WO1995010600A1 - In vitro human epidermal sun-tanning test - Google Patents

In vitro human epidermal sun-tanning test Download PDF

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WO1995010600A1
WO1995010600A1 PCT/FR1993/000997 FR9300997W WO9510600A1 WO 1995010600 A1 WO1995010600 A1 WO 1995010600A1 FR 9300997 W FR9300997 W FR 9300997W WO 9510600 A1 WO9510600 A1 WO 9510600A1
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tanning
epidermis
culture
keratinocytes
reconstituted
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PCT/FR1993/000997
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French (fr)
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Martin Rosdy
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Martin Rosdy
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Priority to PCT/FR1993/000997 priority Critical patent/WO1995010600A1/en
Priority to AU51165/93A priority patent/AU5116593A/en
Publication of WO1995010600A1 publication Critical patent/WO1995010600A1/en

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • G01N33/5088—Supracellular entities, e.g. tissue, organisms of vertebrates
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/33—Insulin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/30—Hormones
    • C12N2501/38—Hormones with nuclear receptors
    • C12N2501/39—Steroid hormones
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/091—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells melanocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/09—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells
    • C12N2502/094—Coculture with; Conditioned medium produced by epidermal cells, skin cells, oral mucosa cells keratinocytes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2502/00—Coculture with; Conditioned medium produced by
    • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
    • C12N2502/1323—Adult fibroblasts
    • C—CHEMISTRY; METALLURGY
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    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/04—Screening or testing on artificial tissues
    • C12N2503/06—Screening or testing on artificial skin

Definitions

  • the culture conditions In order to be able to reproduce in vitro all the characteristics and stages of terminal epidermal differentiation, the culture conditions must be as close as possible to the natural environment of the epidermis. Therefore, after confluence, the cultures must be exposed to air and the nutrient medium must pass through the substrate to reach the proliferative cells of the first layer from below.
  • Several culture systems include the use of dermal or pseudodermal substrates, while others are satisfied with substrate constituted by chemically inert filters, covered or not with an extracellular matrix which can promote cell attachment.
  • the Bell system The dermis equivalent is prepared by including living fibroblasts in a collagen matrix which is modified and contracted by these resident cells. Keratinocytes are cultured on the surface of these substrates which are then exposed to the air by means of stainless steel grids (Bell et al., 1983). These reconstructed skins, in the presence of 10% serum in the medium, express all the main markers of epidermal differentiation, but their precise location in the different cell layers does not entirely correspond to that observed in the epidermis in vivo ( Asselineau et al., 1985). In addition, some basal cell antigens are not polarized as in vivo (Asselineau et al., 1986). Recently the system has been improved by adjusting the acid concentration
  • REPLACEMENT SHEET factors and therefore have a chemically defined culture environment (medium and support), where interference due to molecules present in the serum is impossible.
  • a fully differentiated epithelium having the characteristics of the epidermis was obtained in vitro by culturing normal human keratinocytes (KHN) in a chemically defined medium on inert filter substrates at the air-liquid interface. Double-colored vertical sections and indirect immunofluorescence studies have shown correct stratification and expression of protein markers of differentiation; analysis by electron microscopy shows the presence of desmosomes, keratohyaline granules, lamellar bodies extruding their lipid content and the formation of a ten-layer stratum corneum. In addition, all the lipids typical of the stratified human epidermis were present in these cultures, in particular ceramides, which are probably responsible for the relative impermeability of the stratum corneum.
  • a suspension consisting mainly of keratinocytes, but also of a small number of melanocytes, is collected.
  • the ratio between the two cell types that one obtains varies a lot according to the different skin samples.
  • melanogenesis On such organ cultures, ie growths from a whole skin biopsy, stimulation of melanogenesis has been described (Bertaux et al., 1988).
  • the proliferation of melanocytes is stimulated by UV-B, UV-A rays, as well as in the presence of psoralens, but the density of melanocytes cannot be controlled, therefore melanogenesis cannot be normalized in this system.
  • REPLACEMENT SHEET Another coculture system (Scott and Haake, 1991; Haake and Scott, 1991), on the other hand, makes it possible to study the consequences of different melanocyte / keratinocyte relationships during sowing.
  • this system also requires 10% serum in the culture medium, as well as an artificial dermis containing fibroblasts from newborns.
  • the cells used are not adults, but come from tissues of newborn donors, constituting epidermis in culture which differ significantly from the adult epidermis.
  • melanogenesis The stimulation of melanogenesis in culture has only been described on immersed cultures of pure melanocytes, that is to say in the absence of keratinocytes, which is very far from the epidermal situation in vivo (Libow et al., 1988) . Indeed, keratinocytes seem to control by paracrine mechanisms the proliferation and differentiation (melanogenesis) of human melanocytes (Yaar et al., 1991), therefore their active presence is essential for the normalized reconstitution of melanogenesis.
  • the invention described here consists of a tanning test using adult human epidermis reconstituted by cell culture in a defined medium.
  • the tanning of epidermis reconstructed by culture in a chemically defined medium at the air-liquid interface is evaluated by the quantitative measurement of the production and secretion of melanin grains (elanosomes) by the melanocytes present in these cultures, and of their transfer and distribution. in the surrounding keratinocytes.
  • the test simulates the topical application on the skin
  • the test simulates the systemic effects, ie after ingestion of the product.
  • the possible cytotoxic effects of a product to be tested on epidermal cells can be detected in parallel.
  • the epidermis equivalents are obtained by seeding on inert or collagenous substrates a given number of normal adult human keratinocytes and a given number of normal adult human melanocytes. These two cell types must be isolated beforehand from samples of healthy human skin, and their number amplified by conventional immersion culture in a chemically defined culture medium which allows their rapid proliferation, on one or more passages. Then these stocks of cells (keratinocytes and melanocytes) are frozen and samples are thawed as required, or else they are used directly for the manufacture of reconstituted epidermis.
  • the cell concentrations of each of the suspensions are adjusted to allow the seeding of the mixture in a small volume of defined medium while respecting the desired ratio of melanocytes to keratinocytes.
