EP3445846A1 - Modèle vollhaut in-vitro, contenant un modèle de culture de cellules tridimensionnel de la schweissdrüse - Google Patents

Modèle vollhaut in-vitro, contenant un modèle de culture de cellules tridimensionnel de la schweissdrüse

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Publication number
EP3445846A1
EP3445846A1 EP17713202.4A EP17713202A EP3445846A1 EP 3445846 A1 EP3445846 A1 EP 3445846A1 EP 17713202 A EP17713202 A EP 17713202A EP 3445846 A1 EP3445846 A1 EP 3445846A1
Authority
EP
European Patent Office
Prior art keywords
sweat gland
equivalent
medium
equivalents
skin
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP17713202.4A
Other languages
German (de)
English (en)
Inventor
Patricia Klaka
Sabine GRÜDL
Melanie Giesen
Thomas Welss
Bernhard Banowski
Lars VIERKOTTEN
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henkel AG and Co KGaA
Original Assignee
Henkel AG and Co KGaA
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henkel AG and Co KGaA filed Critical Henkel AG and Co KGaA
Publication of EP3445846A1 publication Critical patent/EP3445846A1/fr
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0629—Keratinocytes; Whole skin
    • C12N5/063—Kereatinocyte stem cells; Keratinocyte progenitors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0633—Cells of secretory glands, e.g. parotid gland, salivary glands, sweat glands, lacrymal glands
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0697—Artificial constructs associating cells of different lineages, e.g. tissue equivalents
    • C12N5/0698—Skin equivalents
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/54—Collagen; Gelatin

Definitions

  • the present invention relates to an in-vitro full-skin model with a dermis and an epidermis equivalent, wherein in the dermis and / or Epidermisäquivalent 1 to 100 three-dimensional sweat gland equivalency with 500 to 500,000 sweat gland cells and a diameter of 100 to 6000 ⁇ , are included.
  • the sweat gland equivalents into the in vitro full-skin models according to the invention, the in-vivo situation in the human skin can be adjusted very well.
  • the cell-cell interactions present in vivo between the different cell types can be very well mapped since the three-dimensional sweat gland equivalents retain their function even after incorporation into the full-thickness model.
  • the present invention relates to a method for producing an in vitro full-skin model, in which first a dermis equivalent is provided on a carrier layer. Subsequently, an epidermis equivalent is applied to this dermis equivalent.
  • the introduction of the three-dimensional sweat gland equivalents in the in vitro full skin model takes place during the provision of dermis and / or Epidermisäquivalents.
  • the present invention relates to the use of an in vitro full-skin model according to the invention in cosmetics and personal care, in particular for testing, identifying and testing cosmetic active ingredients, preferably with regard to their effectiveness in inhibiting sweat secretion and / or body odor, and also Evaluation of the influence of cosmetic agents on the inhibition and / or reduction of sweat secretion and / or body odor.
  • the present invention relates to a system, in particular test system, which comprises an in vitro full-skin model according to the invention.
  • washing, cleansing and caring for one's own body is a basic human need, and modern industry is constantly trying to meet these needs of man in a variety of ways.
  • Especially important for daily hygiene is the permanent elimination or at least reduction of body odor and underarm wetness.
  • Underarm wetness and body odor are caused by the secretion of eccrine and apocrine sweat glands in the human axilla. While the eccrine glands serve to thermoregulate the body and cause the onset of underarm wetness, the apocrine glands secrete viscous secretions in response to stress, resulting in unpleasant body odor due to bacterial decomposition.
  • Sweat glands can then be divided into apocrine and eccrine sweat glands and a mixed form of apocrine and eccrine sweat glands (also referred to as apoekkrine sweat gland).
  • the aforementioned forms can be distinguished by morphological and characteristic features.
  • the eccrine sweat gland in particular the human eccrine sweat gland, is one of the unbranched tubular tubular glands and can be in the secretory tail (also referred to as a coil), the dermal Ausfgang (also referred to as a duct) and the epidermal output (also known as Acrosyringium) divide.
  • the cells present in these glandular sections have different functions and functions, such as, for example, secretion in the coil, reabsorption of ions in the duct and release of the secretion, in particular of sweat, to the surrounding skin by the acrosyringium.
  • the eccrine sweat glands are stimulated by the neurotransmitter acetylcholine (ACh).
  • cosmetic compositions which are able to reliably prevent underarm wetness and / or body odor and which contain neither antibacterial agents nor the aluminum and / or aluminum-zirconium salts used in the prior art.
  • One way to provide such agents is to use substances that effectively inhibit the stimulation of the sweat glands and thus reduce or prevent sweat secretion.
  • in-vivo tests can be carried out with test participants.
  • such assays are expensive and do not allow high throughput screening methods.
  • sweat gland cell models can be used to study the influence of test substances on sweat gland stimulation. Such models must simulate the in-vivo situation as accurately as possible, be standardized and cost-effective and enable screening methods with high throughput rates.
  • a three-dimensional sweat gland model known in the art is exemplified by Li (Li et al., "Matrigel the Membrane Matrix Induces eccrine Sweat Gland Cells to Reconstitute Sweat Gland-like Structures in Nude Mice”; Experimental Cell Research, 2015, 332, pages 67 to 77)
  • primary sweat gland cells are cultured with growth factors in a gel-like substance (Matrigel®) and then implanted into the back of living mice. After implantation, spheroid structures are formed which express sweat gland specific marker proteins. Since the use of mice is required to form the differentiated structures, this model can not be used in cosmetic and pharmaceutical research where the use of experimental animals is prohibited.
  • full-skin models which in addition to a dermis and an epidermis also contain sweat glands known.
  • models By punching out samples of native adult human skin, models are obtained which contain all the skin appendages in this area of the skin, including sweat glands.
  • Such models are also referred to as ex-vivo full-skin models.
  • a disadvantage of these models is their low standardizability, since control of the exact number of sweat glands is not possible and also the characteristics of the model depend on the particular donor.
  • in vitro full-skin models which have similar, preferably identical, histological architecture to human skin and which can be prepared using only in vitro methods. Furthermore, it is desirable if these in-vitro full-skin models can be standardized and produced inexpensively and are suitable for use in high-throughput screening methods.
  • the present invention therefore an object of the invention to provide an in vitro full skin model (hereinafter referred to as skin equivalent) whose cell structure and cell-cell interactions have a high similarity to the cell structure and cell-cell interactions of native skin and which can only be produced by in-vitro methods. Furthermore, this model should be inexpensive to produce, be standardized and are suitable for use in screening methods with high throughput.
  • models can be obtained by introducing three-dimensional sweat gland equivalents into a dermis and / or epidermis equivalent of a skin equivalent, in which the cell-cell interactions between the different cell types almost correspond to those in the native skin.
  • the production takes place exclusively by in-vitro methods and allows a high standardizability as well as an economical production of the skin equivalents. Therefore, these skin equivalents are particularly suitable for use in high throughput screening methods for testing antiperspirant substances for use in cosmetics and pharmacy.
  • a first subject of the present invention is thus an in vitro full-skin model comprising a) at least one carrier layer comprising at least one collagen matrix
  • the dermis equivalent b) and / or the epidermis equivalent c) comprises 1 to 100 three-dimensional sweat gland equivalent (s), the three-dimensional sweat gland equivalents each comprising 500 to 500,000 sweat gland cells and having a diameter of 100 to 6,000 ⁇ each.
  • the introduction of the three-dimensional sweat gland equivalents replaces the cell-cell interactions present in human skin. Therefore, these models map the in-vivo situation very well, so the results obtained with these models are more relevant than the results obtained from full-skin models without sweat gland equivalents.
  • the eccrine sweat gland cells contained in the three-dimensional sweat gland equivalents have a potential stem cell activity in wound healing, so that further processes occurring in the skin can be investigated with the skin equivalent according to the invention.
  • cultured cells to prepare the skin equivalent high standardization can be achieved since a variety of skin equivalents having the same property can be produced from the cultured cells.
