DE10327879A1 - Reconstructed dermal papilla - Google Patents

Abstract

The present invention relates to a reconstructed dermal papilla (pseudo-papilla), their preparation and their use, in particular in the field of medicine, pharmacy and cosmetics, and a skin / hair equivalent containing such reconstructed papillae (pseudo-papillae), in particular a skin - / hair model with reconstructed papillae in a reconstructed dermis (pseudo-dermis), and its preparation and use, in particular in the field of medicine, pharmacy and cosmetics.

Description

The The present invention relates to a reconstructed dermal papilla (Pseudo-optic disc), their preparation and their use, especially in the field medicine, pharmacy and cosmetics as well as a skin / hair equivalent containing such reconstructed papillae (pseudo-papillae), in particular a skin / hair model with reconstructed papillae in a reconstructed dermis (pseudo-dermis), and its preparation and its use, in particular in the field of medicine, pharmacy and cosmetics.

Around To find agents that, for example, a biological effect exercise on the hair follicle and thus influence hair pigmentation, hair growth as well as hair structure can, have to suitable in vitro test systems exist where such Effect can be evaluated. Ideally, these test systems should the screening of a larger number of substances, standardizable and cost-effective be as well as - if these are in vitro systems acts - the possible in vivo situation to come close.

In hair research currently has no suitable in vitro models, for example, hair growth, hair pigmentation and hair texture to investigate. A compilation of existing methods as well a description of the disadvantages of these systems can be found z. B. at K. S. Stenn "Laboratory Assessment of Hair Follicle Growth "in Skin Pharmacol. Appl. Skin Physiol. 1999; 12: 154-157. These Systems range from monolayer cell cultures to animal models and ex vivo systems to in vitro systems.

Monolayer cultures of hair follicle cells have the disadvantage that the cells, when taken out of their complex three-dimensional structures will behave differently than they would in the overall organ. Therefore are statements about the effect of substances on hair follicle cells acting as monolayer were cultivated for the in vivo situation of little relevance. Animal models are for development of cosmetic products prohibited under the Cosmetics Directive. Ex vivo models using in vitro methods with in vivo animal methods combine, therefore, are also out of the question. Similar Problems of availability Material and the standardizability arise in the Use of skin explants with hair in culture. In vivo human studies Although carried out to hedge the effect, but recommend only when a potent drug is discovered and put into a formulation was incorporated, since these studies are correspondingly expensive and expensive are. For screening substances that affect the hair color, also lack appropriate in vitro test systems.

In vitro tests to change hair pigmentation by influencing melanin production The melanocytes are also mainly on single cell cultures carried out. Often For this purpose, epidermal melanocytes or B16 melanoma cells are used and from the results on this cell type on the hair marmocytes extrapolated, as the latter are difficult to isolate and to cultivate are. In addition, in these systems the complex interaction the melanocyte with the hair follicle is missing and relevant to the situation Therefore, in vivo on the hair follicle must be questioned. The same applies to reconstructed skin models containing (epidermal) melanocytes. Animal models, also a popular test model for substances, which has an influence have the hair pigmentation, pose, forbid after the Cosmetics Directive, if the substances for Cosmetic products are to be used. In vivo human studies are tedious and expensive and therefore recommend, if a potent drug was found.

at the technique of "tissue Engineering " various cell types of tissue, z. B. the skin, isolated and multiplied in cell culture as so-called monolayer or single layer. The tissue is then reconstructed from the individual cells. In the case of the skin can to z. As fibroblasts in a collagen gel or other matrix seeded so that they proliferate and form a pseudo-dermis. On this you can epidermal keratinocytes are applied, which also proliferate and form a pseudo-epidermis. By raising the culture to the Air (so-called "air-liquid interface") begin the cells to differentiate and form a stratum corneum.

In the hair research has been mainly with cell cultures, z. B. keratinocytes of the outer root sheath (ORS keratinocytes, ORS = Outer Root Sheath), dermal papilla cells etc. worked. But there were also repeated attempts Hair follicles or parts of hair follicles as a three-dimensional Model, z. B. in a collagen gel to cultivate or whole hair follicles to reconstruct by combining different hair follicle cells.

By M. Michel et al. "Characterization of a new tissue-engineered human skin equivalent with hair" in In Vitro Cell. Dev. Biol. Animal 35: 318-326, June 1999, reports for the first time the insertion of a hair follicle into a reconstructed skin model for use in penetration studies. Here are Whole hair follicles used, which must first be prepared from hairy skin. Apart from the limited availability of the material, the standardizability when using prepared hair is poor because the biological variations are large.

In the EP 0 285 471 A1 and the EP 0 285 474 A1 It also describes the production of an artificial skin consisting of a dermal layer of contractile cells (fibroblasts) and extracellular matrix components into which whole hair follicles or follicular segments are inserted. This is then additionally coated with keratinocytes, which form an epidermal layer. Disadvantage here is that no reconstruction of the papillae takes place, but only part of the hair follicle without papilla is used.

A Japanese working group (M. Inamatsu et al., Hair Follicle Development in Organotypic Culture, "Third Intercontinental Meeting of Hair Research Societies (Abstract), Tokyo, 2001) used as a model for the early phase of hair development freshly isolated dermal papillae from rat whiskers that between a collagen gel with fibroblasts and an epidermal Layer of rat keratinocytes are inserted. This organotypic Culture is at the air-liquid interface (Air-liquid interface) cultured. After 7 days there should be a thickening of the epidermis near the dermal papillae come. First, there are no humane ones here Used cells or papillae, secondly, it is whole, Papillae isolated from hair follicles and not reconstructed Papillae, and third, the papillae are not inserted into the dermis (eg sprayed or grafted), but between the dermal and the epidermal layer is laid. When using isolated papillae The same problems of availability and standardizability arise as with isolated hair follicles.

something similar applies to the work of S.A.W. Watson et al. "Sheep vibrissa dermal papillae induce hair follicle formation in heterotypic skin equivalents "in British Journal of Dermatology (1994) 131, 827-835. Here also dermal papillae or in a collagen gel cultured papilla cells between dermis and epidermis of a dermal equivalent placed with the aim of developing a model of the hair follicle to obtain. However, the dermal papilla cells migrate among them Conditions in the dermal matrix and do not form too a papilla, so that the model must be modified by the papillae or papilla cells are applied to a dermal substrate which will be followed covered by a fetal mouse epidermis and transplanted to nude mice becomes.

A. B. Jahoda et al. "Dermal-Epidermal Interactions - Follicle-Derived Cell Populations in the Study of Hair-Growth Mechanisms "in J. Invest. Dermatol. 101: 33S-38S, In 1993, in 1993, a follicle-like structure from a To produce combination of hair cells by first outer Root Sheath cells (ORS cells) in the collagen capsule of the follicle of a rat whisker and subsequently a mixture of different hair follicle cells - dermal Papilla cells, dermal sheath cells and matrix cell additions. You needed However, the stable collagen capsule of the rat whisker Stabilization of the structure.

A. Limat et al. "The Outer root sheath (ORS) cells organize into epidermoid cyst-like spheroids When cultured inside Matrigel ^®: a light-microscopic and immunohistological comparison between human ORS cells and keratinocytes interfollicular" in Cell Tissue Res (1994). 275: 169-176 add also different hair follicle cells and skin cells in a three-dimensional structure together to study their interaction. They use a collagen (I) gel as base and embed it, a layer of Matrigel ^®, a mixture of basement membrane components containing various cells or cell mixtures containing. The epidermal or ORS keratinocytes (= keratinocytes of the outer root sheath of the hair) form in the Matrigel ^® spheroidal structures, which are not follicular. Here, layers containing different cells are layered on top of each other. However, no new structure is built up within these layers, such as the dermal papilla. Limat et al. describe that ORS keratinocytes can form cystoid structures that internally corrode, but this is not the physiological nature of the hair shaft formation, but the differentiation and stratum corneum formation by cystoid keratinocytes. The Haarschaftbildung but usually done by matrix cells, which are above the dermal papilla.