  • this ratio must be less than or equal to 1/2. This is due to the fact that, unlike keratinocytes, melanocytes only rarely divide in reconstituted epidermis cultures (as in the epidermis in vivo), therefore their seeding density must be determined with precision.
  • the melanocytes can be seeded before the keratinocytes.
  • REPLACEMENT SHEET Melanocytes from another skin sample (from a different donor) than keratinocytes can be co-seeded with the latter. After reaching confluence, the cultures are exposed to the air / culture medium interface by hoisting the culture substrates on inert supports allowing the flow of the medium to the underside of these substrates. After at least 2 days of culture on a chemically defined medium, the cultures are irradiated with UV rays using a laboratory irradiator: Repeated irradiations of UV-A and / or UV-B are carried out in the presence of the products to be tested as well as negative and positive controls.
  • the negative control can be a conventional cosmetic cream which does not interact with the stimulation of tanning by UV rays
  • the positive control can for example be a cosmetic protective cream filtering UV rays or well, on the contrary reinforcing the effects of UV rays (for example: containing psoralens).
  • the effects of the stimulations (example: UV rays) on the epidermis reconstituted under these different experimental conditions are then characterized and / or quantified according to several methods: -measurement of the number of melanin grains in a certain number of colored vertical sections of the cultures by analysis computer-aided image processing, -measurement of the melanin content of culture homogenates by biochemical techniques.
  • the intensities and durations of stimulation necessary to induce an increased detectable synthesis of melanin grains and their transfer in the surrounding keratinocytes vary according to the race and age of the initial cell donor: The calibration of the test is therefore necessary each once another donor's cells are used. To reduce these variations due to skin donors, it is possible to establish cell stocks (keratinocytes and melanocytes separately) where the cells of several donors are mixed. This also brings the advantage of having larger cell stocks, therefore being able to carry out a greater number of cultures from the same stock. However, each of the series of reconstituted epidermis cultures must nevertheless be characterized and controlled by the use of negative and positive controls.
  • Samples of healthy adult skin are provided by a surgical department after a breast reduction operation.
  • the epidermis is separated from the dermis with 0.25% trypsin in a saline solution, first by the action of dispase or collagenase.
  • the cell suspension is diluted in a defined culture medium promoting attachment and growth either of melanocytes (Pittelkow and Shipley, 1989) or of keratinocytes (Boyce and Ham, 1983).
  • the proliferation of melanocytes is stimulated in an environment defined by the presence of growth factors such as basic-FGF (fibroblast growth factor), insulin, and TPA (12-O-tetradecanoyl phorbol 13-acetate) which, moreover, inhibits the proliferation of keratinocytes present by inducing their irreversible terminal differentiation.
  • growth factors such as basic-FGF (fibroblast growth factor), insulin, and TPA (12-O-tetradecanoyl phorbol 13-acetate) which, moreover, inhibits the proliferation of keratinocytes present by inducing their irreversible terminal differentiation.
  • the growth of keratinocytes is stimulated by insulin, EGF (epidermal growth factor) and hydrocortisone. This latter type of medium, however, allows the melanocytes to survive: this makes it possible to isolate the melanocytes after a few days of mixed culture using differential trypsination: the melanocytes detach themselves much faster than keratinocytes.
  • the defined medium used is either MCDB 153 (Boyce and Ham, 1983), or a medium prepared from DMEM (Dulbecco's modified Eagle's Medium) and Ham F-12 medium. Before the last pass, the melanocytes are weaned from TPA for several days. After a number of passages and
  • the cells are trypsinized, counted, and seeded in a melanocyte / keratinocyte ratio 1/2 at a density of 10 5 per cm2
  • Masson makes it possible to distinguish the different cellular layers formed and to visualize and count (by computer-assisted image analysis) the melanin grains present.
  • melanocytes containing melanin grains are mainly located in the first cell layer, as in vivo, while keratinocytes do not contain them.
  • the stimulation by UV-B irradiation induces a much more homogeneous distribution of the melanin grains, since practically all the cells of the first two or three layers contain it.
  • REPLACEMENT SHEET parallels shows an increase in the amount of melanin present in the order of 45 times the control level.
  • the topical prior application of a drop of 20_1 of sunscreen with total UV filter before each irradiation prevents 89% (+/- 5%) of this melanogenesis induced by UV-B.
  • the UV-A irradiation (4 times 2.5 Joules / day) in the presence and absence of psoralen (10 ⁇ M of 8-MOP) in the culture medium produces a weaker tanning reaction: 5 times more melanin grains , but in . plus the number of melanocytes loaded with triple melanin in the basal cell layer.
  • the cultures frozen in parallel made it possible to specifically mark and count with the aid of antimelanosome antibodies (Tomita et al., 1991) by indirect immunofluorescence, the melanocytes present according to the experimental conditions.
  • the homogenates in a buffered medium allowed the biochemical quantification of the total melanin present in each of the cultures: the same increase by a factor of 45 (+/- 6) was measured after irradiation with UV-B.
  • the tyrosinase activity (Halaban et al., 1991) is increased by a factor of three after the first UV-B irradiation and remains at this level even after 4 irradiations.
  • Boyce ST Ham RG. Calcium regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J. Invest. Dermatol. 81: 33s-40s; 1983
  • Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. J. Cell Biol. 107: 1919-1926; 1988
  • Halaban R P ⁇ merantz SH, Marshall S, Lambert DT, Lerner AB.
  • Rosdy M Terminal epidermal differentiation of human keratinocytes grown in chemically defined permeabilum on inert filter substrates at the air-liquid interface. J. Invest. Dermatol. 95: 409-414; 1990
  • Tsao MC Walthall BJ, Ham RG. Clonal growth of normal human epidermal keratinocytes in a defined medium. J. Cellular Physiology 110: 219-229; 1982

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Abstract

The invention relates to a process for obtaining an epiderm equivalent cultivated in a defined medium, the corresponding epiderm equivalent, and its utilization for pharmaceutical and/or cosmetic sun-tanning tests. The disclosed produced epiderms are capable of sun-tanning, under ultraviolet rays, or in the presence of psoralenes, for example. The melanocytes present in said epiderm equvalents produce melanine grains which are transferred, as in vivo, into the environning keratinocytes as a function of the stimulation intensity. After application to the reproduced epiderms (and/or in the culture medium) a product to be tested, it is possible to measure its effect for retarding, inhibiting or stimulating said sun-tanning reaction, through various technics.