  • the introduction of a precisely determinable number of three-dimensional sweat gland equivalents also ensures a high degree of standardizability.
  • an in-vitro full-skin model (hereinafter also referred to as skin equivalent) is a model of the skin which was produced exclusively using in vitro methods and has both a stratified epidermis and a dermis, a basal membrane interposed between the skin Dermis and the epidermis is located.
  • carrier layer is understood as meaning a self-supporting layer which serves as a substrate for the dermal equivalent of the dermal equivalent of the invention this layer is a collagen matrix, whereby a collagen matrix is understood as meaning a spatial network of collagen, which preferably has pores, but does not include a matrix which is formed by the collagen produced by the fibroblasts of the dermis.
  • a dermis equivalent in the context of the present invention is understood to mean a fibroblast fibrous-like layer which largely corresponds to the native dermis.
  • an epidermis equivalent in the context of the present invention is understood to mean a preferably multilayered layer of keratinocytes in the form of a stratified epidermis which largely corresponds to the native epidermis.
  • the cohesion between the collagen braid and the laminin network is preferably ensured by Perlecan (a proteoglycan formed from heparin sulfate) and entactin (also referred to as nidogen).
  • a three-dimensional sweat gland equivalent is understood to mean a cell model of sweat gland cells which has an extension in all three spatial directions and in which the cells exhibit a similar function, in particular an identical function, as cells of a native sweat gland.
  • the in vitro full-skin model according to the invention comprises a carrier layer which contains a collagen matrix.
  • the skin equivalent as the carrier layer preferably has a matrix of specific collagen compounds.
  • the collagen matrix comprises a sparingly soluble collagen, in particular a sparingly soluble collagen from tendons of horses, pigs or cattle.
  • sparingly soluble collagen is understood to mean coarse-fiber collagen which has no visible or only slight swelling or no or only slight gel formation, in particular no gel formation, in the aqueous medium for at least 5 hours.
  • Such collagen is preferably obtained from the tendons of animals, in particular mammals, preferably horses, pigs or cattle, most preferably from the Achilles tendon or the skin of wrestlers, in particular from the Achilles tendon of cattle.
  • the matrix used as a carrier layer is formed from lyophilized sparingly soluble collagen.
  • the collagen matrix comprises a lyophilized sparingly soluble collagen, in particular a lyophilized sparingly soluble collagen from tendons of horses, pigs or cattle.
  • lyophilized sparingly soluble collagen is understood to mean a sparingly soluble collagen which has been obtained by freeze-drying a homogeneous suspension of this sparingly soluble collagen in a solvent, preferably water.
  • Freeze-drying is also referred to as sublimation drying and stands for the drying of a deep-frozen material in a high vacuum by freezing the solvent, preferably in the form of water, which then evaporates in the frozen state.
  • This collagen matrix of lyophilized sparingly soluble collagen particularly preferably has a skin with pores on the surface. These pores lead to a Particularly good colonization of the carrier material with fibroblasts during the formation of the dermis equivalent, so that a disturbance during differentiation of the fibroblasts required to form the Dermisäquivalents is avoided.
  • the collagen matrix of the carrier layer contains a specific total amount of collagen.
  • Preferred embodiments of the present invention are therefore characterized in that the collagen matrix has a total amount of collagen, in particular lyophilized sparingly soluble collagen, of from 0.5 to 5.0% by weight, preferably from 0.8 to 3.5% by weight. , preferably from 0.8 to 2.5 wt .-%, in particular from 0.8 to 1, 2 wt .-%, based on the total weight of the collagen matrix contains.
  • Carrier layers which contain the aforementioned total amount of collagen, in particular lyophilized sparingly soluble collagen lead to an effective stabilization of the skin equivalent according to the invention and in this way allow a good handling of the equivalents.
  • the use of crosslinked collagen matrices has proven itself.
  • the avoidance of shrinkage ensures a high degree of standardizability, since the application of the test substances can always take place on an equally large surface. As a result, concentration fluctuations are avoided after application of the test substances.
  • the collagen matrix is a crosslinked collagen matrix.
  • the crosslinking of the collagen matrix can be carried out, for example, by means of chemical or physical methods.
  • chemical crosslinking in particular chemical crosslinking agents are used, the physical crosslinking can be done for example by means of UV irradiation or dehydrothermal crosslinking (also referred to as DHT).
  • the crosslinking of the collagen matrix is effected by means of chemical crosslinking agents. It is therefore preferred in the context of the present invention if the crosslinking of the collagen matrix by means of a chemical crosslinking agent from the group of glutaraldehyde, p-benzoquinone, dimehtyladipimidate, dimethylpimelinidate, dimethylsuberimidate, 1, 4-phenylenediisothiocyanate, polyoxyethylene bis (imidazolyl carbonyl), bis [polyoxyethylene bis (imidazolyl carbonyl)] and suberic acid bis (N-hydroxysuccinimide ester), 1-ethyl-3- (3-dimethyl aminopropyl) carbodiimide, enzymes and mixtures thereof, in particular glutaraldehyde.
  • a chemical crosslinking agent from the group of glutaraldehyde, p-benzoquinone, dimehtyladipimidate, dimethylpimelinidate, dimethylsuberimidate
  • the in vitro full-skin model according to the invention comprises a dermis equivalent.
  • This dermis equivalent is preferably formed from dermal fibroblasts.
  • Preferred embodiments of the present invention are therefore characterized in that the dermis equivalent is formed from primary fibroblasts, in particular human primary fibroblasts.
  • Primary fibroblasts are understood to be naturally occurring fibroblasts, in particular occurring in the human dermis, genetically modified fibroblasts, fibroblasts resulting from spontaneous mutation or their precursors.
  • Precultured human primary fibroblasts are particularly preferably used to form the dermis equivalent.
  • Precultivated human primary fibroblasts can be obtained by cultivating the human primary fibroblasts, wherein the cultivation preferably takes place in vitro with the proliferation of the cells.
  • a DMEM medium Dulbecco 's Modified Eagle Medium
  • the dermis equivalent contains a specific total number of primary fibroblasts. It is therefore preferred in the context of the present invention, when the dermis primary fibroblasts, in particular human primary fibroblasts in a total cell count of 5x10 5 to 6x10 6, preferably 6x10 5 to 5x10 6, more preferably 7x10 5 to 4x10 6, contains.
  • Dermis equivalents containing the aforementioned total cell numbers of primary fibroblasts have a differentiation that is very similar to the differentiation in the dermis of the human skin. This ensures that the in-vitro full-skin models according to the invention simulate the in-vivo situation as accurately as possible.
  • the in vitro full-skin model according to the invention contains at least one epidermis equivalent.
  • This epidermis equivalent is preferably formed from primary keratinocytes.
  • keratinocytes are understood as meaning cells of the epidermis which form a keratinized squamous epithelium, genetically modified keratinocytes, keratinocytes resulting from spontaneous mutation and their precursors. Since the formation of a well-differentiated epidermis equivalent with intact keratinization depends on the proportion of basal stem cells in the keratinocytes used to form the epidermis equivalent, it is preferred to use largely undifferentiated keratinocyte stem cells from untreated biopsy tissue.
  • the epidermis equivalent is formed from primary keratinocytes, in particular human primary keratinocytes.
  • the epidermis equivalent is preferably formed from precultured keratinocytes, which are obtained by culturing primary keratinocytes, preferably by increasing these cells.
  • precultured keratinocytes which are obtained by culturing primary keratinocytes, preferably by increasing these cells.
  • the epidermis equivalent has several mutually different cell layers.
  • the epidermis equivalent comprises a plurality of mutually different cell layers, in particular at least two differently differentiated cell layers and at least one keratinized cell layer.
  • the term "multiple cell layers” is understood to mean at least two mutually different cell layers, in particular 2 to 20 mutually different cell layers.
  • the presence of different cell layers in the epidermis can be determined by means of a light microscope 2 to 10 different cell layers, wherein at least one of these cell layers is selected from stratum corneum, stratum spinosum and stratum granulosum.
