WO2004113514A1

WO2004113514A1 - Reconstructed dermal papillae

Info

Publication number: WO2004113514A1
Application number: PCT/EP2004/006643
Authority: WO
Inventors: Ralf Paus; Albrecht Bettermann
Original assignee: Henkel Kommanditgesellschaft Auf Aktien; Cutech Srl
Priority date: 2003-06-18
Filing date: 2004-06-18
Publication date: 2004-12-29
Also published as: DE10327879A1

Abstract

The invention relates to a reconstructed dermal papilla (pseudopapilla), to its production and to its use, more particularly for medical, pharmaceutical and cosmetic applications, and to a skin/hair equivalent containing such reconstructed papillae (pseudopapillae), more particularly to a skin/hair model with reconstructed papillae in a reconstructed dermis (pseudodermis) and to its production and its use, particularly for medical, pharmaceutical and cosmetic applications.

Description

Reconstructed dermal Papillae

Finding active substances with, for example, a biological effect on the hair follicle, so that they are capable of influencing hair pigmentation, hair growth and hair structure, requires suitable in vitro test systems on which any such effect can be evaluated. These test systems should ideally allow the screening of a relatively large number of substances, should be standardizable and inexpensive and - in the case of in vitro systems - should simulate the in vivo situation.

In hair research, there are at present no suitable in vitro models, for example for studying hair growth, hair pigmentation and hair structure. A summary of existing methods and an illustration of the disadvantages of these systems can be found, for example, in 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 via animal models and ex-vivo systems to in vitro systems.

Monolayer cultures of hair follicle cells have the disadvantage that, when removed from their complex three-dimensional structures, the cells behave differently than they would in the organ as a whole. Because of this, information on the effect of substances on hair follicle cells cultivated as a monolayer is of little relevance to the in vivo situation. Under the guidelines on cosmetics, animal models may not be used for the development of cosmetic products. Accordingly, ex vivo models which combine in vitro methods with in vivo methods on the animal are also out of the question. Similar problems as to the availability of material and standardizability are involved in the use of skin explantates with hairs in culture. Although in vivo studies on human beings are carried out to screen the effect, they are not advisable until a potent active substance has been discovered and incorporated in a formulation because such studies are correspondingly expensive and complex. There are also no suitable in vitro test systems for screening substances which influence hair color.

In vitro tests for modifying hair pigmentation by influencing the melanin production of the melanocytes are also mainly carried out on single cell cultures. Epidermal melanocytes or B16 melanoma cells are often used for this purpose and the results obtained with this cell type are extrapolated to the hair melanocytes because hair melanocytes are difficult to isolate and cultivate. In addition, the complex interaction of the melanocytes with the hair follicle is missing in these systems so that their relevance to the situation in vivo on the hair follicle has to be called into question. The same applies to reconstructed hair models which contain (epidermal) melanocytes. Animal models which are also a popular test model for substances with an effect on hair pigmentation are prohibited under the guidelines on cosmetics where the substances are to be used for cosmetic products. In vivo studies on human beings are laborious and expensive and, accordingly, are only advisable after a potent active substance has been found.

In the technique of tissue engineering, various cell types are isolated from tissue, for example skin tissue, and multiplied in cell culture as a so-called monolayer. The tissue is then reconstructed from the single cells. In the case of skin, fibroblasts, for example, can be "sown" into a collagen gel or other matrix so that they proliferate and form a pseudodermis. Epidermal keratinocytes can be applied to the pseudodermis thus formed where they also proliferate and form a pseudoepidermis. By raising the culture into the air (air/liquid interface), the cells begin to differentiate and to form a stratum corneum.

Hitherto, cell cultures, for example keratinocytes of the outer root sheath (ORS keratinocytes, ORS = outer root sheath), dermal papilla cells, etc., have mainly been used in hair research. However, repeated attempts have also been made to cultivate hair follicles or parts of hair follicles as a three-dimensional model, for example in a collagen gel, or to reconstruct entire hair follicles by combining different hair follicle cells.

In "Characterization of a new tissue-engineered human skin equivalent with hair" published in In Vitro Cell. Dev. Biol. - Animal 35:318-326, June 1999, M. Michel et al. report for the first time on the insertion of a hair follicle into a reconstructed hair model for use in penetration studies. Here, the authors used whole hair follicles which had to be prepared beforehand from hair-covered skin. Apart from the limited availability of the material, standardizability is poor where prepared hairs are used because the biological variations are considerable.

EP 0 285 471 A1 and EP 0 285 474 A1 also describe the production of an artificial skin which consists of a dermal layer of contractile cells (fibroblasts) and extracellular matrix components into which whole hair follicles or follicle segments are inserted. The dermal layer is then additionally coated with keratinocytes which form an epidermal layer. The disadvantage here is that the papillae are not reconstructed, instead only part of the hair follicle with no papilla is used.

The model used by a group of Japanese researchers (M. Inamatsu et al. "Hair Follicle Development in Organotypic Culture", Third Intercontinental Meeting of Hair Research Societies (Abstract), Tokyo, 2001) for the early phase of hair development consists of freshly isolated dermal papillae from rat whiskers which are inserted between a collagen gel containing fibroblasts and an epidermal layer of rat keratinocytes. This organotypic culture is cultivated at the air/liquid interface. After 7 days, the epidermis is said to thicken in the vicinity of the dermal papillae. Firstly, no human cells or papillae are used here, secondly the papillae are not reconstructed papillae but whole papillae isolated from hair follicles and thirdly the papillae are not inserted (for example injected or grafted) into the epidermis, but are placed between the dermal and the epidermal layers. The use of isolated papillae involves the same problems of availability and standardizability as the use of isolated hair follicles.

The same applies to the works of S.A.J. 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, dermal papillae or papilla cells cultivated in a collagen gel are placed between the dermis and epidermis of a skin equivalent with a view to obtaining a model for the development of the hair follicle. Under these conditions, however, the dermal papilla cells migrate into the dermal matrix and do not form themselves into a papilla so that the model has to be modified by applying the papillae or the papilla cells to a dermal substrate and which is then covered by a fetal mouse epidermis and transplanted onto hairless mice.

In 1993, 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, 1993 succeeded in producing a follicle-like structure from a combination of hair cells by first cultivating outer root sheath cells (ORS cells) in the collagen capsule of the follicle of a rat whisker and then adding a mixture of different hair follicle cells - dermal papilla cells, dermal sheath cells and matrix cells. However, they needed the stable collagen capsule of the rat whisker to stabilize the structure.

A. Limat et al. "Outer Root Sheath (ORS) cells organize into epidermoid cyst-like spheroids when cultured inside Matrigel®: a light-microscopic and immu- nohistological comparison between human ORS cells and interfollicular keratinocytes" in Cell Tissue Res. (1994) 275: 169-176 also fit various hair follicle cells and skin cells together in a three-dimensional structure in order to study their interaction. They use a collagen (I) gel as base and, on it, bed a layer of Matrigel® or a mixture of basal membrane components containing various cells or cell mixtures. In the Matrigel®, the epidermal or ORS keratinocytes (= outer root sheath keratinocytes) form spheroidal, but non-follicular structures. Here, layers containing various cells are placed one above the other. However, no new structure is built up in these layers like the dermal papilla. Although Limat et al. report that ORS keratinocytes are capable of forming cystoidal structures which turn horny internally, it is not a question here of the physiological nature of hair shaft formation, but rather of the differentiation and stratum corneum formation by cystoidally arranged keratinocytes. However, hair shafts are normally formed by matrix cells which lie above the dermal papilla.