Japanese Laid-Open Patent Publication No. 10-136977 of Toyobo Co., Ltd., Japan, describes an artificial tissue and its reconstruction having hair-shaft-like structures at the boundary between a layer of fibroblasts in a collagen layer and a collagen layer containing hair papilla cells. This model is intended as a test system for determining the compatibility and efficacy of drugs and cosmetics. Here, the same applies as in the publication of A. Limat et al .: In this model, no dermal papillae are reconstructed, the three-dimensional structure of the hair follicle Instead, the cells are aligned one above the other in layers, and no new structure is reproduced. Melanocytes are obviously also not used in the described model. As an application for the model efficacy tests are called. However, it is not indicated which endpoints should be assessed on the model, ie how the application of active ingredients affects the structure of the reconstructed model and what can be read thereon in terms of the effect of this substance on hair growth and hair structure.

The "Philpott Model" (M.P.Phlpott "Human Hair Growth in vitro ", J. Cell. Sci. 97, 463-471, 1990), in which isolated hair follicles were kept in culture for 9 days have, on the one hand, the disadvantage of the great variability between the individual follicles and thus a poor standardizability and second, the poor availability of hair follicles. Therefore, only a very limited amount of active ingredients within a assessed period. In addition, only substances or formulations be administered, which are soluble in the medium, but not water-insoluble Substances or formulations such. B. creams.

So to say as a further development of the "Philpott model" was also tried isolated hair follicle segments in reconstructed skin models too insert (see M. Inamatsu et al., and M. Michel et al.). In order to Although there are follicles, follicular segments or dermal papillae in contact with the dermis, which is closer to the in vivo situation than other models the prior art; however, the disadvantages of these systems are if they are to be used to study drugs, much like that of the "Philpott model" (poor availability the hair follicle, no standardizability, high effort etc.).

Problematic it is also enough to achieve high cell density of the cultured papilla cells, thus Conclusions on the natural ones Cell-cell interactions within the dermal papilla possible are.

The The problem underlying the present invention is therefore in the provision of a reconstructed papilla or a skin / hair equivalent ("Skin model"), especially one Skin / hair model with reconstructed papillae in a reconstructed Dermis, which the previously described disadvantages of the prior Technology at least partially avoids.

Further Object of the present invention is the discovery or provision of a skin / hair equivalent or -modells, which is suitable as an in-vitro model system, in particular for Testing and / or evaluation of active substances, in particular on Hair follicles. In particular, such a model is intended for discovery and testing pharmaceutical-pharmaceutical as well as cosmetic or active substances are suitable. Furthermore, such a system should also an in vitro assessment of the influence of the hair follicle, the Hair growth, hair pigmentation, hair texture and the like allow by such agents.

The The present invention relates to a reconstructed dermal papilla (Pseudo-optic disc), their preparation and their use, especially in the field medicine, pharmacy and cosmetics as well as a skin / hair equivalent containing such reconstructed papillae (pseudo-papillae), in particular a skin / hair model with reconstructed papillae in a reconstructed dermis (pseudo-dermis), and its preparation and its use, in particular in the field of medicine, pharmacy and cosmetics.

object The present invention thus provides a process for the production a reconstructed papilla (pseudo-papilla), characterized that a suspension comprising cultured papilla cells in a dispersing solution is dropped, in which the suspension is reconstructed to a Papilla (pseudo-papilla) solidified or formed and their use, especially for test purposes, preferably in the field of medicine, pharmacy and cosmetics.

With the term "solidification" is in the context of the invention meaning that the suspension starts after dropping into the dispersing solution, a less liquid, tougher, tougher or more solid consistency. The drops are therefore gelatinous (gelled), densified, crosslinked, polymerized or hardened. So becomes a papilla like Tissue homolog formed.

there Preference is given to the formation of a matrix, preferably a gel a, which then optionally with the discharge of any existing Medium contracted or contracted by the cells.

A particular advantage of the resulting pseudo-papilla is that it achieves a much higher cell density compared to a gel or matrix containing a normal suspension of cultured papilla cells can be. Furthermore, the physiological shape of the papilla is reproduced very well by the technique (in the form of three-dimensional droplets or spheres).

A Such high cell density is especially important to the natural situation in the hair follicle, especially with regard to the cell-cell contacts and / or the resulting formation of typical proteins within the papilla, especially the high concentration of growth factors and / or extracellular Matrix proteins within the follicle, particularly good to reconstruct. About that In addition, a higher cell density correspondingly better signals during use, in particular of the so reconstructed papilla containing reconstructed hair follicle model, allow as a test system.

The flexible internal structure of the matrix used allows further advantageously a self-organization of the cells to physiological compatible or realistic (ie physiological) aggregates that freely organize themselves in it can, wherein the formed matrix as a flexible structural framework for training of characteristic proteins, in particular a cell-own new matrix, serves.

The thus obtained pseudo-papilla advantageously has a comparable Size or a comparable volume as the natural human papilla on, so that the reconstruction in particular of the human, dermal papilla a reconstructed hair follicle or a hair model better the natural one Situation can be modeled.

in the Contrary to the reconstruction of the papilla on solid supports it simply by appropriate choice of the composition of the dripping Suspension possible, the hair-specific extracellular Simulate matrix. This can be done especially by the use a mixture of different components are modeled. It can additionally also the degree of crosslinking (of the components) by the corresponding Gelling conditions freely selected become. For example, by increasing the collagen concentration the "hardness" or the rigidity the reconstructed papilla are also increased.

By Variations of the matrix molecules used are beyond that possible, the biological activity the cells, for example the relationship between apoptosis and Proliferation of cells to influence or modulate.

simultaneously is the inventively prepared Making pseudo-papilla with high reproducibility and allows so a high degree of standardizability by means of the reconstructed hair or hair follicle model.

The cultured dermal papilla cells (hair papilla cells) leave to produce in a conventional manner, for example by one dermal papilla cells (hair papilla cells) from the hair follicles more human or animal skin, then cultured and then extracted the resulting single-layer cultures (Monolayer cultures), which gains dermal papilla cells, in particular through gentle trypsinization. The number of passages should be as possible be low, in general, the number of passengers for the dermal Papillae in the range of 1 to 10, preferably 1 to 3. The Cultivation of the dermal papilla cells takes place in a suitable broth where as a nutrient medium in particular essential minimal medium MEM, for example DME medium (Dulbecco's modified Eagle Medium) and / or RPMI medium (from the Roswell Park Memorial Institute developed medium) and / or Chang medium, if appropriate together with other components, such as fetal calf serum (FCS), collagen, especially type I collagen, and the like. examples for according to the invention suitable Culture Media for the Cultivation of the dermal papilla cells are all based on RPMI or DMEM based cell culture media, such. B. RPMI 1640 (Sigma) with 20% FCS, Chang medium (Irvine Scientific) with 10% FCS and the like. Preferred are for the In particular, passage numbers of pseudo-papillae produced by dripping 1 to 3.

According to a preferred embodiment of the invention, the suspension to be used is obtained by mixing the papilla cells obtained from single-layer cultures, optionally in a suitable nutrient medium with a matrix-forming, in particular gel-forming medium MBM _S , which at least one matrix former MB _S , in particular gelling agent, and optionally further Contains ingredients;

The matrix-forming, in particular gel-forming medium MBM _S contains as a matrix former MB _S , in particular gelling agent, at least one collagen, preferably collagen type I, and optionally other ingredients which are in particular selected from the group of matrix and / or scleroproteins, esp laminin, gelatin, chitosan, glucosamines, glucosaminoglycans (GAG), heparan sulfate proteoglycans, sulfated glycoproteins such as nidogen (in particular entactin), tissue-plasmimogen activator and growth factors such as tissue growth factor beta (TGF-β), fibroblast growth factor, and other growth factors from the Engelbreth-Holm-Swarm tumor (EHS tumor), and / or the human placenta and mixtures of the aforementioned ingredients. As according to the invention suitable matrix materials in the slurry for forming the pseudo papillae (PP), for example, the following substances can be used: Matrigel ^® Basement membrane matrix (Matrigel ^®), collagen, gelatine, collagen / chitosan / GAG (GAG = glucosaminoglycan), glucosamine or any other type of matrix, as well as mixtures of these substances.