  • the epidermis equivalent contains a specific total cell number of primary keratinocytes. It is therefore preferred within the scope of the present invention for the epidermis equivalent to contain primary keratinocytes, in particular human primary keratinocytes, in a total cell number of 4x10 5 to 5 ⁇ 10 6 , preferably of 5.5 ⁇ 10 5 to 4 ⁇ 10 6 , in particular of 6.5 ⁇ 10 5 to 3, 5x10 6 , contains.
  • Epidermis equivalents containing the aforementioned total cell numbers of primary keratinocytes have a differentiation which is very similar to the differentiation in the epidermis of the human skin. This ensures that the in-vitro full-skin models according to the invention simulate the in-vivo situation as accurately as possible.
  • the full-skin model according to the invention comprises a basal membrane, which is located between the dermis and the epidermis equivalent.
  • the basal membrane of the full-skin model comprises certain proteins.
  • Preferred embodiments of the present invention are therefore characterized in that the basal membrane comprises proteins from the group of laminin, collagen type IV and their mixture. Basal membranes containing the previously mentioned proteins lead to a particularly effective connection between the dermis and the epidermis equivalent and in this way ensure good stability and handling of the full-skin models according to the invention.
  • the full-skin models according to the invention preferably contain 1 to 100 three-dimensional sweat gland equivalents.
  • the incorporation of the three-dimensional sweat gland equivalents allows an improved representation of the in vivo situation in human skin compared to full-skin models containing no sweat glands or sweat gland equivalents.
  • the incorporation of an equal number of discrete sweat gland equivalents also allows the production of standardizable full-skin models compared to the use of punch biopsies with a varying number of sweat glands in each of these biopsies.
  • the dermis equivalent b) and / or the epidermis equivalent c) comprises 2 to 100 three-dimensional sweat gland equivalents, preferably 5 to 100 three-dimensional sweat gland equivalents, preferably 20 to 100 three-dimensional sweat gland equivalents, in particular 50 to 100 three-dimensional sweat gland equivalents.
  • the discrete three-dimensional sweat gland equivalents contained in the full-skin model according to the invention can be prepared for example by means of the hanging drop method from primary sweat gland cells using only in vitro methods.
  • sweat glands from skin biopsies in particular native eccrine and / or apocrine sweat glands from skin biopsies, are first isolated.
  • the isolation can be carried out by enzymatic digestion of the human skin using a mixture of 2 to 3 mg / ml collagenase II and 0.1 to 0.2 mg / ml thermolysin for 3 to 6 hours at 35 to 40 ° C, especially at 37 ° C, done.
  • primary sweat gland cells can be obtained.
  • the three-dimensional sweat gland equivalents are preferably contained in the dermis equivalent.
  • the incorporation of these sweat gland equivalents into the dermis equivalent allows a better readjustment of the in vivo situation. It is therefore advantageous according to the invention if the dermis equivalent b) comprises the three-dimensional sweat gland equivalents.
  • the dermis equivalent b) comprises the three-dimensional sweat gland equivalents.
  • Three-dimensional sweat gland equivalents which have specific diameters, are preferably contained in the full-thickness skin model according to the invention. It is therefore advantageous according to the invention if the three-dimensional sweat gland equivalents each have a Diameter of 100 to 4,000 ⁇ , preferably from 100 to 3,000 ⁇ , in particular from 200 to 2,500 ⁇ have.
  • the indication of the diameter here refers to the diameter of a single three-dimensional sweat gland equivalent.
  • the diameter of the spherical sweat gland equivalents according to the invention can be carried out for example by means of microscopic measurement using the software "CellSens".
  • the sweat gland equivalents contained in the full-skin model according to the invention are free of matrix compounds and / or carriers.
  • matrix compounds are meant compounds which are capable of forming spatial networks. However, this does not include the substances produced and excreted by the cells themselves, which are capable of forming spatial networks.
  • carriers within the meaning of the present invention are understood as meaning self-supporting substances which can serve as a support or framework for the sweat gland cells.
  • the three-dimensional sweat gland equivalents are each free of matrix compounds and / or carriers, in particular free of matrix compounds and carriers.
  • the term "free of” is understood in accordance with the invention to mean that the three-dimensional sweat gland equivalents contain less than 1% by weight, based on the total weight of the three-dimensional sweat gland equivalent, of matrix compounds and / or carriers It is therefore advantageous for the purposes of the present invention the three-dimensional sweat gland equivalent 0 to 1 wt .-%, preferably 0 to 0.5 wt .-%, preferably 0 to 0.2 wt .-%, in particular 0 wt .-%, based on the total weight of the three-dimensional sweat gland equivalent, of matrix compounds and carriers.
  • the three-dimensional sweat gland equivalents are free of certain matrix compounds and carriers. It is therefore preferred if the three-dimensional sweat gland equivalent contains no matrix compounds and / or carriers which are selected from the group of collagens, in particular collagen type I and / or type III and / or type IV, scleroproteins, gelatins, chitosans, glucosamines, glucosaminoglucans (GAG), heparin sulfate proteoglucans, sulfated glycated proteins, growth factors, cross-linked polysaccharides, cross-linked polypeptides and mixtures thereof.
  • collagens in particular collagen type I and / or type III and / or type IV, scleroproteins, gelatins, chitosans, glucosamines, glucosaminoglucans (GAG), heparin sulfate proteoglucans, sulfated glycated proteins, growth factors, cross-linked polysacc
  • the three-dimensional sweat gland equivalents contained in the skin equivalent according to the invention are equivalents of the eccrine and / or apocrine human sweat gland.
  • Preferred embodiments of the present invention are therefore characterized in that the three-dimensional sweat gland equivalents are each three-dimensional sweat gland equivalents of the eccrine and / or apocrine human sweat gland.
  • Full-skin models containing such sweat gland equivalents are particularly suitable for the identification of antiperspirant active ingredients for in vivo use in humans.
  • At least one carrier layer comprising at least one collagen matrix, the collagen matrix containing a total amount of lyophilized sparingly soluble collagen of from 0.8 to 1.2% by weight based on the total weight of the collagen matrix, and wherein the collagen matrix is a crosslinked collagen matrix .
  • the dermis equivalent b) and / or the epidermis equivalent c) comprises 1 to 100 three-dimensional sweat gland equivalent (s), the three-dimensional sweat gland equivalents each comprising 500 to 500,000 sweat gland cells and having a diameter of 100 to 6,000 ⁇ each.
  • a particularly preferred embodiment of this subject of the invention is thus an in vitro full-skin model comprising
  • At least one carrier layer comprising at least one collagen matrix, the collagen matrix containing a total amount of lyophilized sparingly soluble collagen of from 0.8 to 1.2% by weight based on the total weight of the collagen matrix, and wherein the collagen matrix is a crosslinked collagen matrix .
  • the dermis equivalent b) and / or the epidermis equivalent c) comprises 1 to 100 three-dimensional sweat gland equivalent (s), the three-dimensional sweat gland equivalents each comprising 500 to 500,000 sweat gland cells and having a diameter of 100 to 6,000 ⁇ each.
  • a particularly preferred embodiment of this subject of the invention is an in vitro full-skin model comprising a) at least one carrier layer comprising at least one collagen matrix, the collagen matrix containing a total amount of lyophilized sparingly soluble collagen of from 0.8 to 1.2% by weight based on the total weight of the collagen matrix, and wherein the collagen matrix is a crosslinked collagen matrix .
  • the epidermis equivalent is formed from human primary keratinocytes and wherein the epidermis equivalent comprises at least two differently differentiated cell layers and at least one keratinized cell layer
  • the dermis equivalent b) and / or the epidermis equivalent c) comprises 1 to 100 three-dimensional sweat gland equivalent (s), the three-dimensional sweat gland equivalents each comprising 500 to 500,000 sweat gland cells and having a diameter of 100 to 6,000 ⁇ each.
  • a particularly preferred embodiment of this subject of the invention is an in vitro full-skin model comprising
  • At least one carrier layer comprising at least one collagen matrix, the collagen matrix containing a total amount of lyophilized sparingly soluble collagen of from 0.8 to 1.2% by weight based on the total weight of the collagen matrix, and wherein the collagen matrix is a crosslinked collagen matrix .