Description

TEST DE BRONZAGE EPIDERMIQUE HUMAIN IN VITROIN VITRO HUMAN EPIDERMAL TANNING TEST
ETAT DE LA TECHNIQUE;STATE OF THE ART;
A) LES EPIDERMES RECONSTITUEES IN VITROA) EPIDERMAS RECONSTITUTED IN VITRO
Afin de pouvoir reproduire in vitro toutes les caractéristiques et étapes de la différenciation épidermique terminale, les conditions de culture doivent se rapprocher le plus possible de l'environnement naturel de l'épiderme. Par conséquent, après confluence, les cultures doivent être exposées à l'air et le milieu nutritif doit traverser le substrat pour parvenir par le bas aux cellules prolifératives de la première couche. Plusieurs systèmes de culture comprennent l'utilisation de substrats dermiques ou pseudodermiques, tandis que d'autres se contentent de substrat constitués par des filtres chimiquement inertes, recouverts ou non d'une matrice extracellulaire pouvant favoriser l'attachement cellulaire.In order to be able to reproduce in vitro all the characteristics and stages of terminal epidermal differentiation, the culture conditions must be as close as possible to the natural environment of the epidermis. Therefore, after confluence, the cultures must be exposed to air and the nutrient medium must pass through the substrate to reach the proliferative cells of the first layer from below. Several culture systems include the use of dermal or pseudodermal substrates, while others are satisfied with substrate constituted by chemically inert filters, covered or not with an extracellular matrix which can promote cell attachment.
Dans ces systèmes, on obtient la formation d'un vrai stratum corneum, c'est à dire une différenciation épidermique apparemment complète avec présence de toutes les couches cellulaires successives caractéristiques, et l'expression des marqueurs biochimiques essentiels.In these systems, the formation of a true stratum corneum is obtained, that is to say an apparently complete epidermal differentiation with the presence of all the successive characteristic cellular layers, and the expression of essential biochemical markers.
Le système de culture sur derme désépidermisé présente l'avantage de garder une membrane basale intacte comme surface d'attachement des keratinocytes tout en éliminantThe de-epidermized dermis culture system has the advantage of keeping an intact basement membrane as the attachment surface for keratinocytes while eliminating
- 1 - FEUILLE DE REMPLACEMENT les interférences dues aux fibroblastes du derme, puisque ces derniers sont "tués" par congélations successives (Régnier et coll., 1981,1989; Pruniéras et coll., 1983a,b).- 1 - REPLACEMENT SHEET interference due to fibroblasts of the dermis, since the latter are "killed" by successive freezing (Régnier et al., 1981, 1989; Pruniéras et al., 1983a, b).
Le système de Bell: L'équivalent de derme est préparé en incluant des fibroblastes vivants dans une matrice de collagène qui est modifiée et contractée par ces cellules résidentes. Des keratinocytes sont cultivés à la surface de ces substrats qui sont alors exposés à l'air au moyen de grilles en acier inoxydable (Bell et coll., 1983). Ces peaux reconstruites, en présence de 10% de sérum dans le milieu, expriment tous les principaux marqueurs de la différenciation épidermique, mais leur localisation précise dans les différentes couches cellulaires ne correspond pas tout à fait à celle observée dans l'épiderme in vivo (Asselineau et coll., 1985). De plus, certains antigènes des cellules basales ne sont pas polarisés comme in vivo (Asselineau et coll., 1986). Récemment le système a été perfectionné en ajustant la concentration en acideThe Bell system: The dermis equivalent is prepared by including living fibroblasts in a collagen matrix which is modified and contracted by these resident cells. Keratinocytes are cultured on the surface of these substrates which are then exposed to the air by means of stainless steel grids (Bell et al., 1983). These reconstructed skins, in the presence of 10% serum in the medium, express all the main markers of epidermal differentiation, but their precise location in the different cell layers does not entirely correspond to that observed in the epidermis in vivo ( Asselineau et al., 1985). In addition, some basal cell antigens are not polarized as in vivo (Asselineau et al., 1986). Recently the system has been improved by adjusting the acid concentration
_9 rétinoïque à 10 M, grâce à l'utilisation d'un sérum délipidisé: l'expression des différents marqueurs ressemble alors fortement à la situation normale in vivo (Asselineau et coll. , 1989) ._9 10 M retinoic, thanks to the use of a delipidized serum: the expression of the different markers then strongly resembles the normal situation in vivo (Asselineau et al., 1989).
Le même substrat de derme-équivalent a servi pour l'obtention, à partir de follicules de cheveux humains exposé à l'interface air-liquide, d'un épiderme in vitro présentant tous les aspects de la différenciation épidermique (Lenoir et coll., 1988). Ce travail est enThe same dermis-equivalent substrate was used to obtain, from human hair follicles exposed to the air-liquid interface, an epidermis in vitro having all the aspects of epidermal differentiation (Lenoir et al., 1988). This work is in
- 2 - FEUILLEDE REMPLACEMENT accord avec l'observation courante que les racines des poils qui subsistent dans une plaie après un traumatisme sont capables de régénérer un épiderme interfolliculaire normalement différencié. D'autres chercheurs cultivent des keratinocytes sur ces mêmes lattices de Bell, mais en culture d'organe, c'est à dire en y incorporant une biopsie de peau humaine (Coulomb et coll. ,1986; 1989).- 2 - REPLACEMENT SHEET agree with the common observation that the hair roots that remain in a wound after trauma are capable of regenerating a normally differentiated interfollicular epidermis. Other researchers cultivate keratinocytes on these same Bell lattices, but in organ culture, that is to say by incorporating a biopsy of human skin (Coulomb et al., 1986; 1989).