Particularly preferred are collagen, Matrigel ^® and mixtures thereof. In the reconstructed papillae thus prepared, the cells can be further cultured with a comparatively low apoptosis or necrosis rate. Particularly preferred are mixtures of collagen, particularly collagen I, and Matrigel ^® in a ratio of between 0.1: 1 to 10: 1 (collagen: Matrigel ^®.

Very particular preference suspensions is between 1: 1 and 6: 1, for example 3: 1, 4: 1 or 5: 1 ((collagen: Matrigel ^®).

In the suspension can additionally be included further reagents. For example, such Reagents are used, which promote solidification, in particular crosslinking reagents, such as thrombin and / or chemical Crosslinking agents, such as carbodiimides or glutaraldehyde.

These Suspension is dropped into a dispersing solution. It may be preferred be, the dispersing solution while to move the dripping, in particular to stir.

The Size of the dripping device, in particular their outlet diameter or the outlet size, and the outlet pressure influence besides the composition of the suspension the size of the resulting Pseudo-papilla. The diameter of the reconstructed papilla is reduced with increasing contraction of the matrix, which is all the stronger the more cells and / or matrix formers are contained.

The Dripping preferably takes place through the cannula (for example a syringe) or similar devices. This is a cannula diameter from 0.4 to 1.8 mm, in particular from 0.6 to 1 mm, particularly preferred there are so pseudo-papillae with a diameter, in particular from about 0.5 to 3 mm in diameter (on the day of manufacture) can be obtained.

Depending on the size, a pseudopapilla according to the invention may preferably contain from 10 ² to 10 ⁹ , in particular 10 ² to 10 ⁷ cells / ml (on the day of preparation). The number of cells per ml continuously increases due to the contraction of the matrix of the papilla and the proliferation of the cells.

advantageously, is it possible over the Outlet size of the dripping device the size of the pseudo-papillae to vary and adapt to the requirements, in particular at solid carriers not or only with difficulty possible is.

The is dispersing solution according to the invention especially not aqueous. Particularly suitable are low-viscosity neutral oils, the also contain other, in particular non-aqueous components can.

According to one preferred embodiment is the dispersing solution selected from liquid Fats, in particular paraffin oils or triglycerides, which are preferably independent of one another of fatty acids with 4 to 18, in particular carry 6 to 12 C atoms.

It is also possible two immiscible liquids if they are chosen in particular so that the reconstructed Papillae are no deeper than at the phase boundary.

When Another component is in particular liquid, unreactive, optionally substituted, in particular perfluorinated hydrocarbons, in particular with a chain length from 8 to 16 carbon atoms, which also preferably has one or more hydroxy groups can carry suitable.

The pseudo-papillae thus prepared can then be freed from the dispersion solution and in egg ner physiological solution or nutrient medium washed or stored. In order to separate the pseudo-papillae from the dispersing solution, suitable auxiliaries and procedures known to the person skilled in the art may be used, which should be particularly gentle, so as not to impair, damage and / or destroy the structure and functioning of the pseudo-papillae. For example, corresponding tweezers, pipettes and nets, sieves, membranes or filters with appropriate pore size are suitable.

These reconstructed dermal papillae can be used in the field of pharmacy, Medicine or cosmetics and body care, in particular for the discovery, investigation and / or (re) testing of pharmaceutical or cosmetic active ingredients, in particular in terms of compatibility and / or efficacy, especially with respect to the hair follicle, especially with regard to hair pigmentation, hair growth, the hair structure, hair color and the like are used.

Of the Use in preferably automated screening methods, in particular for the discovery, investigation and / or (re) testing of pharmaceutical or cosmetic agents, preferably for in vitro evaluation of the influence hair follicle, hair pigmentation, hair growth, hair structure, the hair color and the like, in particular by pharmaceutical or cosmetic agents.

• (a) Bereitstellung einer geeigneten rekonstruierten Dermis (Pseudo-Dermis; PD) oder eines Pseudo-Dermis-Ansatzes;
• (b) Bereitstellung rekonstruierter Papillen (Pseudo-Papillen; PP), die kultivierte Papillenzellen, vorzugsweise dermale Papillenzellen (Haarpapillenzellen), in einer geeigneten Matrix, insbesondere Gelmatrix, umfassen, oder aber Bereitstellung entsprechender Vorläufer solcher rekonstruierten Papillen (Pseudo-Papillen; PP), die kultivierte Papillenzellen, insbesondere dermale Papillenzellen (Haarpapillenzellen), in einem geeigneten matrixbildenden, insbesondere gelbildenden Medium MBM_PP umfassen, wobei das matrixbildende, insbesondere gelbildende Medium MBM_PP in situ, insbesondere in der rekonstruierten Dermis (Pseudo-Dermis; PD), eine Matrix, insbesondere Gelmatrix, ausbilden kann;
• (c) Einbringung oder Insertion der unter Schritt (b) bereitgestellten rekonstruierten Papillen (Pseudo-Papillen; PP) oder deren Vorläufer in die unter Schritt (a) bereitgestellte Pseudo-Dermis (PD) bzw. den Pseudo-Dermis-Ansatz;
• (d) gegebenenfalls Aufbringung einer rekonstruierten Epidermis (Pseudo-Epidermis; PE oder eines rekonstruierten Periderms (Pseudo-Periderm; PI) auf die Pseudo-Dermis (PD), dadurch gekennzeichnet, dass die unter b) bereitgestellte rekonstruierte Papille (Pseudo-Papille, PP) gebildet wird, in dem eine Suspension, umfassend kultivierte Papillenzellen, in eine Dispergierlösung eingetropft wird, in der sich die Suspension zu einer rekonstruierten Papille verfestigt bzw. polymerisiert.

The present invention furthermore relates to a method for producing a skin / hair equivalent, in particular a skin / hair model with reconstructed papillae (pseudopapillae; PP) in a reconstructed dermis (pseudo-dermis; PD), the method comprising the following method steps comprising:

• (a) providing a suitable reconstructed dermis (pseudo-dermis; PD) or a pseudo-dermis approach;
• (b) providing reconstructed papillae (pseudopapillae; PP) comprising cultured papilla cells, preferably dermal papilla cells (hair papilla cells), in a suitable matrix, in particular gel matrix, or else providing corresponding precursors to such reconstructed papillae (pseudo-papillae; PP) comprising cultured papilla cells, in particular dermal papilla cells (hair papilla cells), in a suitable matrix-forming, in particular gel-forming medium MBM _PP , wherein the matrix-forming, in particular gel-forming medium MBM _PP in situ, in particular in the reconstructed dermis (pseudo-dermis, PD), a Matrix, in particular gel matrix, can form;
• (c) introduction or insertion of the reconstructed papillae (pseudopapillae; PP) or their precursors provided under step (b) into the pseudo-dermis (PD) or the pseudo-dermis approach provided under step (a);
• (d) optionally applying a reconstructed epidermis (pseudo-epidermis, PE or a reconstructed periderm (pseudo-periderm, PI) to the pseudo-dermis (PD), characterized in that the reconstructed papilla (pseudo-papilla, PP) is formed by dropping a suspension comprising cultured papilla cells into a dispersing solution in which the suspension solidifies or polymerizes into a reconstructed papilla.

in the Process step (a) is thus the provision of a reconstructed Dermis or pseudo-dermis or a pseudo-dermis approach. According to the invention, each pseudo-dermis known from the prior art can be used, provided for the inventive method is suitable, this means in particular that it is compatible with the other constituents of the skin / hair equivalent according to the invention is compatible. In particular, the inventively used Pseudo-dermis (PD) cultured contractile cells, in particular Fibroblasts, preferably dermal fibroblasts, in a suitable Matrix. The matrix may in particular be a matrix based on collagen, preferably collagen type I and / or type III, and optionally be further components. The cultured contractile cells for the Pseudo-dermis (PD) or the pseudo-dermis approach be obtained in a conventional manner, for example by the fact that isolating dermal fibroblasts from human or animal skin, subsequently cultivated and then from the resulting single-layer cultures (Monolayer cultures) which gains contractile cells (e.g. by gentle trypsinization). For the cultivation of contractile Cells are used a suitable nutrient medium, in particular should be compatible with the contractile cells; as suitable according to the invention broth For example, one can use essential minimal medium MEM (Modified Eagle Medium).