  • the epidermis equivalent is formed from human primary keratinocytes and wherein the epidermis equivalent comprises at least two differently differentiated cell layers and at least one keratinized cell layer
  • this basement membrane being between the dermis equivalent and the epidermis equivalent and the basal membrane comprising proteins from the group of laminin, collagen type IV and their mixture,
  • the dermis equivalent b) and / or the epidermis equivalent c) comprises 1 to 100 three-dimensional sweat gland equivalent (s), the three-dimensional sweat gland equivalents each comprising 500 to 500,000 sweat gland cells and having a diameter of 100 to 6,000 ⁇ each.
  • a particularly preferred embodiment of this subject of the invention is an in vitro skin model comprising a) at least one carrier layer comprising at least one collagen matrix, the collagen matrix containing a total amount of lyophilized sparingly soluble collagen of from 0.8 to 1.2% by weight based on the total weight of the collagen matrix, and wherein the collagen matrix is a crosslinked collagen matrix .
  • the epidermis equivalent is formed from human primary keratinocytes and wherein the epidermis equivalent comprises at least two differently differentiated cell layers and at least one keratinized cell layer
  • this basement membrane being between the dermis equivalent and the epidermis equivalent and the basal membrane comprising proteins from the group of laminin, collagen type IV and their mixture,
  • the dermis equivalent b) comprises 50 to 100 three-dimensional sweat gland equivalents, the three-dimensional sweat gland equivalents each comprising 500 to 500,000 sweat gland cells having a diameter of 200 to 2,500 ⁇ each and wherein the three-dimensional sweat gland equivalents are each three-dimensional sweat gland equivalents of the eccrine and / or apocrine human sweat gland.
  • the in vitro full-skin models according to the invention have a higher standardizability and availability than the punch biopsies currently used and have a greater proximity to the in-vivo situation than full-skin models which contain no sweat glands and / or sweat gland equivalents. Furthermore, these skin equivalents represent a cost-effective alternative to in vivo studies in humans, since these skin models, the antiperspirant effect of test substances can be examined, for example by comparing gene expression or protein expression in a stimulation with acetylcholine (ACh) in the presence and absence a specific test substance.
  • the full-skin models of the invention simulate human skin in vivo both in their structure and in their histological composition, so that the information obtained with these models is readily transferable to humans and can also be compared with data for compounds already tested in vivo.
  • a second aspect of the present invention is a method for producing an in vitro full-skin model, the method comprising the following steps in the order given:
  • step b) preparation of the carrier layer by lyophilization of the collagen suspension provided in step a), c) production of the dermis equivalent by application of primary fibroblasts, in particular human primary fibroblasts, to the carrier layer produced in step b) and cultivation of these fibroblasts over a period of 7 to 28 days,
  • step d) application of primary keratinocytes, in particular human primary keratinocytes, to the dermis equivalent produced in step c) and cultivation of these keratinocytes over a period of 1 to 10 days,
  • step d) cultivation of the model obtained according to step d) at the air-medium boundary over a period of 7 to 42 days,
  • the term "suspension of sparingly soluble collagen” is understood as meaning a homogeneous mixture of solid collagen in a solvent, preferably water.
  • a solvent preferably water.
  • devices known to the person skilled in the art such as static mixers or Ultra Turrex mixers, can be used.
  • the term “culturing” means maintaining, preferably in vitro, the vital functions of cells, in particular of fibroblasts and keratinocytes, in a suitable environment, for example by adding and removing metabolic factors and products, in particular also by increasing the number of cells.
  • the suspension provided in process step a) contains a specific total amount of collagen.
  • Preferred embodiments of the method according to the invention are therefore characterized in that the suspension of sparingly soluble collagen in step a) a total amount of collagen from 0.2 to 4.0 wt .-%, preferably from 0.3 to 3.0 parts by weight. %, preferably from 0.4 to 2.0 wt .-%, in particular from 0.5 to 1, 5 wt .-%, based on the total weight of the suspension contains.
  • the use of suspensions with the above-mentioned total amounts of collagen leads to the formation of particularly stable carrier layers, so that a high stability and good handling of the full-skin models resulting from the process according to the invention is ensured.
  • the suspension of sparingly soluble collagen in step a) has a pH of from pH 0.1 to pH 6.9, preferably from pH 2.0 to pH 5, 0, preferably from pH 3.0 to pH 4.5, in particular from pH 3.5 to pH 4.0.
  • the use of suspensions with the abovementioned pH values leads to the formation of particularly stable carrier layers.
  • the collagen suspension is preferably added to containers whose dimensions correspond to those of the desired carrier layer.
  • Suitable containers are, for example, wells of microtiter plates.
  • suitable agents are, for example, gelatin, polylysine, fibrin or fibrinogen / thrombin or fibronectin.
  • the inner vessel walls are first wetted with the abovementioned agents and then dried, so that a layer of the agents adheres to the inner vessel surface. Subsequently, the collagen suspension is filled.
  • the lyophilization carried out in process step b) preferably takes place in these vessels. Furthermore, it is preferred according to the invention if the lyophilization takes place at a certain cooling rate. It is therefore preferred in the context of the present invention if the lyophilization of the collagen suspension in step b) at a cooling rate of 5 ° C to 40 ° C per hour, preferably from 10 ° C to 30 ° C per hour, preferably from 18 ° C. to 23 ° C per hour, in particular from 20 ° C to 22 ° C per hour, takes place.
  • the slow cooling rate results in the formation of a pellicle on the surface of the lyophilized carrier layer, the pellicle having pores on the surface.
  • a crosslinked collagen matrix is to be used as the carrier, the crosslinking of this matrix takes place after the lyophilization described in process step c).
  • the crosslinking agents described above in connection with the first subject of the invention are suitable for this purpose.
  • Glutaraldehyde is particularly preferably used for crosslinking. Due to the cross-linking, a shrinkage process of the full-skin models during production is avoided, so that a high reproducibility can be ensured due to the uniform size and nature.
  • fibroblasts of a suitable passage are pre-cultured in a cell culture flask and detached from the soil by trypsinization immediately prior to their use.
  • a nutrient medium for culturing the fibroblasts for example DMEM, which contains 10 wt .-% FCS can be used. It has been found to be advantageous according to the invention when certain total concentrations of fibroblasts are used to produce the dermis equivalent.
  • Preferred embodiments of the present invention are therefore characterized in that for the preparation of the dermis equivalent in step c) primary fibroblasts, in particular human primary fibroblasts, in a total concentration of 2x10 5 to 2x10 6 cells per ml of medium, preferably from 3x10 5 to 1x10 6 cells per ml of medium, preferably from 4x10 5 to 7 ⁇ 10 5 cells per ml of medium, in particular from 4.5 ⁇ 10 5 to 5, 5 ⁇ 10 5 cells per ml of medium.
  • the medium used is preferably a DMEM medium which contains 10% by weight of FCS.
  • human fibronection and / or laminin may additionally be added to the culture medium.
  • Fibronection is a structural or adhesion protein produced in fibroblasts, whose function in vivo consists in the binding of other macromolecules, for example collagen, and in the attachment of cells to neighboring cells.
  • Laminin is a protein of the basement membrane to which cells can adhere.
  • the addition of fibronection and / or laminin to the culture medium enhances the binding of the fibroblasts to the support layer as well as to one another.
  • the use of the aforementioned total concentrations ensures sufficient colonization of the carrier layer with the fibroblasts.
  • the cell count of the primary fibroblasts can be determined using a classical counting chamber and trypan blue.
  • the suspension of cultured primary fibroblasts is added undiluted with a trypan blue solution and determines the number of cells in the corresponding corner squares. From these values, the arithmetic mean is formed. Taking into account the volume of the counting chamber, the dilution factor and the chamber factor, the cell count per ml or ⁇ is determined from this mean value.
  • the cell number used in method step c) here refers to the number of living cells (live number).