D'autres systèmes consistent à inoculer des cellules épidermiques sur des films ou gels de collagène, des filtres en nylon ou de nitrocellulose couverts ou non de collagène ou d'équivalent de membrane basale déposé par des cellules endotheliales de boeuf, puis d'exposer ces cultures à l'air pour 14 à 21, ou même 65 jours (Fusenig et coll. ,1983; Bernstam et coll. ,1990; Vaughan et coll.,1986; Lillie et coll. ,1980,1988).Other systems consist in inoculating epidermal cells on collagen films or gels, nylon or nitrocellulose filters covered or not with collagen or equivalent of basement membrane deposited by beef endothelial cells, then exposing these air cultures for 14 to 21, or even 65 days (Fusenig et al., 1983; Bernstam et al., 1990; Vaughan et al., 1986; Lillie et al., 1980,1988).
Un des points communs de tous ces systèmes de culture épidermique tridimensionnelle est l'exposition à l'air, qui semble essentielle pour la formation d'une couche cornée in vitro; mais surtout toutes ces cultures ont été réalisées en présence de sérum dans le milieu de culture, estimée nécessaire pour arriver à la différenciation terminale complète. Or, pour estimer ou mesurer l'influence de facteurs ou produits cosmétiques ou pharmaceutiques (tels que les retinoïdes, ou des extraits organiques de toutes origines) sur le métabolisme et la différenciation épidermiques en culture, il faut pouvoir utiliser des concentrations parfois infimes (10 -12 M, par exemple) de cesOne of the common points of all these three-dimensional epidermal culture systems is exposure to air, which seems essential for the formation of a stratum corneum in vitro; but above all all of these cultures were carried out in the presence of serum in the culture medium, considered necessary to arrive at complete terminal differentiation. However, to estimate or measure the influence of factors or cosmetic or pharmaceutical products (such as retinoids, or organic extracts of all origins) on epidermal metabolism and differentiation in culture, it is necessary to be able to use concentrations that are sometimes minute (10 -12 M, for example) of these
- 3 -- 3 -
FEUILLE DE REMPLACEMENT facteurs, et donc disposer d'un environnement (milieu et support) de culture chimiquement défini, où les interférences dues aux molécules présent dans le sérum sont impossible.REPLACEMENT SHEET factors, and therefore have a chemically defined culture environment (medium and support), where interference due to molecules present in the serum is impossible.
Une différenciation épidermique terminale complète de keratinocytes humains cultivés en milieu chimiquement défini sur des substrats constitués par des filtres inertes à l'interface air-liquide est décrite dans la demande de brevet français n° FR2665175 du 27.07.1990 (Rosdy et Clauss, 1990):A complete terminal epidermal differentiation of human keratinocytes cultivated in a chemically defined medium on substrates constituted by inert filters at the air-liquid interface is described in French patent application No. FR2665175 of 07.27.1990 (Rosdy and Clauss, 1990) :
Un épithélium totalement différencié ayant les caractéristiques de l'épiderme a été obtenu in vitro en cultivant des keratinocytes humains normaux (KHN) dans un milieu chimiquement défini sur des substrats de filtres inertes à l'interface air-liquide. Des coupes verticales doublement colorées et des études d'immunofluorescence indirecte ont montré la stratification et l'expression correctes de marqueurs protéiques de la différenciation; l'analyse par microscopie électronique montre la présence de desmosomes, de granules de kératohyaline, de corps lamellaires extrudant leur contenu lipidique et la formation d'un stratum corneum de dix couches. De plus, tous les lipides typiques de l'épiderme humain stratifié étaient présents dans ces cultures, notamment les céramides, qui sont probablement responsables de la relative imperméabilité du stratum corneum.A fully differentiated epithelium having the characteristics of the epidermis was obtained in vitro by culturing normal human keratinocytes (KHN) in a chemically defined medium on inert filter substrates at the air-liquid interface. Double-colored vertical sections and indirect immunofluorescence studies have shown correct stratification and expression of protein markers of differentiation; analysis by electron microscopy shows the presence of desmosomes, keratohyaline granules, lamellar bodies extruding their lipid content and the formation of a ten-layer stratum corneum. In addition, all the lipids typical of the stratified human epidermis were present in these cultures, in particular ceramides, which are probably responsible for the relative impermeability of the stratum corneum.
- 4 - FEUILLE DE REMPLACEMENT B) LA PRESENCE DE MELANOCYTES FONCTIONNELS DANS LES EPIDERMES RECONSTITUES- 4 - REPLACEMENT SHEET B) THE PRESENCE OF FUNCTIONAL MELANOCYTES IN RECONSTRUCTED EPIDERMALS
Lorsqu'on isole des cellules épidermiques à partir d'un échantillon de peau, on recueille une suspension constituée en grande majorité de keratinocytes, mais aussi d'un petit nombre de melanocytes. Le rapport entre les deux types cellulaires que l'on obtient varie beaucoup selon les différents échantillons de peau.When epidermal cells are isolated from a skin sample, a suspension consisting mainly of keratinocytes, but also of a small number of melanocytes, is collected. The ratio between the two cell types that one obtains varies a lot according to the different skin samples.
Ainsi, dans les cultures de keratinocytes sur couche nourritrice de fibroblastes irradiés et en présence de 10% de sérum (système développé par Howard Green à Boston; Rheinwald et Green,1975) on peut détecter des melanocytes fonctionnels, c'est à dire qui sont capables de transférer des mélanosomes aux keratinocytes (DeLuca et coll., 1988). Ces cultures forment des feuillets de 3 à 4 couches cellulaires où le nombre de melanocytes présents est aléatoire, donc n'est pas contrôlé. C'est le cas également pour le système de culture d'organe entier, bien qu'il permette la constitution d'équivalents épidermiques plus complets, toujours en présence de 10% de sérum dans le milieu de culture (Topol et coll., 1986). Sur de telles cultures d'organe, c'est à dire des excroissances à partir d'une biopsie de peau entière, la stimulation de la mélanogénèse a été décrite (Bertaux et coll., 1988). La prolifération des melanocytes y est stimulée par les rayons UV-B, UV-A, ainsi qu'en présence de psoralenes, mais la densité de melanocytes ne peut être contrôlée, donc la mélanogénèse ne peut être normalisée dans ce système.Thus, in keratinocyte cultures on a nourishing layer of irradiated fibroblasts and in the presence of 10% serum (system developed by Howard Green in Boston; Rheinwald and Green, 1975) functional melanocytes can be detected, that is to say which are capable of transferring melanosomes to keratinocytes (DeLuca et al., 1988). These cultures form sheets of 3 to 4 cell layers where the number of melanocytes present is random, therefore is not controlled. This is also the case for the whole organ culture system, although it allows the constitution of more complete epidermal equivalents, always in the presence of 10% of serum in the culture medium (Topol et al., 1986 ). On such organ cultures, ie growths from a whole skin biopsy, stimulation of melanogenesis has been described (Bertaux et al., 1988). The proliferation of melanocytes is stimulated by UV-B, UV-A rays, as well as in the presence of psoralens, but the density of melanocytes cannot be controlled, therefore melanogenesis cannot be normalized in this system.