The pseudo-dermis (PD) can then be obtained by mixing the obtained from single-layer cultures, optionally in a suitable nutrient medium contractile cells with a matrix-forming, in particular gel-forming medium MBM _PD , which at least one Ma trixbildner MB _PD , ins special gelling agent, and optionally contains other ingredients.

The resulting mixture is referred to in the context of the invention as a pseudo-dermis approach. The pseudo-dermis approach forms depending on the concentration of the matrix-forming medium faster (higher concentration) or slower (lower concentration) a matrix, preferably a gel matrix, finally, and finally, possibly with the emission of existing nutrient medium, too a pseudo-dermis (PD). All phases or states of the matrix formation and the Contraction is encompassed by the term pseudo-dermis approach. The pseudo-dermis therefore becomes after completion of matrix formation and contraction of the pseudo-dermis approach.

The matrix former MB _PD , in particular gel former, of the matrix-forming, in particular gel-forming medium MBM _PD may in particular be a matrix former based on collagen, preferably collagen type I and / or type III, and optionally further components (eg constituents the dermis extracellular matrix, preferably matrix and / or scleroproteins such as laminin).

On the thus provided or generated pseudo-dermis (PD) one can optionally a suitable nutrient medium, in particular essential minimal medium MEM, and optionally apply additional components.

The Pseudo-papillae (PP) provided in step (b) include cultured ones Papilla cells, in particular dermal papilla cells (hair papilla cells), in a suitable matrix. In the process, the pseudo-papillae become smaller one of the above-described processes for the preparation of reconstructed papilla (pseudo-papilla) produced. In particular, the matrix can be a matrix based on collagen, in particular type I collagen, and optionally act on other components.

It may be preferable to the so over the dripping and hardening produced pseudo-papillae directly or in a physiological solution, in particular a nutrient solution and only after several days, in particular 1 to 14, preferably 4 into the pseudo-dermis for up to 10 days.

The Advantages of using the method according to the invention reconstructed papilla, as already described, in particular in the high cell density, the good handling of these pseudo-papillae and the good properties in terms of strength at the same time Flexibility, great resemblance to natural Model and good biological activity of the papillary cells within the reconstructed papilla.

The Pseudo-papillae, which are caused by the grafting (hereinafter may also be referred to as individual pseudo-papillae for distinction) used to make a macroscopic pseudo-papilla with multiple contained therein according to the invention To produce pseudo-papillae.

The macroscopic pseudo-papillae are then obtained by mixing the pseudo-papillae optionally produced in a suitable nutrient medium according to the invention with a matrix-forming, in particular gel-forming medium MBM _PP containing at least one matrix former MB _PP , in particular gelling agent, and optionally further constituents; the resulting mixture forms a matrix, preferably a gel, which subsequently, with the expulsion of the nutrient medium which may be present, contracts. The macroscopic pseudo-papillae can then be formed from the contracted matrix or the contracted gel (eg by punching out or punching out).

As described below, the formation of the macroscopic pseudo-papillae (PP) but also in situ, especially in the pseudo-dermis (PD), take place. The matrix-forming, in particular gel-forming medium MBM _PP contains as matrix former MB _PP , in particular gelling agent, at least one collagen, preferably collagen type IV, and optionally other constituents which are in particular selected from the group of matrix and / or scleroproteins, in particular laminin, Gelatin, chitosan, glucosamines, glucosaminoglycans (GAG), heparan sulfate proteoglycans, sulfated glycoproteins such as nidogen (especially entactin), tissue-plasmimogen activator, and growth factors such as tissue growth factor beta (TGF-β), fibroblast growth factor, and other growth factors from the Engelbreth-Holm-Swarm tumor (EHS tumor), and / or the human placenta and mixtures of the aforementioned ingredients. As according to the invention suitable matrix materials for forming the pseudo papillae (PP), for example, the following substances can be used: Matrigel ^® Basement membrane matrix (Matrigel ^®), collagen, gelatine, collagen / chitosan / GAG (GAG = glucosaminoglycan), glucosamine or any other type of matrix as well as mixtures of these substances are used. Particularly preferred are collagen, Matrigel ^® and mixtures thereof. Very particular preference Matrigel ^® and mixtures in a ratio of between 0.1: 1 to 10: 1 (collagen: Matrigel ^®

As previously mentioned, can the macroscopic pseudo-papillae (PP) then out of the matrix, in particular the gel, being shaped, the shaping of these pseudo-papillae either before or after the introduction made in step (c) or insertion of the macroscopic pseudo-papillae (PP) or their precursors (For example, the matrix containing the individual pseudo-papillae, the formed into macroscopic pseudo-papilla) can be done.

Both the matrix generator MB _PD for the formation of the pseudo-dermis (PD) and the matrix formers MB _S for the formation of the individual pseudo-papillae and MB _PP for the formation of the macroscopic pseudo-papillae should be capable of being heated, in particular under heating at temperatures of 20 ° C to 40 ° C, to gel, for example, under polymerization, and to promote the growth and differentiation of cells. The formation of the matrix of the pseudo-dermis (PD) and the formation of the matrix of the macroscopic pseudo-papilla are generally carried out in three-dimensional structures. The matrix formation, in particular gelation, is preferably irreversible.

in the others are under the term pseudo-papilla (PP) both by the inventive method prepared reconstructed papilla (single pseudo-papilla) as well to understand the macroscopic pseudo-papilla.

The introduction or insertion of the pseudo-papillae (PP) carried out in step (c) can take place in various ways:
According to one embodiment, suitable cavities for receiving the pseudo-papilla (PP), in particular by punching or piercing, can be formed, preferably in the already contracted pseudo-dermis (PD), and then the pseudo-papillae (PP) are formed in the cavities thus formed. containing cultured papilla cells which, in the case of the macroscopic pseudo-papilla, are shaped so that their dimensions correspond to the voids formed in the pseudo-dermis (PD), or inserted (e.g. by grafting). The punching of the pseudo-dermis (PD) or piercing into the pseudo-dermis (PD) can with a z. B. punch (eg., With a diameter of 0.5 to 4 mm, preferably about 2 mm) or with a button cannula or with a conventional cannula.

Before the introduction of the pseudo-papilla (PP) or its precursors, the punched out or punctured cavities in the pseudo-dermis (PD) can still be lined (eg by spraying), in particular with at least one collagen, preferably collagen of the type IV, and / or other matrix proteins, in particular basement membrane proteins such as laminin. Preferably, the liner with mixtures of two or more of the ingredients, in particular Matrigel ^® is.

In this procedure, the pseudo-papillae (PP) are preferably in a number or density of 1 to 50 / cm ² pseudo-dermis (PD), in particular from 3 to 7 / cm ² pseudo-dermis (PD), in the pseudo -Dermis (PD) or the pseudo-dermis approach introduced or advertised.

According to one another embodiment can be carried out in step (c) introducing or inserting perform the pseudo-papilla (s) (PP) in such a way that the pseudo-papilla (s) (PP), directly into the pseudo-dermis (PD) or the pseudo-dermis approach injects or punctures. In particular, this introduction takes place into the pseudo-dermis approach before the contraction to pseudo-dermis is complete is. Particularly preferably, the introduction or insertion takes place before or at the beginning of the contraction phase.

In this approach, the pseudo-papillae (PP) are also preferably in a number or density of 1 to 50 / cm ² pseudo-dermis (PD), in particular from 3 to 7 / cm ² pseudo-dermis (PD), in the Pseudo-dermis (PD) or the pseudo-dermis approach introduced or advertised.

Alternatively, one can also inject or puncture the precursors of macroscopic pseudo-papillae (PP) into the pseudo-dermis (PD) or the pseudo-dermis approach; Such precursors of macroscopic pseudo-papilla consist of a mixture of the papillae produced according to the invention and at least one matrix former MB _PP , as described above, so that this mixture then in situ in the pseudo-dermis (PD) or the pseudo-dermis approach , in particular under gelling, the matrix and thus the macroscopic pseudo-papillae (PP) form, ie the formation of macroscopic pseudo-papillae (PP) takes place according to this embodiment in situ only in the pseudo-dermis (PD) or the Pseu do-dermis approach.