Published Japanese patent application 10-136977 (Toyobo Co., Ltd., Japan) describes an artificial tissue and its reconstruction which comprises 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 said to serve as a test system for determining the compatibility and effectiveness of active substances and cosmetics. The same applies here as to the article by A. Limat et al.: in this model, dermal papillae which share the three-dimensional structure of the hair follicle are not reconstructed, instead the cells are arranged one above the other in layers and no new structure is formed. Melanocytes are apparently not used in the described model either. Effectiveness tests are mentioned as a potential application for the model. However, there is no indication of which end points are to be evaluated on the model, i.e. how the application of active substances affects the structure of the reconstructed model and what can be read into this so far as the effect of this substance on hair growth and hair structure are concerned.

The "Philpott Model" (M.P. Philpott "Human hair growth in vitro", J. Cell. Sci. 97, 463-471, 1990), where isolated hair follicles are kept in culture for 9 days, has the disadvantage on the one hand of significant variability between the individual follicles and hence poor standardizability and, on the other hand, poor availability of the hair follicles. Because of this, only a very limited amount of active substances can be evaluated within a fixed period. In addition, only substances and formulations which are soluble in the medium can be applied, but not water- insoluble substances or formulations, such as creams for example.

In what was virtually a further development of the Philpott Model, an attempt was also made to insert isolated hair follicle segments into reconstructed skin models (see M. Inamatsu et al. and M. Michel et al.). Although follicles, follicle segments or dermal papillae are thus in contact with the dermis, which comes closer to the in vivo situation than other known models do, the disadvantages of these systems where they are to be used for studying active substances are similar to those attending the Philpott Model (poor availability of the hair follicles, no standardizability, high cost, etc.).

Problems are also involved in reaching a sufficiently high density of the cultivated papilla cells to enable conclusions to be drawn as to the natural cell/cell interactions within the dermal papilla.

The problem addressed by the present invention was therefore to provide a reconstructed papilla or a skin/hair equivalent ("skin model"), more particularly a hair/skin model with reconstructed papillae in a reconstructed dermis, which would at least partly avoid the above-mentioned disadvantages of the prior art. Another problem addressed by the present invention was to find or provide a skin/hair equivalent or model which would be suitable as an in vitro model system, more particularly for testing and/or evaluating active substances, more particularly on the hair follicle. Such a model would be particularly suitable for discovering and testing pharmaceutical/medical and cosmetic active principles. It would also allow in vitro evaluation of the effect of such active principles on the hair follicle, hair growth, hair pigmentation, hair structure and the like.

The present invention relates to a reconstructed dermal papilla (pseudopapilla), to its production and to its use, more particularly for medical, pharmaceutical and cosmetic applications, and to a skin/hair equivalent containing such reconstructed papillae (pseudopapillae), more particularly to a skin/hair model with reconstructed papillae in a reconstructed dermis (pseudodermis) and to its production and its use, particularly for medical, pharmaceutical and cosmetic applications.

Accordingly, the present invention relates to a process for the production of a reconstructed papilla (pseudopapilla) which is characterized in that a suspension containing cultivated papilla cells is added dropwise to a dispersion solution in which the suspension is solidified or formed into a reconstructed papilla (pseudopapilla), and to its use, more particularly for test purposes, preferably in the medical, pharmaceutical and cosmetic fields.

In the context of the present invention, the term "solidified" means that, after dropwise addition to the dispersion solution, the suspension assumes a non-liquid, relatively viscous, relatively hard or relatively firm consistency. Accordingly, the drops are gel-like (gelled), compacted, crosslinked, polymerized or hardened. A tissue homolog similar to the papilla is thus formed.

A matrix, preferably a gel, is preferentially formed and then contracts or is contracted by the cells, optionally with ejection of any medium present. A particular advantage of the pseudopapilla thus formed is that a very much higher cell density can be achieved by comparison with a gel or a matrix containing a normal suspension of cultivated papilla cells. In addition, the physiological form of the papilla is reproduced particularly well (in the form of three-dimensional droplets or spheres) by the technique.

A high cell density is particularly important for a particularly good reconstruction of the natural situation in the hair follicle, particularly in relation to cell/cell contacts and/or the resulting formation of typical proteins within the papilla, more especially the high concentration of growth factors and/or extracellular matrix proteins in the follicle. In addition, a higher cell density can correspondingly allow better signals when the reconstructed hair follicle model, more particularly the reconstructed hair follicle model thus containing the reconstructed papilla, is used as a test system.

In addition, the flexible inner structure of the matrix used advantageously enables the cells to organize themselves into physiologically compatible or "near-to-reality" (i.e. physiological) aggregates which can freely organize themselves therein, the matrix formed serving as a flexible structural framework for the development of characteristic proteins, more especially a new matrix unique to the cell.

The pseudopapilla thus obtained is advantageously comparable in size or volume to the natural human papilla so that the reconstruction of, in particular, the human dermal papilla of a reconstructed hair follicle or a hair model can be better assimilated to the natural situation.

In contrast to the reconstruction of the papilla on solid supports, it is readily possible to simulate the hair-specific extracellular matrix by suitably selecting the composition of the suspension to be added dropwise. This can be reproduced in particular by using a mixture of various components. In addition, the degree of crosslinking (of the components) can also be freely selected through the corresponding gelation conditions. For example, the "hardness" or the rigidity of the reconstructed papilla can be increased by increasing the collagen concentration.

In addition, the biological activity of the cells, for example the ratio between apoptosis and proliferation of the cells, can be influenced or modulated by variations of the matrix molecules used.

At the same time, the pseudopapilla produced in accordance with the invention can be produced with a high degree of reproducibility and thus provides for a high level of standardizability of processes to be carried out with the reconstructed hair or hair follicle model.

The cultivated dermal papilla cells (hair papilla cells) may be produced in known manner, for example by isolating dermal papilla cells (hair papilla cells) from the hair follicles of human or animal skin and, after cultivation, recovering the dermal papilla cells from the resulting monolayer cultures, more particularly by moderate trypsinization. The number of passages should be small, the number of passages for the dermal papillae generally being between 1 and 10 and preferably between 1 and 3. The dermal papilla cells are cultivated in a suitable nutrient medium, the nutrient medium used being in particular essential minimal medium MEM, for example DMEM (Dulbecco's Modified Eagle Medium) and/or RPMl medium (medium developed by the Roswell Park Memorial Institute) and/or Chang medium, optionally together with other components, for example fetal calf serum (FCS), collagen, more particularly type I collagen, and the like. Examples of nutrient media suitable in accordance with the invention for cultivating the dermal papilla cells are any RPMl- or DMEM-based cell culture media such as, for example, RPMl 1640 (Sigma) containing 20% FCS, Chang medium (Irvine Scientific) containing 10% FCS and the like. 1 to 3 passages are particularly preferred for the pseudopapilla produced by droplet formation.

In a preferred embodiment of the invention, the suspension to be used is obtained by mixing of the papilla cells obtained from monolayer cultures and optionally present in a suitable nutrient medium with a matrix-forming, more particularly gel- forming, medium MFM_S which contains at least one matrix former MFs, more particularly gel former, and optionally other constituents.