  • the cultivation of the fibroblasts applied to the carrier layer in process step c) is preferably carried out in a submerged culture. This is understood to mean the cultivation of nutrient-solution-covered fibroblasts. By altering the cultivation conditions or by adding chemical substances, such as ceramides and vitamin C, to the medium, a barrier function can additionally be produced. It is preferred according to the invention if the cultivation of the primary fibroblasts, in particular the human primary fibroblasts, in step c) over a period of 8 to 25 days, preferably from 10 to 22 days, preferably from 1 1 to 20 days, in particular from 12 to 18 days, at a temperature of 30 to 40 ° C.
  • Keratinocytes used for the production of the epidermis equivalent is carried out by the methods known to the person skilled in the art. Keratinocytes of the first and / or second passage in a cell culture flask are preferably detached from the bottom by trypsinization immediately prior to their use.
  • a nutrient medium for cultivating the fibroblasts for example, a mixture of DMEM and Ham 's F12, which contains 1 to 30 wt .-% FCS and other serum products and additives can be used.
  • method step d) the application of keratinocytes to the dermis equivalent produced in method step c) takes place.
  • the use of the aforementioned total concentrations ensures sufficient colonization of the dermis equivalent with the keratinocytes.
  • the cell number can be determined as previously described in connection with the cell count of the fibroblasts.
  • the keratinocytes applied in method step d) are preferably cultured for a certain time in a submersed culture. It is therefore preferred in the context of the present invention if the cultivation of the primary keratinocytes, in particular of the human primary keratinocytes, in step d) in a submersed culture over a period of 1 to 8 days, preferably 1 to 6 days, preferably 1 to 4 days, in particular from 1 to 2 days, at a temperature of 30 to 40 ° C.
  • the keratinocytes are preferably seeded onto the carrier layer in a cell culture medium, particularly preferably DMEM / F12 medium, which contains about 1 to 30% by weight, based on the total weight of the medium, fetal calf serum (FCS), NCS, defined serum or Contains serum replacement products and various additives that promote the proliferation and differentiation of the cells.
  • a cell culture medium particularly preferably DMEM / F12 medium, which contains about 1 to 30% by weight, based on the total weight of the medium, fetal calf serum (FCS), NCS, defined serum or Contains serum replacement products and various additives that promote the proliferation and differentiation of the cells.
  • FCS fetal calf serum
  • NCS defined serum
  • the carrier layer with DMEM medium containing in particular EGF from the mouse or comparable preparations from other animals epidermal growth factor (hEGF) (eg in a concentration of 0.2 igl ⁇
  • Complete differentiation of the keratinocyte layers is achieved by culturing the keratinocytes at the medium-air interface. This type of cultivation is also referred to as an airlift culture, using DMEM without hEGF and BPE (bovine pituitary extract) as the culture medium.
  • an “airlift culture” is understood as meaning a culture in which the level of the nutrient medium level is precisely matched to the level of the dermis equivalent, while the cell layers formed by the keratinocytes lie above the nutrient medium level and are not covered by the nutrient medium, ie the cultivation takes place
  • the models are lifted from the wells of the microtiter plate and placed on filter papers which rest on metal spacers in Petri dishes, the medium is filled so high in the Petri dishes that it does not completely cover the filter paper, but that a liquid collar is present around the base of the skin models (also referred to as air-liquid interface)
  • a typical skin epidermis equivalent is formed s method is therefore characterized characterized in that the cultivation of the model obtained according to step d) at the air-medium limit over a period of 8 to 35 days, preferably from 9 to 30 days, preferably from 10 to 20 days, in particular from 10 to 13 days, at a temperature of 30 to 40 ° C.
  • the three-dimensional sweat gland equivalents are introduced in the process according to the invention in process step c) and / or d).
  • the incorporation of the sweat gland equivalents in process step c) during the production of the dermis equivalent is preferred according to the invention, since this allows the in vivo situation to be adjusted very well. It is therefore preferred according to the invention, if the introduction of the three-dimensional sweat gland equivalents in step c), wherein the primary fibroblasts, in particular the human primary fibroblasts, mixed with the three-dimensional sweat gland equivalents and then applied to the carrier layer produced in step b).
  • step d) it can also be provided according to the invention to introduce the three-dimensional sweat gland equivalents during step d) of the method according to the invention. It is therefore also preferred according to the invention if the introduction of the three-dimensional sweat gland equivalents takes place in step d), wherein the three-dimensional sweat gland equivalents are seeded 1 to 3 hours before application of the primary keratinocytes, in particular human primary keratinocytes.
  • the term "sewn" here is to be understood as meaning the application of the three-dimensional sweat gland equivalents to the surface of the dermis equivalent.
  • the introduction of 1 to 100 discrete sweat gland equivalents, each having a cell number of 500 to 500,000 cells and a diameter of 100 to 6,000 ⁇ m, is carried out in the aforementioned method steps c) and / or d).
  • a larger number of sweat gland equivalents with a small diameter is introduced into the full-skin models. It is therefore within the scope of the present invention preferred if in step c) and / or in step d), in particular in step c), 50 to 100 three-dimensional sweat gland equivalents with a cell count of 500 to 500,000 sweat gland cells and a diameter of 500 to 2,500 ⁇ be introduced.
  • a particularly preferred embodiment of this subject matter is accordingly a process for the production of an in vitro full-skin model, the process comprising the following steps in the order given: a) providing a suspension of sparingly soluble collagen, wherein the suspension of poorly soluble collagen in step a) a total amount of collagen from 0.2 to 4.0 wt .-%, preferably from 0.3 to 3.0 parts by weight.
  • % preferably from 0.4 to 2.0 wt .-%, in particular from 0.5 to 1, 5 wt .-%, based on the total weight of the suspension, and wherein the suspension of sparingly soluble collagen in step a) a pH of from pH 0.1 to pH 6.9, preferably from pH 2.0 to pH 5.0, preferably from pH 3.0 to pH 4.5, especially from pH 3.5 to pH 4.0 , having,
  • step c) production of the dermis equivalent by application of primary fibroblasts, in particular human primary fibroblasts, to the carrier layer produced in step b) and cultivation of these fibroblasts over a period of 7 to 28 days,
  • step d) application of primary keratinocytes, in particular human primary keratinocytes, to the dermis equivalent produced in step c) and cultivation of these keratinocytes over a period of 1 to 10 days,
  • step d) cultivation of the model obtained according to step d) at the air-medium boundary over a period of 7 to 42 days,
  • a further particularly preferred embodiment of this subject matter of the invention is accordingly a process for the preparation of an in vitro full-skin model, the process comprising the following steps in the order given:
  • step b) preparation of the carrier layer by lyophilization of the collagen suspension provided in step a), wherein the lyophilization of the collagen suspension in step b) at a cooling rate of 5 ° C to 40 ° C per hour, preferably from 10 ° C to 30 ° C per hour, preferably from 18 ° C. to 23 ° C. per hour, in particular from 20 ° C. to 22 ° C.
  • step c) Production of the dermis equivalent by application of primary fibroblasts, in particular human primary fibroblasts, to the carrier layer produced in step b) and culturing these fibroblasts over a period of 7 to 28 days, d) application of primary keratinocytes, in particular human primary keratinocytes, to the dermis equivalent produced in step c) and cultivation of these keratinocytes over a period of 1 to 10 days,
  • step d) cultivation of the model obtained according to step d) at the air-medium boundary over a period of 7 to 42 days,
  • a further particularly preferred embodiment of this subject matter of the invention is accordingly a process for the preparation of an in vitro full-skin model, the process comprising the following steps in the order given:
  • step b) preparation of the carrier layer by lyophilization of the collagen suspension provided in step a), wherein the lyophilization of the collagen suspension in step b) at a cooling rate of 5 ° C to 40 ° C per hour, preferably from 10 ° C to 30 ° C per hour, preferably from 18 ° C. to 23 ° C. per hour, in particular from 20 ° C. to 22 ° C.