According to a further, particularly preferred embodiment, the introduction or embedding of the pseudo-papillae (PP) carried out in step (c) can be carried out in such a way that the individual pseudopapillas (PP) produced according to the invention by being introduced into a dispersing solution, directly in the production of the pseudo-dermis with the contractile cells and the matrix-forming, in particular gel-forming medium MBM _PD , which contains at least one matrix former MB _PD , in particular gelling agent, and optionally further constituents. This early blending of the pseudo-papillae with the pseudo-dermis approach results in a uniform skin / hair equivalent. The single pseudo-papilla can be in a density of 1 to 20 000 PP / cm ³ pseudo-dermis (PD), in particular from 20 to 5000 pseudo-papillae / cm ³ pseudo-dermis (PD), in the pseudo-dermis (PD ) or the pseudo-dermis approach are introduced or advertised.

Around the in vivo system as possible realistically to adjust, carried out in step (c) introduction or insertion, in particular when introducing punches and the Injection of the pseudopapillae (PP) or their precursors into the pseudo-dermis (PD) at an angle of 30 ° to 90 °, in particular 40 ° to 60 °, based to the level of pseudo-dermis (PD).

The Introduction or insertion of the pseudo-papillae (PP) into the pseudo-dermis (PD) can be done on a regular basis.

In front or after the introduction or insertion performed in step (c) the pseudo-papillae (PP) or their precursors may optionally be in Step (d) a reconstructed epidermis (pseudo-epidermis; PE) or a reconstructed periderm (pseudo-periderm; PI) on the Pseudo-dermis (PD) can be applied. Preferably, the application is such a pseudo-epidermis following the introduction made in step (c) or insertion of the pseudo-papillae (PP) or their precursors. The Pseudo-epidermis (PE) or the pseudo-periderm (PI) can be cultured from Keratinocytes, hair follicle keratinocytes (ORS and / or matrix keratinocytes) in particular keratinocytes of the outer hair root sheath (ORS keratinocytes) and / or epidermal keratinocytes, and optionally also from melanocytes, in particular melanocytes of the outer hair root sheath (ORS melanocytes) and / or epidermal melanocytes, and optionally other Consist of components. It can the keratinocytes and the optionally present melanocytes each for in separate, single or multi-layered layers or together in mixture as a single or multi-layer on the pseudo-dermis (PD) are applied. Dependent on from the medium used in particular the keratinocytes of the outer hair root sheath (ORS keratinocytes) Form pseudo-periderm (PI). The pseudo-periderm with its peridermal Structure corresponds to the conditions in embryonic follicular development. This has the advantage that so the natural conditions, in particular the direct and indirect cell-cell and / or cell-matrix interactions in observed, studied or influenced in a three-dimensional geometry can.

According to one embodiment of the method according to the invention, the following procedure can be followed: To produce the model, a pseudo-dermis (PD) of epidermal fibroblasts can first be produced in a collagen matrix. These can then be punched at a certain angle (eg 30-90 °) "holes", which are then optionally lined with basement membrane proteins (eg, laminin, collagen IV, etc.) or z. B. with Matrigel ^® , a mixture of basement membrane proteins, or mixtures of Matrigel with collagen (in particular collagen type I), can be lined; Reconstructed papillae (pseudo-papillae, PP) are used in these "holes", both single and macroscopic ones. These macroscopic pseudo-papillae can be obtained, for example, by the individual, produced according to the invention via the dripping, pseudo-papillae with a matrix and / or gelling agent, for. B. Matrigel ^® , mixed and gelled; The macroscopic pseudo-papillae (PP) can be punched out of the gel after gelation and inserted into the pseudo-dermis (PD). The gelling can also be done in situ after introduction into the pseudo-dermis. Alternatively, the pseudo papillae can, in particular embedded in Matrigel ^® individual pseudo papillae was (were reconstructed on the dripping which via individually), are introduced directly into the pseudo-dermis (PD) without first "holes" are punched are, or will be in the Matrigel ^® - introduced embedded or collagen solution single pseudo papillae via a branch duct in the pseudo-dermis (PD). The embedding in relatively liquid Matrigel or collagen solutions allows better handling of the reconstructed papilla when used in the pseudo-dermis.

It is also possible that cells of other cell types, in particular keratinocytes and / or melanocytes, preferably obtained from the outer root, are also present in the pseudo-papilla in addition to dermal papilla cells Sheath, are included.

According to one preferred embodiment can be the macroscopic pseudo-papilla in addition to the reconstructed individual pseudo-papilla also more Contain cell types. In particular, such cell types are analogous to the method already described for the preparation of the reconstructed To make papilla also by dripping in spährischer form and use. It is particularly preferred that the cell types selected are preferably obtained from keratinocytes and / or melanocytes from the outer root sheath. The existing in the macroscopic pseudo-papilla narrow spatial Contact enables one particularly good reconstruction of a hair follicle.

On The pseudo-dermis (PD) can eventually make a cell layer out Hair follicle melanocytes and / or hair follicle keratinocytes (ORS and / or matrix keratinocytes) be applied. It may also be preferred, especially for the hair model be the individual individuals put into the pseudo-dermis or macroscopic pseudo-papillae targeted with one or more Cell layers of hair follicle keratinocytes and / or melanocytes to overlay, without covering the entire pseudo-dermis with it. Thus, the natural Conditions of a hair follicle (papilla with surrounding or layered keratinocytes and / or melanocytes) in a dermal tissue even better become.

Especially it is preferred to use the individual pseudo-papillae that over the Dropping process according to the invention individually into the pseudo-dermis or the pseudo-dermis approach to insert. these can then either in preformed "holes" in the pseudo-dermis used, or simply integrated by pressure into the pseudo-dermis or directly in the production of the pseudo-dermis with the contractile Cells and the gelling agent (or the pseudo-dermis approach) mixed become.

The Pseudo-papilla (PP) affected the possibly above it Pseudo-epidermis (PE) or the pseudo-periderm (PI) from melanocytes and keratinocytes in its structure: It forms under these conditions one haarfollikelähnliche Structure. The model produced in this way can be used, for example, for Examination of substances (pharmacological, cosmetic etc.) on hair growth and hair texture as well as for examination the effect of substances on hair pigmentation.

In This three-dimensional hair / skin model then forms follicle-like or folliculoid (follicular) structures, including those the earliest Stages of hair morphogenesis (morphogenesis stages I to III) recall (such as the periderm).

Another The present invention relates to the process according to the invention available Hair / skin equivalent.

at the skin / hair equivalent according to the invention in particular, it is a skin / hair model with particular three-dimensionally shaped and spatially demarcated, reconstructed papillae (pseudo-papillae, PP) with follicle- or folliculoid structures (including those at earliest stages hair morphogenesis, d. H. Morphogenesis stages I to III, remember), where the skin / hair equivalent a reconstructed dermis (pseudo-dermis; PD) into which the pseudo-papillae (PP) are inserted or inserted, the pseudo-papillae (PP) cultured papilla cells, especially dermal papilla cells (Hair papilla cells), in a suitable matrix, in particular gel matrix, and, optionally, the pseudo-dermis (PD) a reconstructed epidermis (Pseudo-epidermis; PE) or a pseudo-periderm (PI) applied is, wherein the pseudo-papillae by one of the described method according to the invention getting produced.

The skin / hair equivalent according to the invention, in particular hair follicle model, so in general includes a Pseudo-dermis (PD) with dermal papillae reconstructed in it (pseudo-papillae; PP). About it can one or multiple layers of keratinocytes that become a pseudo-epidermis (PE) or a pseudo-periderm (PI) have formed or formed, and optionally applied one or more layers of melanocytes be. This in vitro model is suitable, for example, for efficacy and compatibility tests in Pharmaceuticals, medicine and cosmetics. In the inventive three-dimensional hair / skin model form follicle-like or folliculoid (follicular) structures, including those the earliest Stages of hair morphogenesis (morphogenesis stages I to III) recall.