The matrix-forming, more particularly gel-forming, medium MFMs contains as matrix former MF_S, more particularly gel former, at least one collagen, more particularly type I collagen, and optionally other constituents selected in particular from the group of matrix and/or scleroproteins, more particularly laminin, gelatine, chitosan, glucosamines, glucosaminoglycans (GAG), heparane sulfate proteoglycans, sulfated glycoproteins, such as nidogen (more particularly 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 human placenta and mixtures of the above-mentioned constituents. According to the invention, the following substances, for example, are suitable matrix materials in the suspension for forming the pseudopapillae (PP): Matrigel® Basement Membrane Matrix (Matrigel®), collagen, gelatine, collagen/chitosan/GAG (GAG = glucosaminoglycan), glucosamine or any other type of matrix and mixtures of these substances.

Collagen, Matrigel® and mixtures thereof are particularly preferred. In the reconstructed papillae thus produced, the cells can be further cultivated with a comparatively low apoptosis or necrosis rate. Mixtures of collagen, more particularly type I collagen, and Matrigel^® in a ratio of 0.1 :1 to 10:1 (collagen:Matrigel^®) are especially preferred.

Suspensions between 1 :1 and 6:1 , for example 3:1 , 4:1 or 5:1 (collagen:Matrigel@) are most particularly preferred.

The suspension may additionally contain other reagents, for example reagents which promote solidification of the suspension, more particularly crosslinking reagents, such as thrombin for example, and/or chemical crosslinking agents, such as carbodiimides or glutaraldehyde for example.

This suspension is added dropwise to a dispersion solution. In a preferred embodiment, the dispersion solution is agitated, more particularly stirred, during the dropwise addition.

Besides the composition of the suspension, the size of the device used for the dropwise addition, more particularly its outlet diameter or outlet size, and the outlet pressure determine the size of the resulting pseudopapilla. The diameter of the reconstructed papilla decreases with increasing contraction of the matrix which is greater, the larger the number of cells and/or matrix formers present.

The dropwise addition is preferably made through the cannula (for example of a syringe) or similar devices. A cannula diameter of 0.4 to 1.8 mm and more especially 0.6 to 1 mm is particularly preferred because pseudopapillae with a diameter of, in particular, about 0.5 to 3 mm (on the day of production) can thus be obtained.

Depending on its size, a pseudopapilla according to the invention may contain preferably from 10² to 10⁹ and more particularly from 10² to 10⁷ cells/ml (on the day of production). The number of cells per ml increases continuously through the contraction of the matrix of the papilla and the proliferation of the cells.

It is advantageously possible in this way to vary the size of the pseudopapillae and to adapt it to requirements through the outlet size of the device used for the dropwise addition which is only possible with difficulty, if at all, in the case of solid supports in particular.

The dispersion solution according to the invention is, in particular, not aqueous. Low-viscosity neutral oils, which may also contain other, more particularly nonaqueous components, are particularly suitable.

In a preferred embodiment, the dispersion solution is selected from liquid fats, particularly paraffin oils or triglycerides which, independently of one another, preferably carry residues of C₄-₁8 and more particularly C₆-₁₂ fatty acids.

Two immiscible liquids may also be used providing they are selected in particular so that the reconstructed papillae do not stay any deeper than at the phase boundary. A suitable additional component is, in particular, a liquid unreactive, optionally substituted, more particularly perfluorinated hydrocarbon, more particularly with a chain length of 8 to 16 carbon atoms, which preferably may also carry one or more hydroxy groups.

The pseudopapillae thus produced may then be separated from the dispersion solution and washed or stored in a physiological solution or nutrient medium. The pseudopapillae may be separated from the dispersion solution using agents and procedures known to the expert which, above all, should be gentle in order not to impair, damage and/or destroy the structure and mode of operation of the pseudopapillae. For example, appropriate tweezers, pipettes and nets, sieves, membranes or filters with a suitable pore size are suitable for this purpose.

These reconstructed dermal papillae may be used in the pharmaceutical, medical or cosmetics field and for body care, more particularly for discovering, investigating and/or monitoring/testing pharmaceutical or cosmetic agents, more particularly for compatibility and/or effectiveness, more especially in relation to the hair follicle, more particularly in regard to hair pigmentation, hair growth, hair structure, hair color and the like.

They may preferably be used in automated screening processes, more particularly for discovering, investigating and/or monitoring/testing pharmaceutical or cosmetic agents, preferably for in vitro evaluation of the influence which pharmaceutical or cosmetic agents in particular have on the hair follicle, hair pigmentation, hair growth, hair structure, hair color and the like.

The present invention also relates to a process for the production of a skin/hair equivalent, more particularly a skin/hair model with reconstructed papillae (pseudopapillae; PP) in a reconstructed dermis (pseudodermis; PD), the process comprising the following steps:

(a) providing a suitable reconstructed dermis (pseudodermis; PD) or a pseudodermis preparation; (b) providing reconstructed papillae (pseudopapillae;PP) comprising cultivated papilla cells, preferably dermal papilla cells (hair papilla cells), in a suitable matrix, more particularly gel matrix, or providing corresponding precursors of such reconstructed papillae (pseudopapillae; PP) comprising cultivated papilla cells, more particularly dermal papilla cells (hair papilla cells), in a suitable matrix-forming, more particularly gel-forming, medium MFM_Pp which is capable of forming a matrix, more particularly a gel matrix, in situ, more particularly in the reconstructed dermis (pseudodermis; PD);

(c) introducing or inserting the reconstructed papillae (PP) or their precursors provided in step (b) into the pseudodermis (PD) or the pseudodermis preparation provided in step (a);

(d) optionally applying a reconstructed epidermis (pseudoepidermis; PE) or a reconstructed periderm (pseudoperiderm; PI) to the pseudodermis (PD),

characterized in that the reconstructed papilla (pseudopapilla, PP) provided in step b) is formed by adding a suspension containing cultivated papilla cells dropwise to a dispersion solution in which the suspension solidifies or polymerizes to form a reconstructed papilla.

Accordingly, step (a) of the process comprises providing a reconstructed dermis or pseudodermis or a pseudodermis preparation. According to the invention, any pseudodermis known from the prior art may be used providing it is suitable for the process according to the invention which means in particular that it is compatible with the other constituents of the skin/hair equivalent according to the invention. In particular, the pseudodermis (PD) used in accordance with the invention comprises cultivated contractile cells, more particularly fibroblasts, preferably dermal fibroblasts, in a suitable matrix. The matrix may be, in particular, a matrix based on collagen, preferably type I and/or type III collagen, and optionally other components. The cultivated contractile cells for the pseudodermis (PD) or the pseudodermis preparation may be obtained in known manner, for example by isolating dermal fibroblasts from human or animal skin and, after cultivation, recovering the contractile cells from the resulting monolayer cultures (for example by moderate trypsinization). A suitable nutrient medium which should be compatible with the contractile cells in particular is used for cultivating those cells. According to the invention, one example of a suitable nutrient medium is essential minimal medium MEM (Modified Eagle Medium).

The pseudodermis (PD) may then be obtained by mixing the contractile cells recovered from monolayer cultures and optionally present in a suitable nutrient medium with a matrix-forming, more particularly gel-forming, medium MFMPD which contains at least one matrix former MF_PD, more particularly gel former, and optionally other constituents.

In the context of the invention, the resulting mixture is referred to as the pseudodermis preparation. Depending on the concentration of the matrix-forming medium, the pseudodermis preparation forms a matrix, preferably a gel matrix, relatively quickly (higher concentration) or relatively slowly (lower concentration) and finally contracts, optionally with ejection of any nutrient medium present, to a pseudodermis (PD). All phases or states of the matrix forming process and the contraction process are encompassed by the term "pseudodermis preparation". Accordingly, the pseudodermis is obtained on completion of the matrix formation and contraction of the pseudodermis preparation.

