visible or only slight swelling or gel formation in the aqueous medium over several hours, preferably poorly soluble collagen Collagen from the tendons of animals, in particular of mammals, preferably of horses, pigs or cattle, most preferably from the Achilles tendon of cattle.

24. The method according to any one of claims 18 to 23, characterized in that the lyophilization process in step c) with a cooling rate of up to 50 "C per hour, in particular 5 ⁰ C to 40 ⁰ C per hour, preferably 10 ⁰ C to 30 ⁰ C per hour, more preferably 15 ⁰ C to 25 ⁰ C per hour, most preferably from 18 ⁰ C to 23 ⁰ C and even more preferably from 20 ⁰ C to 22 ⁰ C per hour is performed.

25. Whole skin model, obtainable by a process according to one of claims 21 to 24.

26. Whole skin model according to claim 25, characterized in that typical skin proteins, in particular elastin, are expressed.

27. Dermis equivalent, obtainable by a process according to claim 19.

28. Epidermis equivalent, obtainable by a process according to claim 20.

29. Use of a skin model according to any one of claims 1 to 17, a whole skin model according to claims 25 or 26, a dermis equivalent according to 27 or an epidermis equivalent according to claim 28 for product testing, in particular with regard to efficacy, undesired side effects, irritant, toxicity and inflammatory effects or sensitizing or allergenic effects, or tolerability of substances.

30. A method for detaching the epidermis from a skin model, preferably an epidermis equivalent, more preferably a whole skin model, characterized in that the epidermis of the skin model with proteolytic enzymes, preferably thermolysin, is treated.