Another object of the present invention is the use of the skin / hair equivalent according to the invention, as described in claims 33 to 38 and also for the reconstructed papilla itself, is described. However, the skin / hair equivalent is particularly suitable for answering these questions because of its natural-like and at the same time more manageable structure. For further details, reference may also be made to the above statements on the method according to the invention and the skin / hair equivalent according to the invention, which also apply correspondingly to the use according to the invention.

One Another object of the present invention is a system, in particular Test system (for example a screening system) which contains the skin / hair equivalent according to the invention includes. For further Details can also to the above statements to the method according to the invention, the skin / hair equivalent according to the invention and the use according to the invention, be referenced, which also corresponding to the system according to the invention be valid.

There are a number of advantages associated with the present hair / skin equivalent:
The skin / hair equivalent according to the invention is a reconstructed model which is more standardized than the isolated hair follicle. It reduces the need for hair follicles and is closer to the in vivo situation than monolayer systems. It also represents an alternative to animal testing.

It is a complex three-dimensional model, which in its structure and its histological structure the hair follicle in vivo, making it highly relevant for effectiveness and tolerability of active ingredients (cosmetics, pharmaceuticals, etc.) results.

Among other things, the following endpoints can be evaluated or measured to make statements about the effectiveness of substances in improving hair structure and influencing hair growth:
Proliferation / apoptosis of keratinocytes over the pseudo-papilla; Structure and arrangement of keratinocytes over the pseudo-papilla; Structure of the epidermis; Structure of the stratum corneum; Volume and structure of dermal papilla; Analysis of certain hair-specific proteins (especially hair-specific keratins); Analysis of cytokines, chemokines and all types of messengers, which are formed, among others, by the dermal papilla; Hair-chip analysis, proteome or expression analysis, etc.

It is a reconstructed hair follicle model, with the influences can be measured for hair pigmentation (eg pigmentation the amelocytic ORS melanocytes; Melaninsysnthese; melanin granules; Arrangement of melanocytes; Migration of melanocytes; change melanocyte markers such as TRP-1, TRP-2, NKI / beteb, etc .; delivery of melanin on keratinocytes). It can also be used for hair-chip analysis, for example be used.

The hair / skin model according to the invention is suitable for the most diverse applications in the field of medicine, pharmacy and cosmetics (eg for drug discovery with biological activity on the hair follicle, influencing hair pigmentation, Hair growth and structure, in in vitro test systems, in screening procedures, for the Development of cosmetic products etc.). The model according to the invention or equivalent allows Statements about the effect of substances on hair follicle cells with in vivo relevance. The model according to the invention or equivalent represents hair follicles or parts in a three-dimensional model prepared by hair follicles. The reconstructed papillae (pseudo-papillae: PP) are - im Unlike isolated hair follicles - always available and standardized. In particular, dermal papillae are reconstructed in this way, that they even better the three-dimensional structure of the hair follicle are modeled. In this way you can evaluate how applied Active ingredients on the structure of the reconstructed three-dimensional Model and what about the effect of these substances on the hair follicle (eg hair growth, hair structure, hair pigmentation etc.) can be read.

Further Embodiments, Embodiments, modifications and variations of the present invention are for those skilled in the reading of the present application readily recognizable and realizable, without giving it the scope of the present Invention leaves.

The The present invention will become apparent from the following embodiment However, which illustrates the present invention in no way restrict should.

WORKING EXAMPLES

in the The following are the abbreviations listed in the table below uses.

Abbreviations:

Example 1: Reconstruction the dermal papilla

Production of the dermal papilla

dermal Papilla cells are taken from the scalp (temporal or occipital region) isolated with intact hair follicles. This will be the upper dermis removed and the follicles together with the dermal papilla with the help of a watchmaker tweezers plucked from the dermis. Under the stereo magnifier, the others took place Isolation of the dermal papilla. With the help of a microcapillary the transfer into a culture flask coated with collagen I took place. Cultivation is either enriched in RPMI 1640 medium with glutamine (Sigma) with 20% fetal calf serum (FCS) (Gibco BRL, Karlsruhe, Germany) and antibiotics or in Chang's medium with 10% fetal calf serum (BIOCHROM KG) [2].

Reconstruction of the dermal Papilla (pseudo-papillae

Sterile, for reconstruction of the dermal papilla is neutralized collagen type I collagen from rat tail tendon (BD Biosciences) with Matrigel ^® in a ratio of 4: 1 and with a cell suspension of dermal papilla cells (10 ⁶ cells, passage 2 and 3 in Chang's Medium) in fetal calf serum.

This mixture is filled into a syringe and added dropwise to a solution of neutral fat (caprylic / caproic acid triglyceride), this non-aqueous phase being stirred permanently at 37 ° C. The droplets which polymerize under these conditions are stirred for a further 10 minutes at a temperature of 37 ° C. and then removed from the fatty phase with the aid of a net. The collagen drops (pseudo-papillae) are transferred to a Petri dish and washed with suitable culture medium. Thereafter, the culture is overlaid with nutrient medium (fibroblast medium) and incubated at 37 ° C, 5% CO ₂ in the incubator for up to 7 days.

Results:

To 7 days could be based on histological examinations a high Cell density and organotypic arrangement of cells detected become.

Example 2: Preparation a skin / hair model with reconstructed dermal papillae

in the The following is the preparation of a skin / hair model with reconstructed dermal papillae (pseudo-papillae; PP), which are in a pseudo-dermis (PD) are embedded or inserted. These are the in Example 1 prepared pseudo-papillae either in the pseudo-dermis (PD) injected, mixed with this or with the help of punching placed in the pseudo-dermis (PD). The pseudo-dermis (PD) can in the further course with a layer of epidermal or hair follicle keratinocytes representing a pseudo-epidermis (PE) or a pseudo-periderm (PI), and possibly covered with melanocytes.

manufacturing the other single cell cultures

Dermal fibroblasts

Dermal fibroblasts are isolated from human foreskin. The epidermis and dermis of the foreskin are separated enzymatically by means of thermolysin (0.5 mg / h HEPES buffer). For extraction of the fibroblasts, the dermis is digested with the aid of collagenase H (0.2 U / ml, Boehringer Mannheim, Mannheim, Germany) (3-4 h at 37 ° C). After the incubation phase, the solution is mixed gently to separate the cells, filtered through a cell strainer and the cells are centrifuged off. The culture was carried out in Dulbecco's modified Eagle's medium (DMEM) with glutamax-I (L-alanyl-L-glutamine) with sodium pyruvate, 4,500 mg L- ¹ glucose and pyridoxine (Gibco BRL, Karlsruhe, Germany), supplemented with 10% fetal calf serum (FCS) (Gibco BRL, Karlsruhe, Germany), 25 μg mL ^-1 gentamicin (Sigma, Taufkirchen, Germany) and 100 μL mL ^-1 penicillin G (Sigma) [1].

Epidermal keratinocytes

dermal Fibroblasts are isolated from human foreskin. Epidermis and Dermis dermis are enzymatically by means of thermolysin (0.5 mg / nl HEPES buffer) from each other separated. For extraction of keratinocytes, the epidermis is Trypsin (Gibco BRL, Karlsruhe, Germany) digested (20 min at 37 ° C). To the incubation phase becomes the solution Carefully mixed to separate the cells through a cell strainer filtered and the cells centrifuged off. The cultivation takes place in a mixture of DMEM Glutamax I and Ham's F12 (Sigma) (3: 1), enriched with Newborn Calf Serum (NCF, fetal clone II, Hyclone), Epidermal Growth Factor (EGF) (Sigma), insulin (Sigma), hydrocortisone (Sigma), triiodo-L-thyronine (Sigma), adenine (Sigma), cholera toxin (Sigma) and antibiotics [1] on feeder layers (Dermal fibroblasts used to inhibit the Proliferation have been irradiated with 60 gray).