The matrix former MFPD, more particularly gel former, of the matrix-forming, more particularly gel-forming, medium MFMPD may be in particular a matrix former based on collagen, preferably type I and/or type III collagen, and optionally other components (for example constituents of the extracellular matrix of the dermis, preferably matrix and/or scleroproteins, such as laminin).

A suitable nutrient medium, more particularly essential minimal medium MEM, and optionally other components may optionally be applied to the pseudodermis (PD) thus provided or generated.

The pseudopapillae (PP) provided in step (b) comprise cultivated papilla cells, more particularly dermal papilla cells (hair papilla cells) in a suitable matrix. The pseudopapillae are produced by one of the above-described processes for producing the reconstructed papilla (pseudopapilla). The matrix may be in particular a matrix based on collagen, more particularly type I collagen, and optionally other components.

It can be of advantage directly to use the pseudopapillae produced by droplet formation and solidification or hardening or initially to store them in a physiological solution, more particularly a nutrient solution, and only to introduce them into the pseudodermis after several days, more particularly 1 to 14 and preferably 4 to 10 days.

As already described, the advantages of using the papillae reconstructed by the process according to the invention lie in particular in the high cell density, the favorable handling behavior of the pseudopapillae and their favorable properties in terms of strength and flexibility, considerable similarity to the natural model and favorable biological activity of the papilla cells in the reconstructed papilla.

The pseudopapillae formed by the dropwise addition (hereinafter referred to for differentiation as individual pseudopapillae) may also be used to produce a macroscopic pseudopapilla with several pseudopapilla produced in accordance with the invention present therein.

The macroscopic pseudopapilla are then obtained by mixing the individual pseudo papilla produced in accordance with the invention and optionally present in a suitable nutrient medium with a matrix-forming, more particularly gel-forming, medium MFMPP which contains at least one matrix former MFpp, more particularly gel former, and optionally other constituents; the resulting mixture forms a matrix, preferably a gel, and then contracts with ejection of the nutrient medium present, if any. The macroscopic pseudopapillae (PP) can then be formed (for example by punching or cutting out) from the contracted matrix or the contracted gel. As described in the following, however, the pseudopapillae (PP) may also be formed in situ, more particularly in the pseudodermis (PD). The matrix-forming, more particularly gel-forming, medium MFMp_P contains as matrix former MF_PP, more particularly gel former, at least one collagen, more particularly type IV collagen, and optionally other constituents selected in particular from the group of matrix and/or scleroproteins, more particularly laminin, gelatine, chitosan, glucosamines, glucosaminoglycans (GAG), heparane sulfate proteoglycans, sulfated glycoproteins, such as nidogen (more particularly 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 human placenta and mixtures of the above-mentioned constituents. According to the invention, the following substances, for example, are suitable matrix materials for forming the pseudopapillae (PP): Matrigel® Basement Membrane Matrix (Matrigel®), collagen, gelatine, collagen/chitosan/GAG (GAG = glucosaminoglycan), glucosamine or any other type of matrix and mixtures of these substances. Collagen, Matrigel® and mixtures thereof are particularly preferred. Matrigel® and mixtures in a ratio of 0.1:1 to 10:1 (collagen:Matrigel®) are most particularly preferred.

As previously mentioned, the macroscopic pseudopapillae (PP) may be formed from the matrix, more particularly the gel, the forming/shaping of these pseudopapillae (PP) taking place either before or after the introduction or insertion in step (c) of the macroscopic pseudopapillae (PP) or their precursors (for example the matrix containing the individual pseudopapillae which develops into macroscopic pseudopapillae).

The matrix former MF_PD for forming the pseudodermis (PD), the matrix former MFs for forming the individual pseudopapillae (PP) and the trix former MF_PP for forming the macroscopic pseudopapillae (PP) should be capable of gelling on heating, more particularly at temperatures of 20°C to 40°C, for example polymerizing in the process, and of promoting the growth and differentiation of cells. The matrix of the pseudodermis (PD) and the matrix of the macroscopic pseudopapillae (PP) are generally formed in three-dimensional structures. Formation of the matrix, more particularly the gel, can be reversible or irreversible although it is preferably irreversible.

In the following, the term pseudopapilla (PP) encompasses both the reconstructed papilla (individual pseudopapilla) produced by the process according to the invention and the macroscopic pseudopapilla.

The introduction or insertion of the pseudopapillae (PP) in step (c) can be carried out in various ways:

In one embodiment, suitable cavities for accommodating the pseudopapillae (PP) are first formed, preferably in the already contracted pseudodermis (PD), more particularly by punching or pricking, and the pseudopapillae (PP), which contain cultivated papilla cells and which are, in case of the macroscopic pseudopapilla, shaped so that their dimensions correspond to the cavities formed in the pseudodermis (PD), are then introduced or inserted (for example by grafting) into those cavities. Punching of the pseudodermis (PD) or pricking of the pseudodermis (PD) may be carried out, for example, with a punch (for example with a diameter of 0.5 to 4 mm, preferably about 2 mm) or with a button cannula or with a conventional cannula.

Before introduction of the pseudopapilla (PP) or their precursors, the cavities formed by punching or pricking of the pseudodermis (PD) may be lined (for example by spraying), more particularly with at least one collagen, preferably type IV collagen, and/or other matrix proteins, more particularly basal membrane proteins, such as laminin. The cavities are preferably lined with mixtures of two or more of the constituents mentioned, more particularly Matrigel^®. Where this procedure is adopted, the pseudopapillae (PP) are preferably introduced or inserted into the pseudodermis (PD) or the pseudodermis preparation in a number or density of 1 to 50/cm² pseudodermis (PD) and more particularly 3 to 7/cm² pseudodermis (PD).

In another embodiment, the introduction or insertion of the pseudopapillae (PP) in step (c) can be carried out by directly injecting or inserting the pseudopapillae (PP), into the pseudodermis (PD) or the pseudodermis preparation. In one particular embodiment, the introduction of the pseudopapillae into the pseudodermis preparation takes place before contraction to the pseudodermis is complete. In a particularly preferred embodiment, the introduction or insertion of the pseudopapillae can take place as soon as the pseudodermis preparation has formed the matrix (or gel), preferably before or at the beginning of the contraction phase.

Where this procedure is adopted, the pseudopapillae (PP) are again preferably introduced or inserted into the pseudodermis (PD) or the pseudodermis preparation in a number or density of 1 to 50/cm² pseudodermis (PD) and, more particularly, 3 to 7/cm² pseudodermis (PD).

Alternatively, the precursors of macroscopic pseudopapillae (PP) may also be injected or inserted into the pseudodermis (PD) or the pseudodermis preparation. These precursors of the macroscopic pseudopapillae consist of a mixture of the (individual) pseudopapillae produced in accordance with the invention (individually reconstructed by droplet formation) and at least one matrix former MFpp, as described above, so that this mixture then forms the matrix and hence the macroscopic pseudopapillae (PP) in situ in the pseudodermis (PD) or the pseudodermis preparation, more particularly by gelling, i.e. in this embodiment, the macroscopic pseudopapillae (PP) are formed in situ in the pseudodermis (PD) or the pseudodermis preparation.