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FEUILLE DE REMPLACEMENT Un autre système de coculture (Scott et Haake, 1991; Haake et Scott, 1991) permet en revanche d'étudier les conséquences de différents rapports mélanocyte/kératinocyte à l'ensemencement. Mais ce système nécessite également 10% de sérum dans le milieu de culture, ainsi qu'un derme artificiel contenant des fibroblastes de nouveau-né. En plus, les cellules utilisées ne sont pas adultes, mais proviennent de tissus de donneurs nouveau-nés, constituant des épidermes en culture qui diffèrent notablement de 1'épiderme adulte.REPLACEMENT SHEET Another coculture system (Scott and Haake, 1991; Haake and Scott, 1991), on the other hand, makes it possible to study the consequences of different melanocyte / keratinocyte relationships during sowing. However, this system also requires 10% serum in the culture medium, as well as an artificial dermis containing fibroblasts from newborns. In addition, the cells used are not adults, but come from tissues of newborn donors, constituting epidermis in culture which differ significantly from the adult epidermis.
La stimulation de la mélanogénèse en culture n'a été décrite que sur des cultures immergées de melanocytes pures, c'est à dire en absence de keratinocytes, ce qui est très loin de la situation épidermique in vivo (Libow et coll., 1988). En effet, les keratinocytes semblent contrôler par des mécanismes paracrines la prolifération et la différenciation (mélanogénèse) des melanocytes humains (Yaar et coll., 1991), donc leur présence active est indispensable à la reconstitution normalisée de la mélanogénèse.The stimulation of melanogenesis in culture has only been described on immersed cultures of pure melanocytes, that is to say in the absence of keratinocytes, which is very far from the epidermal situation in vivo (Libow et al., 1988) . Indeed, keratinocytes seem to control by paracrine mechanisms the proliferation and differentiation (melanogenesis) of human melanocytes (Yaar et al., 1991), therefore their active presence is essential for the normalized reconstitution of melanogenesis.
C) TESTS DE BRONZAGEC) TANNING TESTS
L'efficacité des produits de protection ou de stimulation solaires est testée couramment in vivo: les modèles animaux (cobaye, rat, souris) présentent l'avantage de pouvoir normaliser les séries de sujets, mais la peau des animaux est plus vulnérable à cause de la plus grande finesse de leur couche cornée et de l'absence de pigmentsThe effectiveness of sun protection or stimulation products is commonly tested in vivo: animal models (guinea pig, rat, mouse) have the advantage of being able to normalize the series of subjects, but the skin of animals is more vulnerable because of the greater fineness of their horny layer and the absence of pigments
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FEUILLE DE REMPLACEMENT mélaniques.REPLACEMENT SHEET melanistic.
Les essais chez l'homme restent les plus probants. La diversité de réactivité de la peau humaine aux rayons ultraviolets implique un grand échantillonage de sujets, ce qui revient très cher.Human trials remain the most convincing. The diversity of reactivity of human skin to ultraviolet rays implies a large sampling of subjects, which is very expensive.
L'appréciation clinique des résultats (sur l'animal ou chez l'homme) se fait couramment à l'oeil nu par 2 observateurs indépendants qui ne connaissent pas les conditions d'application et d'irradiations. Cette observation visuelle semble rester le moyen le plus sûr et le plus sensible, mais des techniques de mesure instrumentale de la couleur de la peau ont récemment été publiées (Chardon et coll. 1991).The clinical assessment of the results (on animals or in humans) is commonly done with the naked eye by 2 independent observers who do not know the conditions of application and irradiation. This visual observation seems to remain the safest and most sensitive means, but techniques for the instrumental measurement of skin color have recently been published (Chardon et al. 1991).
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FEUILLE DÉ REMPLACEMENT L'INVENTION:REPLACEMENT SHEET THE INVENTION:
Test de bronzage épidermique humain in vitro.In vitro human epidermal tanning test.
L'invention décrite ici consiste en un test de bronzage utilisant des épidermes humains adultes reconstitués par culture cellulaire en milieu défini. Le bronzage des épidermes reconstruits par culture en milieu chimiquement défini à l'interface air-liquide est évalué par la mesure quantitative de la production et sécrétion de grains de mélanine ( élanosomes) par les melanocytes présents dans ces cultures, et de leur transfert et distribution dans les keratinocytes environnants.The invention described here consists of a tanning test using adult human epidermis reconstituted by cell culture in a defined medium. The tanning of epidermis reconstructed by culture in a chemically defined medium at the air-liquid interface is evaluated by the quantitative measurement of the production and secretion of melanin grains (elanosomes) by the melanocytes present in these cultures, and of their transfer and distribution. in the surrounding keratinocytes.
La grande reproductibilité de l'aspect et des qualités biochimiques des épidermes reconstitués décrits, qui est due à l'emploi exclusif de milieux définis, permet de comparer les effets d'une stimulation (rayons UV, activateur chimique, etc..) avec une grande précision sur un nombre important d'échantillons produits en parallèle. Par cette uniformité des épidermes obtenus on peut en effet surmonter le problême des variations importantes du bronzage in vivo selon les endroits du corps, et donc normaliser le bronzage in vitro. De plus, on peut obtenir un très grand nombre d'équivalents d'épiderme qui ont tous le même comportement, c'est à dire la même intensité de bronzage, après irradiation aux rayons UV par exemple. Ceci permet de tester en parallèle un grand nombre de produits (ou de concentrations de produits) à usage cosmétique ou pharmaceutique censés interférer (freiner, inhiber ou stimuler) avec le bronzage de la peau humaine.The high reproducibility of the appearance and the biochemical qualities of the reconstructed epidermis described, which is due to the exclusive use of defined media, makes it possible to compare the effects of stimulation (UV rays, chemical activator, etc.) with a high precision on a large number of samples produced in parallel. By this uniformity of the epidermis obtained, it is indeed possible to overcome the problem of significant variations in tanning in vivo according to the locations of the body, and therefore to normalize tanning in vitro. In addition, a very large number of skin equivalents can be obtained which all have the same behavior, that is to say the same tanning intensity, after irradiation with UV rays for example. This makes it possible to test in parallel a large number of products (or concentrations of products) for cosmetic or pharmaceutical use intended to interfere (curb, inhibit or stimulate) with the tanning of human skin.