Keratinocytes of the outer root sheath (ORS keratinocytes)

ORS keratinocytes become plucked male Hair of the occiput isolated. For extraction of keratinocytes be first removed the remains of the hair bulb with the help of a scalpel and the follicles by means of trypsin (protease from Gibco BRL, Karlsruhe, Germany) digested (40 min at 37 ° C). After the incubation phase becomes the solution Carefully mixed to separate the cells through a cell strainer filtered and the cells centrifuged off. The cultivation took place in a mixture of DMEM Glutamax I and Ham's F12 (Sigma) (3: 1), enriched with Newborn Calf Serum (NCF, fetal clone II, Hyclone), Epidermal Growth Factor (EGF) (Sigma), insulin (Sigma), hydrocortisone (Sigma), triiodo-L-thyronine (Sigma), adenine (Sigma), cholera toxin (Sigma) and antibiotics Feeder Layers (Dermal fibroblasts used to inhibit proliferation irradiated with 60 gray).

ORS melanocytes are named after Tobin et al. [3] isolated.

Production of the pseudo-dermis (PD)

To establish the hair model, first the pseudo-dermis (PD) is developed. For this purpose dermal fibroblasts of the fourth passage are mixed with collagen I from the rat tail tendon and seeded in multiwell plates. The production takes place according to the following protocol:
1 part of HBSS buffer (Gibco BRL) is mixed with 8 parts of collagen solution (Becton Dickinson) and mixed with 1 M sodium hydroxide solution neutralized. The desired amount of cell was added in 1 part of fetal calf serum (FCS, Gibco BRL). The resulting mixture (gel mixture) is poured into cell culture dishes and incubated for 1 h at 37 ° C in an incubator. After collagen polymerization, the models are overlaid with DMEM supplemented with 10% FCS and penicillin / streptomycin. The medium is changed three times a week over a period of seven days.

The following approaches are preferred:

Production of injection channels for insertion the DPC into the pseudo-dermis (PD)

to Production of injection channels become a button cannula, a conventional one cannula and a 2mm punch. For optical representation of channels becomes a solution Made of 10% Berlin Blue in 1% agarose solution and with the help of the button cannula as well as the conventional one cannula into the dermis equivalent injected. When using the punches are those with the biopsy punch made channels either immediately or after 24 h with the help of the button cannula filled. The preparation of the channels was done with the help of a stereo magnifier. After 24 h the models will be for the preparation of cryosections and histological examination frozen.

Production of the reconstructed Papilla by inserting punches into the pseudodermis (PD)

to Preparation of macroscopic pseudo-papillae were dermal papillae mixed with collagen I from the rat tail and possibly Matrigel and seeded in multiwell plates. The choice of papilla number was based on the production the pseudodermis (PD).

The Production was based on the following protocol: 1 part HBSS Buffer (Gibco BRL) was charged with 4 parts of Matrigel and 8 parts collagen solution (Becton Dickinson) and neutralized with 1 M sodium hydroxide solution. The addition of the desired Papilla amount was in 1 part fetal calf serum (FCS, Fa. Gibco BRL). The resulting mixture was poured into cell culture dishes and 1 h at 37 ° C incubated in the incubator. After polymerization of the collagen were Models overlaid with Chang medium supplemented with 10% FCS. The medium was changed three times a week over a period of eight Days.

To Expiration of this cultivation period was in a seven day old Pseudodermis prepared with the help of a 2 mm punch holes, in each case 5 μl Matrigel injected and each 3 mm biopsies from the polymerized collagen / reconstructed Papillae mixture were used (macroscopic pseudo-papilla). The models were overlaid with Chang medium supplemented with 10% FCS and the medium was changed three times a week for a period of six Days. Subsequently Histological and immunohistochemical evaluation of the models

Production of a skin model from a pseudodermis and an epidermis of epidermal keratinocytes (NHEK) or a pseudo-periderm of hair keratinocytes (ORS keratinocytes) (with and without pseudo-papilla)

To further develop the reconstructed hair follicle model, the pseudodermis was transferred to Snapwell inserts (Corning Costar) after five days of culture and then overlaid with epidermal keratinocytes (500,000 cells / model). After one week of submerged culture in keratinocyte medium (DMEM Glutamax I and Ham's F12 (Sigma) (3: 1) supplemented with Newborn Calf Serum (NCF, fetal clone II, Hyclone), Epidermal Growth Factor (EGF) (Sigma), insulin ( Sigma), hydrocortisone (Sigma), triiodo-L-thyronine (Sigma), adenine (Sigma), cholera toxin (Sigma), ascorbyl-2-phosphate (Sigma) and antibiotics), the models were transferred to the air-liquid interface and two more weeks in DMEM Glutamax I and Ham's F12 (Sigma) (3: 1), enriched with insulin (Sigma), hydrocortisone (Sigma), ascorbyl-2-phosphate (Sigma), bovine serum albumin (BSA) (Sigma) and antibiotics.

In another experiment was to replace the epidermal keratinocytes ORS keratinocytes used from the hair follicle, which were sown with 800,000 cells / model. Culturing was done with keratinocyte medium submerged over 7 days.

The Coating of the pseudo-dermis with ORS keratinocytes in keratinocyte medium (see Appendix) leads to Formation of a pseudo-periderm while coating with epidermal keratinocytes (NHEK) and keratinocytes in airiquid interface medium lead to the formation of a pseudo-epidermis.

Annex I: Composition the cell culture media:

Fibroblast medium

Keratinocyte medium

Papilla Cell Media RPMI 1640 Medium (Gibco, BRL, Karlsruhe)

Chang Medium (Irvine Scientific, Santa Ana, USA)

Composition Chang Medium D

Medium for the Air-Liquid-Interface

Annex II: In the embodiment cited references

• [1] K. Schlotmann et al., Cosmetic Efficacy Claims In Vitro Using a 3D Human Skin Model, Int. J. Cosmet. Sci. 23, 310-319 (2001).
• [2] R. Warren et al., Improved Method for the Isolation and Cultivation of Human Scalp Dermal Papilla Cells, J. Invest. Dermatol. 98, 693-699 (1992).
• [3] D.J. Tobin et al., Isolation and Long-Term Culture of Human Hair Follicle Melanocytes, J. Invest. Dermatol. 104, 86-89 (1995).
• [4] A. Limat et al., Outer root sheath cells organize into epidermoid cyst-like spheroids When cultured in Matrigel ^®. Cell Tissue Res. 275, 169-176 (1994).
• [5] EP 0 218 065 A2 ,

Claims (39)