  • step c) Production of the dermis equivalent by application of primary fibroblasts, in particular human primary fibroblasts, to the carrier layer produced in step b) and cultivating these fibroblasts over a period of 7 to 28 days, wherein for the production of the dermis equivalent in step c) primary fibroblasts, in particular human primary fibroblasts, in a total concentration of 2x10 5 to 2x10 6 cells per ml of medium, preferably from 3x10 5 to 1 x10 6 cells per ml of medium, preferably from 4x10 5 to 7 ⁇ 10 5 cells per ml of medium, in particular from 4.5 ⁇ 10 5 to 5.5 ⁇ 10 5 cells per ml of medium, be used,
  • step d) application of primary keratinocytes, in particular human primary keratinocytes, to the dermis equivalent produced in step c) and cultivation of these keratinocytes over a period of 1 to 10 days,
  • step d) cultivation of the model obtained according to step d) at the air-medium boundary over a period of 7 to 42 days,
  • a further particularly preferred embodiment of this subject matter is accordingly a process for the production of an in vitro full-skin model, the process comprising the following steps in the order given:
  • step b) preparation of the carrier layer by lyophilization of the collagen suspension provided in step a), wherein the lyophilization of the collagen suspension in step b) at a cooling rate of 5 ° C to 40 ° C per hour, preferably from 10 ° C to 30 ° C per hour, preferably from 18 ° C. to 23 ° C. per hour, in particular from 20 ° C. to 22 ° C.
  • step c) Production of the dermis equivalent by application of primary fibroblasts, in particular human primary fibroblasts, to the carrier layer produced in step b) and cultivating these fibroblasts over a period of 7 to 28 days, wherein for the production of the dermis equivalent in step c) primary fibroblasts, in particular human primary fibroblasts, in a total concentration of 2x10 5 to 2x10 6 cells per ml of medium, preferably from 3x10 5 to 1 x10 6 cells per ml of medium, preferably from 4x10 5 to 7 ⁇ 10 5 cells per ml of medium, in particular from 4.5 ⁇ 10 5 to 5.5 ⁇ 10 5 cells per ml of medium, be used,
  • step d) application of primary keratinocytes, in particular human primary keratinocytes, to the dermis equivalent produced in step c) and cultivation of these keratinocytes over a period of 1 to 10 days, wherein in step d) primary keratinocytes, in particular human primary keratinocytes, in a total concentration of 1, 5 ⁇ 10 5 to 1 ⁇ 10 6 cells per ml of medium, preferably from 2.5 ⁇ 10 5 to 9 ⁇ 10 5 cells per ml of medium, preferably from 3.5 ⁇ 10 5 to 7 ⁇ 10 5 cells per ml of medium, in particular from 4x10 5 to 5 ⁇ 10 5 cells per ml Medium, to be used
  • step d) cultivation of the model obtained according to step d) at the air-medium boundary over a period of 7 to 42 days,
  • a further particularly preferred embodiment of this subject matter is accordingly a method for producing an in-vitro full-skin model, the method comprising the following steps in the order given:
  • the suspension of sparingly soluble collagen in step a) comprises a total amount of collagen from 0.2 to 4.0% by weight, preferably from 0.3 to 3.0 wt .-%, preferably from 0.4 to 2.0 wt .-%, in particular from 0.5 to 1, 5 wt .-%, based on the total weight of the suspension contains and wherein the sparingly soluble collagen suspension in step a) has a pH of from pH 0.1 to pH 6.9, preferably from pH 2.0 to pH 5.0, preferably from pH 3.0 to pH 4.5 , in particular from pH 3.5 to pH 4.0,
  • step b) preparation of the carrier layer by lyophilization of the collagen suspension provided in step a), wherein the lyophilization of the collagen suspension in step b) at a cooling rate of 5 ° C to 40 ° C per hour, preferably from 10 ° C to 30 ° C per hour, preferably from 18 ° C. to 23 ° C. per hour, in particular from 20 ° C. to 22 ° C.
  • step c) Production of the dermis equivalent by application of primary fibroblasts, in particular human primary fibroblasts, to the carrier layer produced in step b) and cultivating these fibroblasts over a period of 7 to 28 days, wherein for the production of the dermis equivalent in step c) primary fibroblasts, in particular human primary fibroblasts, in a total concentration of 2x10 5 to 2x10 6 cells per ml of medium, preferably from 3x10 5 to 1 x10 6 cells per ml of medium, preferably from 4x10 5 to 7x10 5 cells per ml of medium, in particular from 4.5x10 5 to 5,5x10 5 cells per ml of medium, be used,
  • step d) application of primary keratinocytes, in particular human primary keratinocytes, to the dermis equivalent produced in step c) and cultivation of these keratinocytes over a period of 1 to 10 days, wherein in step d) primary keratinocytes, in particular human primary keratinocytes, in a total concentration of 1, 5 ⁇ 10 5 to 1 ⁇ 10 6 cells per ml of medium, preferably from 2.5 ⁇ 10 5 to 9 ⁇ 10 5 cells per ml of medium, preferably from 3.5 ⁇ 10 5 to 7 ⁇ 10 5 cells per ml of medium, in particular from 4x10 5 to 5 ⁇ 10 5 cells per ml Medium, to be used
  • step d) cultivation of the model obtained according to step d) at the air-medium boundary over a period of 7 to 42 days,
  • step c wherein the introduction of 50 to 100 three-dimensional sweat gland equivalents with a cell count of 500 to 500,000 sweat gland cells and a diameter of 100 to 6,000 ⁇ by addition of these equivalents in step c).
  • Three-dimensional sweat gland equivalents of the eccrine and / or apocrine human sweat gland are preferably introduced into the previously described methods for producing in-vitro full-thickness skin models.
  • the processes according to the invention have the advantage that no use of in-vivo methods is required for the preparation of in-vitro full-skin models. Consequently, these models can also be used to test substances intended for cosmetic use. Furthermore, the method according to the invention allows a cost-effective production of standardized models which can be used in screening methods with high throughput rates. In addition, this Herste II method results in full-skin models, which have differentiated different cell layers and sweat gland specific markers Thus, a good transferability of the in vitro data generated with these models to the in vivo situation is made possible.
  • a third aspect of the present invention is the use of the in vitro full-skin model according to the invention in cosmetics and personal care, in particular for testing, identifying and testing cosmetic active ingredients, preferably with regard to their effectiveness in inhibiting and / or reducing body sweat and / or or body odor.
  • this full-skin model can be used as an in vitro model for determining the influence of test substances on the sweat glands in the field of cosmetics and in particular personal care. Due to the high standardizability and the good simulation of the in-vivo situation, the full-skin models of the invention can be used in particular for the discovery of new antiperspirant active ingredients.
  • the full-skin model according to the invention is particularly suitable for product testing, for example with regard to the efficacy, unwanted side effects, for example irritation, toxicity and inflammatory effects, allergenic effects or tolerability of substances.
  • the full-skin model according to the invention can also be used for studies on the absorption, transport and / or penetration of substances and for wound healing.
  • the effects on the skin of external environmental influences such as radiation, heat, radioactivity, electric fields or the like can be examined.
  • the effect of such influences can be determined, for example, by evaluating the gene expression, the metabolism, the proliferation, the differentiation and the reorganization of the cells. It can also be provided to colonize the in-vitro full-skin model according to the invention with microorganisms, in particular pathogenic microorganisms, such as fungi, bacteria and viruses, in order to study infection processes and their healing.
  • Another object of the present invention is the use of an in vitro full-skin model according to the invention in preferably automated screening methods, in particular for testing, identification and testing of cosmetic active ingredients, preferably with respect to their effectiveness in inhibiting and / or reducing body sweat and / or body odor.
  • Another object of the present invention is the use of an in vitro full-skin model according to the invention for the in-vitro evaluation of the influence of cosmetic agents on the inhibition and / or reduction of sweat secretion and / or body odor.
  • a further subject of the present invention is a system, in particular test system, comprising an in vitro full-skin model according to the invention.