- 8 - FEUILLEDEREMPLACEMENT L'intensité de la pigmentation des épidermes reconstitués en culture et donc la production et la distribution de mélanine après stimulation par des rayons UV (ou d'autres stimulants physiques ou chimiques) est quantifiée et calibrée par des témoins négatifs et positifs appropriés pour chaque série d'épidermes reconstitués produits. Ainsi standardisés, ces équivalents d'épiderme humain sont utilisés pour des tests comparatifs reproductibles de produits favorisant, freinant ou inhibant le bronzage de la peau. Ces produit à tester peuvent être appliqués directement sur la couche cornée des épidermes reconstitués et/ou ajoutés au milieu de culture qui nourrit les cellules à travers le support qui sert de substrat aux cultures. Dans le premier cas, le test simule l'application topique sur la peau, dans le second cas le test simule les effets systémiques, c'est à dire après ingestion du produit. Les éventuels effets cytotoxiques d'un produit à tester sur les cellules épidermiques peuvent être détectés parallèlement.- 8 - LOCATION SHEET The intensity of the pigmentation of the epidermis reconstituted in culture and therefore the production and distribution of melanin after stimulation by UV rays (or other physical or chemical stimulants) is quantified and calibrated by negative and positive controls appropriate for each series. of reconstituted epidermis produced. Thus standardized, these equivalents of human epidermis are used for reproducible comparative tests of products promoting, slowing down or inhibiting tanning of the skin. These test products can be applied directly to the stratum corneum of the reconstituted epidermis and / or added to the culture medium which nourishes the cells through the support which serves as a substrate for the cultures. In the first case, the test simulates the topical application on the skin, in the second case the test simulates the systemic effects, ie after ingestion of the product. The possible cytotoxic effects of a product to be tested on epidermal cells can be detected in parallel.
9 -9 -
FEUILLE DE REMPLACEMENT METHODES D'OBTENTION:REPLACEMENT SHEET METHODS OF OBTAINING:
Les équivalents d'épiderme sont obtenus par ensemencement sur des substrats inertes ou collagèneux d'un nombre donné de keratinocytes humains adultes normaux et d'un nombre donné de melanocytes humains adultes normaux. Ces deux types cellulaires doivent être isolés auparavant à partir d'échantillons de peau humaine saine, et leur nombre amplifié par culture en immersion classique dans un milieu de culture chimiquement défini qui permet leur prolifération rapide, sur un ou plusieurs passages. Ensuite ces stocks de cellules (keratinocytes et melanocytes) sont congelés et des échantillons sont décongelés selon les besoins, oubien ils sont utilisés directement pour la fabrication des épidermes reconstitués. Après trypsination, les concentrations cellulaires de chacune des suspensions (keratinocytes et melanocytes) sont ajustées pour permettre l'ensemencement du mélange dans un petit volume de milieu défini en respectant le rapport voulu de melanocytes sur keratinocytes. Plus le nombre de cellules totales est petit, plus le rapport melanocytes sur keratinocytes doit tendre vers 1. Quand la densité de l'ensemencement dépasse 5X10 5 cellules par cm2, ce rapport doit être inférieur ou égal à 1/2. Ceci est dû au fait que, contrairement aux keratinocytes, les melanocytes ne se divisent que rarement dans les cultures d'épidermes reconstitués (comme dans 1'épiderme in vivo) , donc leur densité d'ensemencement doit être déterminée avec précision.The epidermis equivalents are obtained by seeding on inert or collagenous substrates a given number of normal adult human keratinocytes and a given number of normal adult human melanocytes. These two cell types must be isolated beforehand from samples of healthy human skin, and their number amplified by conventional immersion culture in a chemically defined culture medium which allows their rapid proliferation, on one or more passages. Then these stocks of cells (keratinocytes and melanocytes) are frozen and samples are thawed as required, or else they are used directly for the manufacture of reconstituted epidermis. After trypsination, the cell concentrations of each of the suspensions (keratinocytes and melanocytes) are adjusted to allow the seeding of the mixture in a small volume of defined medium while respecting the desired ratio of melanocytes to keratinocytes. The smaller the number of total cells, the more the melanocyte to keratinocyte ratio should tend towards 1. When the seeding density exceeds 5 × 10 5 cells per cm 2, this ratio must be less than or equal to 1/2. This is due to the fact that, unlike keratinocytes, melanocytes only rarely divide in reconstituted epidermis cultures (as in the epidermis in vivo), therefore their seeding density must be determined with precision.
Alternativement, les melanocytes peuvent être ensemencés avant les keratinocytes.Alternatively, the melanocytes can be seeded before the keratinocytes.