A method for producing a reconstructed papilla (pseudo-papilla), characterized in that a suspension comprising cultured papilla cells is dropped into a dispersing solution in which the suspension solidifies into a reconstructed papilla. A method according to claim 1, characterized in that the suspension contains a matrix-forming, in particular gel-forming medium MBM _S. A method according to claim 2, characterized in that the matrix-forming, in particular gel-forming medium (MBM _S ) is selected from collagen, in particular collagen type I and / or type III, and Matrigel ^® and mixtures thereof. A method according to claim 3, characterized in that the suspension type I collagen and Matrigel ^® , preferably in a ratio of 0.1: 1 to 10: 1, preferably 1: 1 to 6: 1. Method according to one of claims 1 to 4, characterized that one obtains the cultured dermal papilla cells, that one dermal papilla cells from the hair follicles more human or isolated animal skin, then cultivated and then out the resulting single-layer cultures (monolayer cultures), the dermal papilla cells gain, especially by gentle Trypsinization. Method according to one of claims 1 to 5, characterized that the cultivation of dermal papilla cells in a suitable broth takes place, in particular wherein as a sewing medium essential minimal medium MEM, in particular DME medium and / or RPMI medium and / or Chang medium, used, optionally together with other components, in particular fetal Calf serum (FCS), Collagen, in particular type I collagen, and the like. Method according to one of the preceding claims, characterized characterized in that the dispersing solution is not aqueous. Method according to one of the preceding claims, characterized in that liquid lipids, in particular low-viscosity paraffin oils or oils, are used as the dispersing solution Triglycerides containing mutually independent residues of fatty acids 4 to 18 carbon atoms, in particular 6 to 12 carbon atoms, or mixtures used of it. Method according to one of the preceding claims, characterized in that the suspension is dripped in by means of a capillary becomes. Method according to one of the preceding claims, characterized characterized in that in a further step, the reconstructed Papilla from the dispersing solution Will get removed. Reconstructed dermal papilla, obtainable after one of the claims 1 to 10. Use of the reconstructed papilla according to claim 11 in medicine, pharmacy or cosmetics, in particular as a system for testing cosmetic or pharmaceutic agents. Process for the preparation of a skin / hair equivalent, in particular a skin / hair model with reconstructed papillae (pseudopapillae; PP) in a reconstructed dermis (pseudo-dermis; PD), the process comprising the following steps: (a) providing a reconstructed dermis (pseudo-dermis; PD) or a pseudo-dermis approach; (b) providing reconstructed papillae (pseudopapillae; PP) comprising cultured papilla cells, preferably dermal papilla cells (hair papilla cells), in a suitable matrix, in particular gel matrix, or else providing corresponding precursors to such reconstructed papillae (PP) cultured papilla cells, in particular dermal papilla cells, in a suitable matrix-forming, in particular gel-forming medium MBM _PP , which can form a matrix, in particular gel matrix, in situ, in particular in the reconstructed dermis (pseudo-dermis; PD); (c) introducing or inserting the reconstructed papillae (PP) or their precursors provided under step (b) into the pseudo-dermis (PD) or the pseudo-dermis approach provided under step (a); (d) optionally applying a reconstructed epidermis (pseudo-epidermis; PE) or a reconstructed periderm (pseudo-periderm; PI) to the pseudo-dermis (PD), characterized in that provided under b) reconstructed papilla (pseudo-papilla, PP) in a method according to one of claims 1 to 10 is produced. Method according to claim 13, characterized in that that the pseudo-dermis (PD) or the pseudo-dermis approach cultured contractile cells in a suitable matrix, in particular wherein the matrix is a matrix based on collagen, in particular collagen type I and / or type III, and optionally further Components is. Method according to claim 13 or 14, characterized that as contractile cells for the pseudo-dermis (PD) or the pseudo-dermis approach Fibroblasts, especially dermal fibroblasts. Method according to one of claims 13 to 15, characterized that the cultured contractile cells for the pseudo-dermis (PD) or derives the pseudo-dermis approach by dermal fibroblasts isolated from human or animal skin, then cultured and then from the resulting single-layer cultures (monolayer cultures) the contractile cells gain, especially by gentle trypsinization. Method according to claim 16, characterized in that that the cultivation of the contractile cells, especially fibroblasts, preferably dermal fibroblasts, in a suitable nutrient medium takes place, in particular wherein as a nutrient medium essential minimal medium MEM used. Method according to one of claims 13 to 17, characterized in that the pseudo-dermis mixture is obtained by mixing obtained from single-layer cultures, optionally in a suitable nutrient medium contractile cells with a matrix-forming, in particular gel-forming medium MBM _PD for the pseudo -Dermis (PD), which contains at least one matrix former MB _PD , in particular gelling agent, and optionally further constituents. Method according to claim 18, characterized that the pseudo-dermis approach a matrix, preferably a gel, forms and in particular under Output of the optionally existing nutrient medium contracted to a pseudo-dermis (PD). Method according to one of claims 18 or 19, characterized in that the matrix-forming, in particular gel-forming medium MBM _PD as a matrix former MB _PD , in particular gelling agent, at least one collagen, in particular type I collagen and / or type III, and optionally other components, in particular Components of the dermis extracellular matrix, preferably matrix and / or scleroproteins such as laminin. Method according to one of claims 13 to 20, characterized that on the pseudo-dermis (PD) a suitable nutrient medium, in particular essential Minimal medium MEM, and optionally applied further components become. Method according to one of claims 13 to 21, characterized in that in step b) provided pseudo-papilla is a macroscopic pseudo-papilla, which is obtained by mixing prepared by the method 1 to 10 pseudo-papillae with a matrix-forming, in particular gel-forming Medium MBM _PP for the pseudo-papillae (PP), which contains at least one matrix former MB _PP , in particular gelling agent, and optionally further constituents, wherein the resulting mixture forms a matrix, preferably a gel, and if necessary contracts under expulsion of the nutrient medium which may be present , A method according to claim 22, characterized in that the matrix-forming, in particular gel-forming medium MBM _PP as matrix former MB _PP , in particular gelling agent, at least one collagen, in particular collagen type IV, and optionally contains further constituents, which are in particular selected from the group of matrix and / or scleroproteins, in particular laminin, gelatin, chitosan, glucosamines, glucosaminoglycans (GAG), heparan sulfate proteoglycans, sulfated glycoproteins such as nidogen (in particular entactin) and growth factors such as tissue growth factor beta (TGF-β), fibroblast growth factor, Tissue-Plasmimogen Activator and other growth factors from the Engelbreth-Holm-Swarm tumor (EHS tumor) and / or the human placenta and mixtures of the aforementioned ingredients. Method according to one of claims 22 or 23, characterized that the formation of the contracted matrix, in particular of the gel, and / or the pseudo-papillae (PP) in situ in the pseudo-dermis (PD) he follows. Method according to one of claims 22 to 24, characterized that the pseudo-papillae (PP) from the matrix, in particular the gel, be formed, the shape of the pseudo-papillae (PP) either before or after the insertion or insertion performed in step (c) pseudo-papillae (PP) or their precursors. Method according to one of claims 13 to 25, characterized that the insertion or insertion of the pseudo-papillae (PP) in Step (c) is carried out by using suitable cavities for Recording of the pseudo-papillae (PP), especially by punching or piercing, in the pseudo-dermis (PD) and then into the thus formed cavities pseudo-papillae (PP), the cultured papilla cells contain, introduce or insert Method according to claim 26, characterized in that that the cavities before introducing the pseudo-papillae (PP) with at least one Collagen, in particular type IV collagen, and / or other matrix proteins, In particular, basement membrane proteins such as laminin have been lined are. Method according to one of claims 13 to 27, characterized that the preformed pseudo-papillae (PP) or the precursors of the Pseudo papillae (PP) directly into the pseudo-dermis (PD) and especially in the pseudo-dermis approach injected or stabbed or directly with the pseudo-dermis especially with the pseudo-dermis approach. Method according to one of claims 13 to 28, characterized that the application in step (d) of the reconstructed Epidermis (pseudo-epidermis, PE) or a reconstructed periderm (Pseudo-Periderm, PI) before or after the introduction made in step (c) or insertion of the pseudopapillae (PP) or their precursors. Method according to claim 29, characterized that the pseudo-epidermis (PE) or the pseudo-periderm; (PI) cultured keratinocytes, in particular Keratinocytes of the outer hair root sheath (ORS keratinocytes) and / or epidermal keratinocytes, and optionally also melanocytes, in particular Melanocytes of the outer hair root sheath (ORS melanocytes) and / or epidermal melanocytes, and optionally contains further ingredients. Method according to one of claims 29 or 30, characterized that the keratinocytes and melanocytes each in separate, single or multi-layered Layers or together in mixture as single or multi-layered Layer can be applied to the pseudo-dermis (PD). Hair / skin equivalent, especially hair model with reconstructed papillae (PP) in one Pseudo-dermis (PD), available according to a method according to claims 1 to 31. Use of a skin / hair equivalent according to claim 32 in the field of pharmacy, medicine or cosmetics and personal care. Use of a skin / hair equivalent according to claim 33 for the discovery, investigation and / or (re) testing of pharmaceutical or cosmetic active ingredients, in particular with regard to compatibility and / or effectiveness. Use according to claim 34 for finding, examining and / or (re) examination of pharmaceutical or cosmetic agents on effect and / or compatibility with regard to the hair follicle, in particular with regard to the Hair pigmentation, hair growth, hair texture, hair color and the same. Use of a skin / hair equivalent according to claim 32 for the Development of pharmaceutical or cosmetic active ingredients. Use of a skin / hair equivalent according to claim 32 in preferably automated screening methods, in particular for the discovery, investigation and / or (re) testing of pharmaceutical or cosmetic agents. Use of a skin / hair equivalent according to claim 32 for in vitro evaluation the influence of the hair follicle, the hair pigmentation, the Hair growth, hair structure, hair color and the like, in particular by pharmaceutical or cosmetic active substances. System, in particular test system, comprising a skin / hair equivalent according to claim 32.