  • the present invention is characterized in particular by the following points:
  • At least one carrier layer comprising at least one collagen matrix
  • the dermis equivalent b) and / or the epidermis equivalent c) comprises from 1 to 100 three-dimensional sweat gland equivalent (s), the three-dimensional sweat gland equivalent (s)
  • Sweat gland equivalents each comprise 500 to 500,000 sweat gland cells and have a diameter of 100 to 6,000 ⁇ .
  • the collagen matrix comprises a sparingly soluble collagen, in particular a poorly soluble collagen from tendons of horses, pigs or cattle.
  • the collagen matrix comprises a lyophilized sparingly soluble collagen, in particular a lyophilized sparingly soluble collagen from tendons of horses, pigs or cattle.
  • in vitro full skin model characterized in that the collagen matrix, a total amount of collagen, in particular lyophilized poorly soluble collagen, from 0.5 to 5.0 wt .-%, preferably from 0.8 to 3, 5 wt .-%, preferably from 0.8 to 2.5 wt .-%, in particular from 0.8 to 1, 2 wt .-%, based on the total weight of the collagen matrix contains.
  • the collagen matrix is a crosslinked collagen matrix.
  • the dermis equivalent is formed from primary fibroblasts, in particular human primary fibroblasts.
  • the dermis equivalent primary fibroblasts in particular human primary fibroblasts, in a total cell number of 5x10 5 to 6x10 6 , preferably from 6x10 5 to 5x10 6 , in particular from 7x10 5 to 4x10 6 , contains.
  • the epidermis equivalent is formed from primary keratinocytes, in particular human primary keratinocytes.
  • the epidermis equivalent comprises a plurality of mutually different cell layers, in particular at least two differently differentiated cell layers and at least one keratinized cell layer.
  • the epidermis equivalent primary keratinocytes in particular human primary keratinocytes, in a total cell count of 4x10 5 to 5x10 6, preferably from 5,5x10 5 to 4x10 6, more preferably from 6,5x10 5 to 3.5x10 6 , contains.
  • the basal membrane comprises proteins from the group of laminin, collagen type IV and their mixture.
  • In-vitro full-skin model according to one of the preceding points characterized in that the dermis equivalent b) comprises the three-dimensional sweat gland equivalents.
  • In-vitro full skin model according to one of the preceding points characterized in that the three-dimensional sweat gland equivalents each have a diameter of 100 to 4000 ⁇ , preferably from 100 to 3,000 ⁇ , in particular from 200 to 2,500 ⁇ have.
  • In-vitro full skin model according to one of the preceding points, characterized in that the three-dimensional sweat gland equivalents are each free of matrix compounds and / or carriers, in particular free of matrix compounds and carriers.
  • the matrix compounds and / or carriers are selected from the group of collagens, in particular collagen type I and / or type III and / or type IV, scleroproteins, gelatins, chitosans, glucosamines, glucosaminoglucans (GAG), heparin sulfate proteoglucans, sulfated glycated proteins, growth factors, cross-linked polysaccharides, cross-linked polypeptides and mixtures thereof.
  • In-vitro full-skin model according to one of the preceding points, characterized in that the three-dimensional sweat gland equivalents are each three-dimensional sweat gland equivalents of the eccrine and / or apocrine human sweat gland.
  • a method of making an in vitro full-skin model comprising the following steps in the order given:
  • step c) production of the dermis equivalent by application of primary fibroblasts, in particular human primary fibroblasts, to the carrier layer produced in step b) and cultivation of these fibroblasts over a period of 7 to 28 days; d) application of primary keratinocytes, in particular human primary keratinocytes the dermis equivalent produced in step c) and cultivation of these keratinocytes over a period of 1 to 10 days,
  • step d) cultivation of the model obtained according to step d) at the air-medium boundary over a period of 7 to 42 days,
  • the suspension of sparingly soluble collagen in step a) contains a total amount of collagen of from 0.2 to 4.0% by weight, preferably from 0.3 to 3.0% by weight, preferably from 0.4 to 2.0 wt .-%, in particular from 0.5 to 1, 5 wt .-%, based on the total weight of the suspension contains.
  • Step c) primary fibroblasts, in particular human primary fibroblasts, in a total concentration of 2x10 5 to 2x10 6 cells per ml of medium, preferably from 3x10 5 to 1x10 6 Cells per ml of medium, preferably from 4x10 5 to 7x10 5 cells per ml of medium, in particular from 4.5 ⁇ 10 5 to 5, 5 ⁇ 10 5 cells per ml of medium.
  • primary fibroblasts in particular human primary fibroblasts, in a total concentration of 2x10 5 to 2x10 6 cells per ml of medium, preferably from 3x10 5 to 1x10 6 Cells per ml of medium, preferably from 4x10 5 to 7x10 5 cells per ml of medium, in particular from 4.5 ⁇ 10 5 to 5, 5 ⁇ 10 5 cells per ml of medium.
  • a method according to any one of items 19 to 24, characterized in that primary in step d), keratinocytes, in particular human primary keratinocytes, in a total concentration of 1, 5x10 5 to 1x10 6 cells per ml of medium, preferably from 2.5x10 5 to 9x10 5 cells per ml of medium, preferably from 3.5x10 5 to 7x10 5 cells per ml of medium, in particular from 4x10 5 to 5x10 5 cells per ml to medium used.
  • Method characterized in that the cultivation of the primary keratinocytes, in particular of the human primary keratinocytes, in step d) in a submersed culture over a period of 1 to 8 days, preferably 1 to 6 days, preferably from 1 to 4 days, in particular from 1 to 2 days, at a temperature of 30 to 40 ° C.
  • Step d Method according to one of the items 19 to 27, characterized in that the introduction of the three-dimensional sweat gland equivalents takes place in step d), wherein the three-dimensional sweat gland equivalents are seeded 1 to 3 hours before application of the primary keratinocytes, in particular human primary keratinocytes.
  • System in particular test system, comprising an in-vitro full-skin model according to one of items 1 to 18.
  • the native sweat glands were obtained from human tissue samples, called biopsies, obtained from plastic surgery of patients who have consented to the use of the material for research purposes.
  • the tissue used was removed for upper arm tightening and facial tautening. From this, the eccrine and apocrine sweat glands were isolated from the underarm area.
  • the respective biopsy was cut into small pieces and then cut into pieces of a maximum of about 1 cm x 1 cm.
  • the skin was digested with a mixture of equal parts Collagenase II (5 mg / ml) and Thermolysin (0.25 mg / ml) at 37 ° C in the incubator for about 3.5 to 5 hours until the connective tissue almost was completely digested.
  • This mixture was then centrifuged at 1200 rpm for 5 minutes and the supernatant discarded to remove the enzyme solution and the excess fat.
  • the resulting pellet was taken up in DMEM solution and the solution transferred to a Petri dish. Using a microcapillary intact sweat glands were isolated under a binocular and transferred into fresh DMEM medium.
  • the sweat glands isolated in step 1.1 were placed in collagen-I coated culture bottles and then added to 25 ml of nutrient medium. After cultivation for 7 to 21 days in the Incubator at 37 ° C and 5% CO2, the growing sweat gland cells were peeled off and re-cultivated on Collagen-I coated culture bottles to confluency (monolayer culture of primary sweat gland cells).
  • composition of the nutrient medium used is as follows:
  • the SD sweat gland equivalents are harvested by adding 50 to 200 [iL of nutrient medium and transferred to a "GravityTRAP®” plate (Insphero AG, Switzerland) before harvesting the "GravityTRAP®” plate moistened with 60 to 100 [iL of keratinocyte medium using a multichannel pipette to minimize the formation of air bubbles and avoid the loss of three-dimensional sweat gland equivalents. After harvesting, plate is centrifuged for 1 to 5 minutes at 100 to 300 xg to remove air bubbles.
  • the preparation of the carrier layer which comprises a crosslinked collagen matrix, is carried out analogously to the method described in Examples 1 and 2 on pages 22 and 23 of the published patent application WO 2006/019147 A1.