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FEUILLE DE REMPLACEMENT Des melanocytes provenant d'un autre échantillon de peau (d'un autre donneur) que les keratinocytes peuvent être coensemencés avec ces derniers. Après avoir atteint la confluence, les cultures sont exposées à l'interface air/milieu de culture en hissant les substrats de culture sur des supports inertes permettant le flux du milieu jusqu'à la face inférieure de ces substrats. Après au minimum 2 jours de culture sur un milieu chimiquement défini, les cultures sont irradiées aux rayons UV à l'aide d'un irradiateur de laboratoire: Des irradiations répétées de UV-A et/ou UV-B sont effectuées en présence des produits à tester ainsi que de témoins négatifs et positifs. Le témoin négatif peut être une crème cosmétique classique qui n'interagit pas avec la stimulation du bronzage par les rayons UV, tandis que le témoin positif peut être par exemple une crème cosmétique de protection filtrant les rayons UV oubien, au contraire renforçant les effets des rayons UV (par exemple: contenant des psoralenes).REPLACEMENT SHEET Melanocytes from another skin sample (from a different donor) than keratinocytes can be co-seeded with the latter. After reaching confluence, the cultures are exposed to the air / culture medium interface by hoisting the culture substrates on inert supports allowing the flow of the medium to the underside of these substrates. After at least 2 days of culture on a chemically defined medium, the cultures are irradiated with UV rays using a laboratory irradiator: Repeated irradiations of UV-A and / or UV-B are carried out in the presence of the products to be tested as well as negative and positive controls. The negative control can be a conventional cosmetic cream which does not interact with the stimulation of tanning by UV rays, while the positive control can for example be a cosmetic protective cream filtering UV rays or well, on the contrary reinforcing the effects of UV rays (for example: containing psoralens).
Les effets des stimulations (exemple: rayons UV) sur les épidermes reconstitués dans ces différentes conditions expérimentales sont ensuite caractérisés et/ou quantifiés selon plusieurs méthodes: -mesure du nombre de grains de mélanine dans un certain nombre de coupes verticales colorées des cultures par analyse d'images assistée par ordinateur, -mesure du contenu en mélanine des homogénats de cultures par des techniques biochimiques.The effects of the stimulations (example: UV rays) on the epidermis reconstituted under these different experimental conditions are then characterized and / or quantified according to several methods: -measurement of the number of melanin grains in a certain number of colored vertical sections of the cultures by analysis computer-aided image processing, -measurement of the melanin content of culture homogenates by biochemical techniques.
- 11 - FEUILLE DE REMPLACEMENT -mesure de l'activité de la tyrosinase épidermique contenue dans les cultures par techniques biochimiques.- 11 - REPLACEMENT SHEET -measurement of the activity of epidermal tyrosinase contained in cultures by biochemical techniques.
-mesure du nombre de melanocytes présents et de leur localisation dans les cultures par coloration à la DOPA, par des techniques d'immunofluorescence indirecte ou de microscopie électronique.-measurement of the number of melanocytes present and their location in cultures by staining with DOPA, by indirect immunofluorescence techniques or by electron microscopy.
-mesure instrumentale de la couleur par colorimétrie qui tient compte de l'équilibre variable entre les différents pigments et leur états physico-chimiques.- instrumental measurement of color by colorimetry which takes into account the variable balance between the different pigments and their physico-chemical states.
-toute autre technique permettant d'évaluer précisément l'intensité de bronzage obtenu.-any other technique allowing to precisely assess the intensity of tanning obtained.
Les intensités et les durées de stimulation nécessaires pour induire une synthèse accrue détectable de grains de mélanine et leur transfert dans les keratinocytes environnants varient en fonction de la race et de l'âge du donneur de cellules initial: La calibration du test est donc nécessaire chaque fois que les cellules d'un autre donneur sont utilisées. Pour réduire ces variations dues aux donneurs de peau, on peut établir des stocks de cellules (keratinocytes et melanocytes séparément) où les cellules de plusieurs donneurs sont mélangées. Ceci apporte aussi l'avantage de disposer de stocks cellulaires plus importants, donc de pouvoir effectuer un plus grand nombre de cultures à partir d'un même stock. Mais chacune des séries de cultures d'épidermes reconstitués doit néanmoins être caractérisée et contrôlée par l'utilisation de témoins négatifs et positifs.The intensities and durations of stimulation necessary to induce an increased detectable synthesis of melanin grains and their transfer in the surrounding keratinocytes vary according to the race and age of the initial cell donor: The calibration of the test is therefore necessary each once another donor's cells are used. To reduce these variations due to skin donors, it is possible to establish cell stocks (keratinocytes and melanocytes separately) where the cells of several donors are mixed. This also brings the advantage of having larger cell stocks, therefore being able to carry out a greater number of cultures from the same stock. However, each of the series of reconstituted epidermis cultures must nevertheless be characterized and controlled by the use of negative and positive controls.
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FEUILLEDEREMPLACEMENT EXEMPLE :LOCATION SHEET EXAMPLE:
Des échantillons de peau adulte saine sont fournis par un service chirurgical après une opération de réduction mammaire. L'épiderme est séparé du derme avec 0.25% de trypsine dans une solution saline oubien d'abord par l'action de la dispase ou de la collagènase. Après comptage, la suspension cellulaire est diluée dans un milieu de culture défini favorisant l'attachement et la croissance soit des melanocytes (Pittelkow et Shipley, 1989), soit des keratinocytes (Boyce et Ham, 1983). La prolifération des melanocytes est stimulée dans un milieu défini par la présence de facteurs de croissance tels que le basic-FGF (fibroblast growth factor), l'insuline, et le TPA (12-O-tetradecanoyl phorbol 13-acetate) qui par ailleurs inhibe la prolifération des keratinocytes présents en induisant leur différenciation terminale irréversible. La croissance des keratinocytes est stimulée par l'insuline, l'EGF (epidermal growth factor) et 1'hydrocortisone. Ce dernier type de milieu permet cependant la survie des melanocytes: ceci permet d'isoler les melanocytes après quelques jours de culture mixte à l'aide d'une trypsination différentielle: les melanocytes se détachent en effet beaucoup plus rapidement que les keratinocytes. Le milieu défini utilisé est soit le MCDB 153 (Boyce et Ham,1983), soit un milieu préparé à partir de DMEM (Dulbecco's modified Eagle's Médium) et de milieu de Ham F-12. Avant le