In another particularly preferred embodiment, the introduction or embedding of the pseudopapillae (PP) in step (c) may be carried out by directly mixing the individual pseudopapillae (PP), individually reconstructed by droplet formation, with the contractile cells and the matrix-forming, more particularly gel-forming, medium MFMpp, which contains at least one matrix former MFp_D, more particularly gel former, and optionally other constituents, during the preparation of the pseudodermis. This early mixing of the pseudopapillae with the pseudodermis preparation leads to a uniform skin/hair equivalent. The individual pseudopapilla may be introduced or inserted into the pseudodermis (PD) in a density of 1 to 20,000 PP/cm³ pseudodermis (PD) and more particularly in a density of 20 to 5,000 pseudopapillae/cm³ pseudodermis (PD).

In order realistically to adjust the in vivo system, the introduction or insertion in step (c), more particularly where punches are inserted and the pseudopapillae (PP) or their precursors are injected into the pseudodermis (PD), takes place at an angle of 30° to 90° and more particularly 40° to 60°, based on the plane of the pseudodermis (PD).

The introduction or insertion of the pseudopapillae (PP) into the pseudodermis (PD) may be carried out at regular intervals.

Before or after the introduction or insertion of the pseudopapillae (PP) or their precursors in step (c), a reconstructed epidermis (pseudoepidermis; PE) or a reconstructed periderm (pseudoperiderm; PI) may optionally be applied to the pseudodermis (PD) in step (d). The pseudoepidermis is preferably applied after the introduction or insertion of the pseudopapillae (PP) or their precursors in step (c). The pseudoepidermis (PE) or the pseudoperiderm (PI) may consist of cultivated keratinocytes, hair follicle keratinocytes (ORS and/or matrix keratinocytes), more particularly outer root sheath keratinocytes (ORS keratinocytes) and/or epidermal keratinocytes and optionally of melanocytes, more particularly outer root sheath melanocytes (ORS melanocytes) and/or epidermal melanocytes, and optionally other constituents. The keratinocytes and the melanocytes present, if any, may be applied to the pseudodermis (PD) individually in separate monolayers or multilayers or together in admixture as a monolayer or multilayer. Depending on the medium used, the outer root sheath keratinocytes (ORS keratinocytes) in particular can form a pseudoperiderm (PI). The pseudoperiderm with its periderm-like structure corresponds to the conditions in the embryonic follicle morphogenesis. This has the advantage that the natural conditions, more particularly the direct and indirect cell/cell and/or cell/matrix interactions observed in a three-dimensional geometry, can thus be investigated or influenced. One embodiment of the process according to the invention may be carried out as follows:

To produce the model, a pseudodermis (PD) is first prepared from epidermal fibroblasts in a collagen matrix. "Holes" can then punched into the pseudodermis (PD) at a certain angle (for example 30-90°) and may then optionally be lined with basal membrane proteins (for example laminin, collagen IV, etc.) or, for example, with Matrigel^®, a mixture of basal membrane proteins, or mixtures of Matrigel^® with collagen (more particularly with type I collagen). Reconstructed papillae (pseudopapillae; PP) - both individual and macroscopic - are then inserted into the "holes". These macroscopic pseudopapillae may be obtained, for example, by mixing the individual pseudopapillae produced in accordance with the invention by droplet formation with a matrix and/or gel former, for example Matrigel®, and gelling the mix. After gelation, the macroscopic pseudopapillae (PP) can be punched from the gel and inserted into the pseudodermis (PD). However, gelation may also take place in situ after introduction into the pseudodermis. Alternatively, however, the pseudopapillae, more particularly the individual pseudopapillae (individually reconstructed by droplet formation) embedded in Matrigel® may also be directly introduced into the pseudodermis (PD) without "holes" having been punched beforehand or the individual pseudopapillae embedded in Matrigel® or collagen solution are introduced into the pseudodermis (PD) via a pricking channel. Embedding in comparatively liquid Matrigel or collagen solutions provides for better handling of the reconstructed papilla when it is introduced into the pseudodermis.

Besides dermal papilla cells, the pseudopapilla may also contain cells of other cell types, more particularly keratinocytes and/or melanocytes, preferably obtained from the outer root sheath.

In a preferred embodiment, the macroscopic pseudopapilla may also contain other cell types besides the reconstructed individual pseudopapillae. More particularly, these cells types may be also be produced and used in spherical form by droplet formation analogously to the already described process for producing the reconstructed papilla. In a particularly preferred embodiment, the cell types are selected from keratinocytes and/or melanocytes preferably obtained from the outer root sheath. The close spatial contact in the macroscopic pseudopapilla provides for particularly good reconstruction of a hair follicle.

Finally, a cell layer of hair follicle melanocytes and/or hair follicle keratinocytes (ORS and/or matrix keratinocytes) can be applied to the pseudodermis (PD). It can also be of advantage, particularly for the hair model, selectively to cover the individual or macroscopic pseudopapillae inserted into the pseudodermis with one or more cell layers of hair follicle keratinocytes and/or melanocytes without coating the entire pseudodermis in the process. The natural features of a hair follicle (papilla with surrounding or covering layer(s) of keratinocytes and/or melanocytes) in a skin tissue can be even better simulated in this way.

In a particularly preferred embodiment, the individual pseudopapilla produced by the dropwise addition process according to the invention are individually inserted into the pseudodermis or the pseudodermis preparation. They may then either be inserted into preformed "holes" in the pseudodermis or may simply be integrated in the pseudodermis by pressure or directly mixed with the contractile cells and the gel former (or the pseudodermis preparation) during the production of the pseudodermis.

The pseudopapilla (PP) influences the structure of the optionally overlying pseudoepidermis (PE) or the pseudoperiderm (PI) keratinocytes and, optionally, melanocytes. A hair-follicle-like structure is formed under these conditions. The model produced in this way may be used, for example, to study substances (pharmacological, cosmetic, etc.) for hair growth and hair structure and to study the effect of substances on hair pigmentation.

Follicle-like or follicular structures, including those reminiscent of the earliest stages of hair morphogenesis (morphogenesis stages I to III), such as the periderm for example, are then formed in this three-dimensional hair/skin model. The present invention also relates to the hair/skin equivalent obtainable by the process according to the invention.

The skin/hair equivalent according to the invention is in particular a skin/hair model with, in particular, three-dimensionally formed, optionally spatially demarcated, reconstructed papillae (pseudopapillae; PP) with follicle-like or follicular structures, including those reminiscent of the earliest stages of hair morphogenesis, i.e. morphogenesis stages I to III), the skin/hair equivalent comprising a reconstructed dermis (pseudodermis; PD) into which the pseudopapillae (PP) are introduced or inserted, the pseudopapillae (PP) comprising cultivated papilla cells, more particularly dermal papilla cells (hair papilla cells), on a suitable carrier and/or in a suitable matrix, more particularly gel matrix, and a reconstructed epidermis (pseudoepidermis; PE) or a pseudoperiderm (PI) optionally being applied to the pseudodermis (PD), whereas the pseudopapillae are formed in accordance with one of the methods described herein.

Accordingly, the skin/hair equivalent, more particularly the hair follicle model, according to the invention generally comprises a pseudodermis (PD) with dermal papillae reconstructed therein (pseudopapillae; PP). One or more layers of keratinocytes, which form or have formed themselves into a pseudoepidermis (PE) or a pseudoperiderm (PI), and optionally one or more layers of melanocytes can be applied over the pseudodermis (PD). This in vitro model is suitable, for example, for effectiveness and compatibility tests in the pharmaceutical, medical and cosmetics fields. Follicle-like or follicular structures, including those reminiscent of the earliest stages of hair morphogenesis (morphogenesis stages I to III), are formed in the three-dimensional hair/skin model according to the invention.