  • 1 ml of a mixture of precultured fibroblasts (6 ⁇ 10 5 fibroblasts per ml of medium, DMEM containing 10% by weight of FCS, 100 U / ml of penicillin G, 25 ⁇ g / ml gentamicin) is applied to this carrier layer in microtiter plates and 1 mM ascorbyl-2-phosphate) were added with 60 to 100 of the three-dimensional sweat gland equivalents prepared under point 1.3 (cell count per equivalent of about 25,000 sweat gland cells).
  • the fibroblasts of the third or fourth passage precultured in culture flasks are dissolved by adding a trypsin solution from the bottom of the culture flasks, washed with medium and centrifuged off. After determining the number of cells, the cell suspension is adjusted to the previously mentioned concentration and the aforementioned number of discrete sweat gland equivalents added. After applying the mixture of fibroblasts and sweat gland equivalents to the carrier layer, place the plate lid on and incubate the plate for 16 days at 37 ° C and 5% v / v CO2 submerged, changing medium every 3 days. After 16 days, a dermis equivalent has formed in which the three-dimensional sweat gland equivalents are incorporated.
  • Keratinocytes are then seeded on this dermis equivalent.
  • keratinocytes of the first or second passage are pre-cultured in cell culture bottles with nutrient medium mentioned under 1.2 and then detached from the bottom by addition of a trypsin solution. After washing with nutrient medium and centrifuging, the cell count of the keratinocyte suspension is adjusted to 1 -6 ⁇ 10 5 keratinocytes per ml of medium. From the wells of the microtiter plate, which contain the dermis equivalents, the medium is aspirated so far that the surface of dermis equivalents remains moist. Subsequently, 1 ml of the previously prepared keratinocyte suspension is added and placed on the slab ceilings.
  • Cultivation in Submers culture is for 2 days at 37 ° C and 5% v / v CO2.
  • the skin models are retrieved from the wells and placed on filter papers lying on metal spacers in a Petri dish.
  • the Petri dish is now filled with the nutrient medium mentioned under point 1.2 so far that the medium reaches the upper edge of the filter paper and distributed around the base of the skin models.
  • the surface of the models is not covered by medium (airlift culture).
  • the in vitro full-skin models according to the invention are obtained, which have a multilayered epidermis and a keratinized tissue surface.
  • the preparation of the carrier layer which carries a dermis equivalent, is carried out analogously to Examples 1 to 3 described in the publication WO 2006/018147 A1 on pages 22 to 24, wherein the cultivation of the fibroblasts is carried out for 16 days.
  • the three-dimensional sweat gland equivalents prepared under point 1.3 are added to this dermis equivalent by suspending them in the nutrient medium described under point 1.2 and then applying this suspension to the dermis equivalent.
  • the keratinocytes are sown, as described under point 1 .4.
  • the models are converted to the culture format of the Air-Liquid interface and cultured for a further 11 days under these conditions.
  • the in vitro full-skin models produced in this way have a complex epidermis and a keratinized tissue surface.
  • Immunofluorescent staining was performed on histological sections to demonstrate protein expression of sweat gland-specific marker proteins.
  • the in vitro full-skin models prepared under items 1 .4 and 1.5 were cut into 1 x 0.5 cm pieces and placed in paraffin or frozen with embedding medium. Subsequently, the objects were cut and stained according to a standard fluorescence protocol for histological sections (e.g., from Abcam, Cambridge, UK). The stained samples can then be analyzed under a fluorescence microscope or a confocal laser scanning microscope.
  • sweat gland-specific marker proteins could be detected: carcinoembryonic antigen (CEA), alpha smooth muscle actin (alpha-SMA), muscarinic acetylcholine receptor M3 (M-ACh-R3), sodium-potassium chloride cotransporter 1 (NKCC1), Galanin receptor 2 (GalR2).
  • CEA carcinoembryonic antigen
  • alpha-SMA alpha smooth muscle actin
  • M-ACh-R3 muscarinic acetylcholine receptor M3
  • NKCC1 sodium-potassium chloride cotransporter 1
  • GalR2 Galanin receptor 2

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Abstract

La présente invention concerne un modèle Vollhautmodell in-vitro qui compote sur une couche formant substrat un ein Dermis- und Epidermisäquivalent sowie 1 bis 100 dreidimensionale Schweißdrüsenäquivalente mit jeweils 500 bis 500.000 Schweißdrüsenzellen sowie einem Durchmesser von jeweils 100 bis 6.000 μιη. La présente invention concerne également la fabrication d'un modèle Vollhautmodells ainsi que l'utilisation de ce modèle en tant que modèle in-vitro, dans le procédé de Screening ainsi que pour l'évaluation in-vitro des principes actifs de l'influence cosmétique sur l'inhibition des sécrétions sudatives ainsi que les odeurs corporelles.
EP17713202.4A 2016-04-22 2017-03-20 Modèle vollhaut in-vitro, contenant un modèle de culture de cellules tridimensionnel de la schweissdrüse Withdrawn EP3445846A1 (fr)

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DE102016206862.8A DE102016206862A1 (de) 2016-04-22 2016-04-22 In-vitro Vollhautmodell, enthaltend dreidimensionale Zellkulturmodelle der Schweißdrüse
PCT/EP2017/056490 WO2017182208A1 (fr) 2016-04-22 2017-03-20 Modèle vollhaut in-vitro, contenant un modèle de culture de cellules tridimensionnel de la schweissdrüse

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DE102018129793B4 (de) * 2018-11-26 2023-10-05 Henkel Ag & Co. Kgaa Dreidimensionales Zellkulturmodell der humanen Schweißdrüse zur Analyse von Stress-assoziierten Schwitzprozessen
JP7236935B2 (ja) * 2019-05-29 2023-03-10 株式会社マンダム 汗腺の識別方法、および汗腺の識別キット
JP7296785B2 (ja) * 2019-05-29 2023-06-23 株式会社マンダム アポクリン汗腺の動態の観察方法、および被験物質の評価方法
US20220236255A1 (en) * 2019-06-28 2022-07-28 Arizona Board Of Regents On Behalf Of The University Of Arizona Self-Contained Responsive Biological Systems and Methods
DE102019135193B4 (de) 2019-12-19 2022-02-03 Henkel Ag & Co. Kgaa Gewebetechnologisch unterstützte Rekonstruktion einer ekkrinen Schweißdrüse
EP4095235A4 (fr) 2020-01-22 2024-03-06 Aist Culture tridimensionnelle de cellules cutanées immortalisées ayant une couche superficielle formée de stratum corneum, procédé de production de ladite culture tridimensionnelle, et procédé d'évaluation d'une substance d'essai à l'aide de ladite culture tridimensionnelle
EP4159249A1 (fr) * 2021-09-30 2023-04-05 Korea University Research and Business Foundation Procédé de culture de peau humaine reconstruite

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US5888720A (en) * 1992-10-27 1999-03-30 Yissum Research And Development Company Of The Hebrew University Of Jerusalem In vitro micro-organs
DE102004039537A1 (de) 2004-08-13 2006-02-23 Phenion Gmbh & Co. Kg Vernetzte Kollagenmatrix zur Herstellung eines Hautäquivalentes
EP1788006B1 (fr) 2004-08-16 2013-12-25 Mitsui Chemicals, Inc. Polymère éthylène et son utilisation
US8105380B2 (en) * 2006-10-23 2012-01-31 Stemedica Cell Technologies, Inc. Cellular scaffold
CN102091352A (zh) * 2009-12-09 2011-06-15 中国人民解放军总医院第一附属医院 一种含有汗腺的组织工程皮肤模型的构建方法
CN101773688B (zh) * 2010-02-05 2013-10-23 中国人民解放军第四军医大学 一种含附属器的组织工程皮肤的制备方法
DE102015222279B4 (de) 2015-11-12 2020-09-17 Henkel Ag & Co. Kgaa Dreidimensionales Zellkulturmodell der Schweißdrüse, insbesondere der humanen Schweißdrüse

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JP6871274B2 (ja) 2021-05-12
US11180728B2 (en) 2021-11-23
DE102016206862A1 (de) 2017-10-26
JP2019514370A (ja) 2019-06-06
WO2017182208A1 (fr) 2017-10-26

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