The present invention also relates to 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. By virtue of its nature-like and at the same time easier-to-handle construction, the skin/hair equivalent is particularly suitable for answering these questions. In addition, for further details, reference may be made to the foregoing observations on the process according to the invention and the skin/hair equivalent according to the invention which also apply accordingly to the use according to the invention.

The present invention also relates to a system, more particularly a test system (for example a screening system), which comprises the skin/hair equivalent according to the invention. In addition, for further details, reference may be made to the foregoing observations on the process according to the invention, the skin/hair equivalent according to the invention and the use according to the invention which also apply accordingly to the system according to the invention.

The particular skin/hair equivalent affords a number of advantages: The skin/hair equivalent according to the invention is a reconstructed model which is more standard izable than the isolated hair follicle. It reduces the demand for hair follicles and is closer to the in vivo situation than monolayer systems. In addition, it is an alternative to animal tests.

The reconstructed model according to the invention is a complex three- dimensional model which simulates the hair follicle in vivo in its structure and its histological composition, resulting in a high level of relevance of the information provided on the effectiveness and compatibility of active substances (cosmetics, pharmaceuticals, etc.).

The following end points inter alia can be evaluated or measured to obtain information on the effectiveness of substances in regard to an improvement in hair structure and the influencing of hair growth: proliferation/apoptosis of the keratinocytes via the pseudopapilla; structure and arrangement of the keratinocytes via the pseudopapilla; structure of the epidermis; structure of the stratum corneum; volume and structure of the dermal papilla; analysis of certain hair-specific proteins (more particularly hair-specific keratins); analysis of cytokines, chemokines and all kinds of messenger substances formed inter alia by the dermal papilla; hair chip(array) analysis, proteom or expression analyses, etc. The reconstructed hair follicle model according to the invention is the a reconstructed hair follicle model with which influences on hair pigmentation can be measured (for example pigmentation of the amelanocytic ORS melanocytes; melanin synthesis; melanin granula; arrangement of the melanocytes; migration of the melanocytes; modification of melanocyte markers, such as TRP-1 , TRP-2, NKI/beteb, etc.; release of melanin to keratinocytes). It may also be used, for example, for hair chip (array) analysis.

The hair/skin model according to the invention is suitable for various applications in the medical, pharmaceutical and cosmetics fields (for example for the discovery of active substances with a biological effect on the hair follicle by influencing hair pigmentation, hair growth and hair structure, in in vitro test systems, in screening processes, for the development of cosmetic products, etc.). The model or equivalent according to the invention provides information on the effect of substances on hair follicle cells with in vivo relevance. The model or equivalent according to the invention provides hair follicles or parts of hair follicles in a three- dimensional model. In contrast to isolated hair follicles, the reconstructed papillae (pseudopapillae; PP) are available at any time and standardizable. Particularly, dermal papillae which share the three-dimensional structure of the hair follicle particularly well are reconstructed. It is possible in this way to evaluate how applied active substances act on the structure of the reconstructed three- dimensional model and what can be read into this with regard to the effect of these substances on the hair follicle (for example hair growth, hair structure, hair pigmentation, etc.).

Other embodiments, modifications and variations of the present invention will be readily apparent to the expert on reading the present specification and can be put into practice without departing from the scope of the invention.

The following Examples are intended to illustrate the invention without limiting it in any way. Examples

The definitions defined in the following Table are used hereinafter.

Abbreviations

Example 1 : reconstruction of the dermal papilla

Production of the dermal papilla cells

Dermal papilla cells are isolated from scalp (temple or occipital region) with intact hair follicles. To this end, the upper dermis is removed and the follicles together with the dermal papilla are plucked from the dermis using watchmaker's tweezers. The further isolation of the dermal papilla is carried out under a stereo magnifying glass. The dermal papilla is then transferred to a culture bottle coated with collagen I with the aid of a microcapillary. Cultivation is carried out either in RPMl 1640 Medium containing glutamine (Sigma) enriched with 20% fetal calf serum (FCS) (Gibco BRL, Karlsruhe, Germany) and antibiotics or in Chang's Medium containing 10% fetal calf serum (BIOCHROM KG) [2].

Reconstruction of the dermal papilla (pseudopapilla)

To reconstruct the dermal papilla, sterile neutralized type I collagen from rat tail tendon (BD Biosciences) is mixed with Matrigel® in a ratio of 4:1 and a cell suspension of dermal papilla cells (10⁶ cells, passage 2 or 3 in Chang's medium) in fetal calf serum is added to the resulting mixture.

The mixture is introduced into a syringe and added dropwise to a solution of neutral fat (caprylic/caproic acid triglyceride), this nonaqueous phase being permanently stirred at 37°C. The drops polymerizing under these conditions are stirred for another 10 minutes at a temperature of 37°C and then removed from the fatty phase with a net. The collagen drops (pseudopapilla) are transferred to a Petri dish and washed with suitable culture medium. The culture is then covered with nutrient medium (fibroblast medium) and incubated in an incubator for up to 7 days at 37°C/5% CO₂.

Results:

After 7 days, a high cell density and organotypical arrangement of the cells were confirmed by histological investigations.

Example 2: production of a skin/hair model with reconstructed dermal papilla

The production of a skin/hair model with reconstructed dermal papillae (pseudopapillae; PP) embedded in or inserted into a pseudodermis (PD) is described in the following. To this end, the pseudopapillae produced in Example 1 are either injected into the pseudodermis (PD), mixed with it and placed by means of punches in the pseudodermis (PD). The pseudodermis (PD) may then be covered with a layer of epidermal or hair follicle keratinocytes, which represent a pseudoepidermis (PE) or a pseudoperiderm (PI), and optionally with melanocytes.

Preparation of the other single cell cultures

Dermal fibroblasts

Dermal fibroblasts are isolated from human foreskin. The epidermis and dermis of the foreskin are enzymatically separated from one another with thermolysin (0.5 mg/nl HEPES buffer). To extract the fibroblasts, the dermis is digested (3-4 h at 37°C) with collagenase H (0.2 U/ml, Boehringer Mannheim, Mannheim, Germany). After the incubation phase, the solution is carefully mixed to thin out the cells, filtered through a cell sieve and the cells are centrifuged off. Cultivation is carried out in Dulbecco's Modified Eagle Medium (DMEM) with Glutamax I (L-alanyl-L- glutamine) and sodium pyruvate, 4,500 mg L^"1 glucose and pyridoxine (Gibco BRL, Karlsruhe, Germany) enriched with 10% fetal calf serum (FCS) (Gibco BRL, Karlsruhe, Germany), 25 μg mL^"1 gentamicin (Sigma, Taufkirchen, Germany) and 100 Ul mL^"1 penicillin G (Sigma) [1].

Epidermal keratinocytes

Dermal fibroblasts are isolated from human foreskin. The epidermis and dermis of the foreskin are enzymatically separated from one another with thermolysin (0.5 mg/nl HEPES buffer). To extract the keratinocytes, the epidermis is digested (20 mins. at 37°C) with trypsin (Gibco BRL, Karlsruhe, Germany). After the incubation phase, the solution is carefully mixed to thin out the cells, filtered through a cell sieve and the cells are centrifuged off. Cultivation is carried out in a mixture of DMEM Glutamax I and Ham's F 12 (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 irradiated with 60 gray to inhibit proliferation).

Outer root sheath keratinocytes (ORS keratinocytes)

ORS keratinocytes are isolated from human hairs plucked from the back of the head. To extract the keratinocytes, the remains of the hair bulb are first removed with a scalpel and the follicles digested (40 mins. at 37°C) with trypsin (protease from Gibco BRL, Karlsruhe, Germany). After the incubation phase, the solution is carefully mixed to thin out the cells, filtered through a cell sieve and the cells are centrifuged off. Cultivation is carried out in a mixture of DMEM Glutamax I and Ham's F 12 (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 irradiated with 60 gray to inhibit proliferation). ORS melanocytes are isolated by Tobin et al.'s method [3].

Production of the pseudodermis (PD)

To establish the hair model, the pseudodermis (PD) is first developed. To this end, dermal fibroblasts of the fourth passage are mixed with collagen I from rat tail tendon and sown in multiwell plates. The object of this is to optimize the number of cells, the cultivation time, the layer thickness and the size of the dermis equivalent (pseudodermis; PD). Production is carried out to the following protocol: 1 part of HBSS buffer (Gibco BRL) is mixed with 8 parts of collagen solution (Becton Dickinson) and neutralized with 1 M sodium hydroxide. The required quantity of cells is added in 1 part of fetal calf serum (FCS, Gibco BRL). The mixture obtained ( pseudodermis preparation) is poured into cell culture dishes and incubated for 1 h at 37°C in an incubation cabinet. After polymerization of the collagen, the models are covered with DMEM supplemented with 10% FCS and penicillin/streptomycin. The medium is changed three times per week over a period of seven days. The following preparations were tested:

Preparation of the injection channels for insertion of the DPC into the pseudodermis (PD)

To prepare the injection channels, a button cannula, a conventional cannula and a 2 mm punch are used. To show up the channels, a solution of 10% Berlin Blue in 1% agarose solution is prepared and injected into the dermis equivalent using the button cannula and the conventional cannula. Where the punches are used, the channels prepared with the biopsy punches are filled either immediately or after 24 h using the button cannula. The channels are prepared with the aid of a stereo magnifying glass. After 24 h, the models are deep-frozen to prepare cryosections and for histological examination.

Production of the reconstructed papilla bv insertion of punches into the pseudodermis (PD)

To produce macroscopic pseudopapillae, dermal papillae were mixed with collagen I from rat rail tendon and optionally Matrigel and sown in multiwell plates. The number of papillae was selected in line with the production of the pseudodermis (PD).

Production was carried out to the following protocol: 1 part of HBSS buffer (Gibco BRL) was mixed with 4 parts of Matrigel and 8 parts of collagen solution (Becton Dickinson) and neutralized with 1 M sodium hydroxide. The required quantity of papillae was added in 1 part of fetal calf serum (FCS, Gibco BRL). The mixture obtained was poured into cell culture dishes and incubated for 1 h at 37°C in an incubating cabinet. After polymerization of the collagen, the models were covered with Chang Medium supplemented with 10% FCS. The medium was changed three times per week over a period of eight days.

After this cultivation period, holes were made in a 7-day-old pseudodermis with the aid of a 2 mm punch. Quantities of 5 μl of Matrigel were injected and 3 mm biopsies from the polymerized collagen/reconstructed papillae mixture inserted into the holes thus formed (macroscopic pseudopapilla).

The models were covered with Chang medium supplemented with 10% FCS and the medium was changed three times per week over a period of six days. The models were then subjected to histological and immunohistochemical evaluation.

Production of the reconstructed papilla by injection of dermal papilla cells into the pseudodermis (PD) or the pseudodermis preparation

To establish very high cell densities, the cells were first mixed with Matrigel® in the required cell concentration and then centrifuged off in a refrigerated centrifuge (5 mins, 1 ,000 r.p.m., T = 1 °C). The excess Matrigel^® was then removed, the cell pellet left behind was taken up with a pipette and directly injected into the pseudodermis (PD) or the pseudodermis preparation. The models thus produced were subjected to histological and immunohistochemical evaluation.

Production of a skin model of a pseudodermis and an epidermis of epidermal keratinocytes (NHEK) or a pseudoperiderm of hair keratinocytes (ORS keratinocytes) (with and without pseudopapilla)

In order further to develop the reconstructed hair follicle model, the pseudodermis was transferred to Snapwell Inserts (Corning Costar) after five days' culture and was first covered with epidermal keratinocytes (500,000 cells/model). After one week's submerse cultivation in keratinocyte medium (DMEM Glutamax I and Hams' 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), ascorbyl-2-phosphate (Sigma) and antibiotics, the models were transferred to the air/liquid interface and cultivated for another two weeks in DMEM Glutamax I and Hams' F12 (Sigma) (3:1 ) enriched with insulin (Sigma), hydrocortisone (Sigma), ascorbyl-2-phosphate (Sigma), bovine serum albumin (BSA) (Sigma) and antibiotics.

In another experiment, the epidermal keratinocytes were replaced by ORS keratinocytes from the hair follicle which were sown with 800,000 cells/model. Cultivation (submerse) was carried out with keratinocyte medium for 7 days.

Appendix I: Composition of the cell culture media Fibroblast medium

Keratinocyte medium

Appendix II: Literature references cited in the Example

[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

Claims:

1. A process for the production of a reconstructed papilla (pseudopapilla), characterized in that a suspension containing cultivated papilla cells is added dropwise to a dispersion solution in which the suspension solidifies to form a reconstructed papilla.

2. A process as claimed in claim 1 , characterized in that the suspension contains a matrix-forming, more particularly gel-forming, medium MFMs.

3. A process as claimed in claim 2, characterized in that the matrix-forming, more particularly gel-forming, medium (MFMs) is selected from collagen, more particularly type I and/or type III collagen, and Matrigel® and mixtures thereof.

4. A process as claimed in claim 3, characterized in that the suspension contains type I collagen and Matrigel®, preferably in a ratio of 0.1 :1 to 10:1 and more particularly in a ratio of 1 :1 to 6:1.

5. A process as claimed in any of claims 1 to 4, characterized in that the cultivated dermal papilla cells are obtained by isolating dermal papilla cells from the hair follicles of human or animal skin, cultivating them and then isolating the dermal papilla cells from the resulting monolayer cultures, more particularly by moderate trypsinization.

6. A process as claimed in any of claims 1 to 5, characterized in that the dermal papilla cells are cultivated in a suitable nutrient medium, more particularly essential minimal medium MEM, more especially DME medium and/or RPMl medium and/or Chang medium, optionally together with other components, more particularly fetal calf serum (FCS), collagen, more particularly type I collagen, and the like.

7. A process as claimed in any of the preceding claims, characterized in that the dispersion solution is not aqueous.

8. A process as claimed in any of the preceding claims, characterized in that liquid fats, more particularly low-viscosity paraffin oils or triglycerides which, independently of one another, carry residues of C₄-₁₈ and more particularly C_δ-₁₂ fatty acids, or mixtures thereof are used as the dispersion solution.

9. A process as claimed in any of the preceding claims, characterized in that the suspension is added dropwise by means of a capillary.

10. A process as claimed in any of the preceding claims, characterized in that the reconstructed papilla is removed from the dispersion solution in another step.

11. A reconstructed dermal papilla obtainable by the process claimed in any of claims 1 to 10.

12. The use of the reconstructed papilla claimed in claim 11 in the medical, pharmaceutical or cosmetics field, more particularly as a system for testing cosmetic or pharmaceutical agents.

13. A process for the production of a skin/hair equivalent, more particularly a skin/hair model with reconstructed papillae (pseudopapillae; PP) in a reconstructed dermis (pseudodermis; PD), the process comprising the following steps:

(a) providing a reconstructed dermis (pseudodermis; PD) or a pseudodermis preparation;

(b) providing reconstructed papillae (pseudopapillae; PP) comprising cultivated papilla cells, preferably dermal papilla cells (hair papilla cells), in a suitable matrix, more particularly gel matrix, or providing corresponding precursors of such reconstructed papillae (pseudopapillae; PP) comprising cultivated papilla cells, more particularly dermal papilla cells, in a suitable matrix- forming, more particularly gel-forming, medium MFMp_P which is capable of forming a matrix, more particularly a gel matrix, in situ, more particularly in the reconstructed dermis (pseudodermis; PD);

characterized in that the reconstructed papilla (pseudopapilla, PP) provided in step b) is produced by the process claimed in any of claims 1 to 10.

14. A process as claimed in claim 13, characterized in that the pseudodermis (PD) or the pseudodermis preparation contains cultivated contractile cells in a suitable matrix, the matrix being in particular a matrix based on collagen, more particularly type I and/or type III collagen, and optionally other components.

15. A process as claimed in claim 13 or 14, characterized in that fibroblasts, more particularly dermal fibroblasts, are used as contractile cells for the pseudodermis (PD) or the pseudodermis preparation.

16. A process as claimed in any of claims 13 to 15, characterized in that the cultivated contractile cells for the pseudodermis (PD) or the pseudodermis preparation are obtained by isolating dermal fibroblasts from human or animal skin and, after cultivation, recovering the contractile cells from the resulting monolayer cultures, more particularly by moderate trypsinization.

17. A process as claimed in claim 16, characterized in that the contractile cells, more particularly fibroblasts, preferably dermal fibroblasts, are cultivated in a suitable nutrient medium, more particularly essential minimal medium MEM.

18. A process as claimed in any of claims 13 to 17, characterized in that the pseudodermis preparation is obtained by mixing contractile cells obtained from monolayer cultures and optionally present in a suitable nutrient medium with a matrix-forming, more particularly gel-forming, medium MFMpo for the pseudodermis (PD) which contains at least one matrix former MF_PD, more particularly gel former, and optionally other constituents.

19. A process as claimed in claim 18, characterized in that the pseudodermis preparation forms a matrix, preferably a gel, and contracts to a pseudodermis (PD), more particularly with ejection of any nutrient medium present.

20. A process as claimed in claim 18 or 19, characterized in that the matrix- forming, more particularly gel-forming, medium MFM_PD contains as matrix former MF_PD, more particularly gel former, at least one collagen, more particularly type I and/or type III collagen, and optionally other components, more particularly constituents of the extracellular matrix of the dermis, preferably matrix and/or scleroproteins, such as laminin.

21. A process as claimed in any of claims 13 to 20, characterized in that a suitable nutrient medium, more particularly essential minimal medium MEM, and optionally other components are applied to the pseudodermis (PD).

22. A process as claimed in any of claims 13 to 21 , characterized in that the pseudopapilla provided in step b) is a macroscopic pseudopapilla obtained by mixing pseudopapillae produced by the process claimed in claims 1 to 10 with a matrix-forming, more particularly gel-forming, medium MFM_PP for the pseudopapillae (PP) which contains at least one matrix former MFpp, more particularly gel former, and optionally other constituents; the resulting mixture forming a matrix, preferably a gel, and then contracting, optionally with ejection of the nutrient medium present, if any.

23. A process as claimed in claim 22, characterized in that the matrix-forming, more particularly gel-forming, medium MFMp_P contains as matrix former MFpp, more particularly gel former, at least one collagen, more particularly type IV collagen, and optionally other constituents selected in particular from the group of matrix and/or scleroproteins, more particularly laminin, gelatine, chitosan, glucosamines, glucosaminoglycans (GAG), heparane sulfate proteoglycans, sulfated glycoproteins, such as nidogen (more particularly 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 human placenta and mixtures of the above-mentioned constituents.

24. A process as claimed in claim 22 or 23, characterized in that the contracted matrix, more particularly the gel, and/or the pseudopapillae (PP) is/are formed in situ in the pseudodermis (PD).

25. A process as claimed in any of claims 22 to 24, characterized in that the pseudopapillae (PP) are formed from the matrix, more particularly the gel, the forming/shaping of the pseudopapillae (PP) taking place either before or after the introduction or insertion of the pseudopapillae (PP) or their precursors in step (c).

26. A process as claimed in any of claims 13 to 25, characterized in that the introduction or insertion of the pseudopapillae (PP) in step (c) is carried out by forming suitable cavities in the pseudodermis (PD) for accommodating the pseudopapillae (PP), more particularly by punching or pricking, and then introducing or inserting the pseudopapillae (PP) which contain cultivated papilla cells into those cavities.

27. A process as claimed in claim 26, characterized in that, before introduction of the pseudopapillae (PP), the cavities are lined with at least one collagen, more particularly type IV collagen, and/or other matrix proteins, more particularly basal membrane proteins, such as laminin.

28. A process as claimed in any of claims 13 to 27, characterized in that the preformed pseudopapillae (PP) or the precursors of the pseudopapillae (PP) are directly injected or inserted into the pseudodermis (PD) and, more particularly, into the pseudodermis preparation or are directly mixed with the pseudodermis and more particularly with the pseudodermis preparation.

29. A process as claimed in any of claims 13 to 28, characterized in that the application of the reconstructed epidermis (pseudoepidermis; PE) or of a reconstructed periderm (pseudoperiderm; PI) optionally carried out in step (d) takes place before or after the introduction or insertion of the pseudopapillae (PP) or their precursors in step (c).

30. A process as claimed in claim 29, characterized in that the pseudoepidermis (PE) or the pseudoperiderm (PI) contains cultivated keratinocytes, more particularly keratinocytes of the outer hair root sheath (ORS keratinocytes) and/or epidermal keratinocytes and optionally even melanocytes, more particularly melanocytes of the outer hair root sheath (ORS melanocytes) and/or epidermal melanocytes, and optionally other constituents.

31. A process as claimed in claim 29 or 30, characterized in that the keratinocytes and melanocytes are applied to the pseudodermis (PD) individually in separate monolayers or multilayers or together in admixture as a monolayer or multilayer.

32. A hair/skin equivalent, more particularly a hair model with reconstructed papillae (PP) in a pseudodermis (PD) obtainable by the process claimed in claims 1 to 31.

33. The use of the skin/hair equivalent claimed in claim 32 in the pharmaceutical, medical or cosmetics and body care fields.

34. The use of the skin/hair equivalent as claimed in claim 33 for discovering, studying and/or monitoring/testing pharmaceutical or cosmetic agents, more particularly for compatibility and/or effectiveness.

35. The use claimed in claim 34 for discovering, studying and/or monitoring/testing pharmaceutical or cosmetic agents for effectiveness and/or compatibility in relation to the hair follicle, more particularly in regard to hair pigmentation, hair growth, hair structure, hair color and the like.

36. The use of the skin/hair equivalent claimed in claim 32 for the development of pharmaceutical or cosmetic agents.

37. The use of the skin/hair equivalent claimed in claim 32 in preferably automated screening processes, more particularly for the discovery, study and/or monitoring/testing of pharmaceutical or cosmetic agents.

38. The use of the skin/hair equivalent claimed in claim 32 for the in vitro evaluation of the influencing of the hair follicle, hair pigmentation, hair growth, hair structure, hair color and the like, more particularly by pharmaceutical or cosmetic agents

39. A system, more particularly a test system, comprising the hair/skin equivalent claimed in claim 32.