dernier passage, les melanocytes sont sevrés de TPA pendant plusieurs jours. Après un certain nombre de passages etSamples of healthy adult skin are provided by a surgical department after a breast reduction operation. The epidermis is separated from the dermis with 0.25% trypsin in a saline solution, first by the action of dispase or collagenase. After counting, the cell suspension is diluted in a defined culture medium promoting attachment and growth either of melanocytes (Pittelkow and Shipley, 1989) or of keratinocytes (Boyce and Ham, 1983). The proliferation of melanocytes is stimulated in an environment defined by the presence of growth factors such as basic-FGF (fibroblast growth factor), insulin, and TPA (12-O-tetradecanoyl phorbol 13-acetate) which, moreover, inhibits the proliferation of keratinocytes present by inducing their irreversible terminal differentiation. The growth of keratinocytes is stimulated by insulin, EGF (epidermal growth factor) and hydrocortisone. This latter type of medium, however, allows the melanocytes to survive: this makes it possible to isolate the melanocytes after a few days of mixed culture using differential trypsination: the melanocytes detach themselves much faster than keratinocytes. The defined medium used is either MCDB 153 (Boyce and Ham, 1983), or a medium prepared from DMEM (Dulbecco's modified Eagle's Medium) and Ham F-12 medium. Before the last pass, the melanocytes are weaned from TPA for several days. After a number of passages and
- 13 - FEUILLE DE REMPLACEMENT avant d'atteindre la confluence, les cellules sont trypsinées, comptées, et ensemencées dans un rapport melanocytes/keratinocytes 1/2 à une densité de 10 5 par cm2- 13 - REPLACEMENT SHEET before reaching confluence, the cells are trypsinized, counted, and seeded in a melanocyte / keratinocyte ratio 1/2 at a density of 10 5 per cm2
sur des filtres en polycarbonate. Les milieux sont changés tous les deux-trois jours. Après avoir atteint la confluence, les cultures sont hissées à l'interface air-liquide sur des grilles en inox ou des pastilles en fibre de verre. Le milieu de culture n'arrive alors plus que par le bas à travers le filtre jusqu'à la couche basale des cultures. Après 14 jours de culture ainsi émergée, certains épidermes reconstruits sont fixées dans une solution de formol, puis inclus dans des blocs paraffine, d'autres sont congelés dans l'azote liquide et stockés à -80°C, d'autres sont inclus dans des blocs de résine pour la microscopie électronique, d'autres encore sont homogénéisés et traités pour la mesure biochimique des activités tyrosinase et de la mélanine. Sur des coupes paraffines la coloration Fontanaon polycarbonate filters. The media are changed every two to three days. After reaching confluence, the cultures are hoisted at the air-liquid interface on stainless steel grids or fiberglass pellets. The culture medium then only reaches the bottom through the filter to the basal layer of the cultures. After 14 days of culture thus emerged, some reconstructed epidermis are fixed in a formalin solution, then included in paraffin blocks, others are frozen in liquid nitrogen and stored at -80 ° C, others are included in resin blocks for electron microscopy, others are homogenized and processed for the biochemical measurement of tyrosinase and melanin activities. Fontana coloring on paraffin cups
Masson permet de distinguer les différentes couches cellulaires formées et de visualiser et de compter (par analyse d'image assistée par ordinateur) les grains de mélanine présents. Dans les cultures non stimulées, les melanocytes contenant des grains de mélanine se situent surtout dans la première couche cellulaire, comme in vivo, alors que les keratinocytes n'en contiennent pas. La stimulation par irradiation aux UV-B (à partir du jour 10, 4 fois lOOmJoules/jour) induit une distribution beaucoup plus homogène des grains de mélanine, puisque pratiquement toutes les cellules des deux trois premières couches en contiennent. L'analyse d'image sur plusieurs coupesMasson makes it possible to distinguish the different cellular layers formed and to visualize and count (by computer-assisted image analysis) the melanin grains present. In unstimulated cultures, melanocytes containing melanin grains are mainly located in the first cell layer, as in vivo, while keratinocytes do not contain them. The stimulation by UV-B irradiation (from day 10, 4 times lOOmJoules / day) induces a much more homogeneous distribution of the melanin grains, since practically all the cells of the first two or three layers contain it. Image analysis on several sections
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FEUILLE DE REMPLACEMENT parallèles montre une augmentation de la quantité de mélanine présente de l'ordre de 45 fois le niveau contrôle. L'application topique préalable d'une goutte de 20_1 de crème solaire à filtre UV total avant chaque irradiation empêche à 89% (+/- 5%) cette mélanogénèse induite par les UV-B. L'irradiation aux UV-A (4 fois 2.5 Joules/jour) en présence et absence de psoralène (10~ M de 8-MOP)dans le milieu de culture produit une plus faible réaction de bronzage: 5 fois plus de grains de mélanine, mais en .plus le nombre de melanocytes chargés de mélanine triple dans la couche cellulaire basale. Les cultures congelées en parallèle ont permis de marquer spécifiquement et de compter à l'aide d'anticorps antimélanosome (Tomita et coll.,1991) par immunofluorescence indirecte, les melanocytes présents selon les conditions expérimentales. Les homogénats en milieu tamponné ont permis la quantification biochimique de la mélanine totale présente dans chacune des cultures: la même augmentation d'un facteur 45 (+/-6) a été mesurée après irradiation aux UV-B. L'activité tyrosinase (Halaban et coll., 1991) est augmentée d'un facteur trois après la première irradiation aux UV-B et reste à ce niveau même après 4 irradiations.REPLACEMENT SHEET parallels shows an increase in the amount of melanin present in the order of 45 times the control level. The topical prior application of a drop of 20_1 of sunscreen with total UV filter before each irradiation prevents 89% (+/- 5%) of this melanogenesis induced by UV-B. The UV-A irradiation (4 times 2.5 Joules / day) in the presence and absence of psoralen (10 ~ M of 8-MOP) in the culture medium produces a weaker tanning reaction: 5 times more melanin grains , but in . plus the number of melanocytes loaded with triple melanin in the basal cell layer. The cultures frozen in parallel made it possible to specifically mark and count with the aid of antimelanosome antibodies (Tomita et al., 1991) by indirect immunofluorescence, the melanocytes present according to the experimental conditions. The homogenates in a buffered medium allowed the biochemical quantification of the total melanin present in each of the cultures: the same increase by a factor of 45 (+/- 6) was measured after irradiation with UV-B. The tyrosinase activity (Halaban et al., 1991) is increased by a factor of three after the first UV-B irradiation and remains at this level even after 4 irradiations.
Ces résultats sont bien entendu relatifs aux cellules utilisées (donneur féminin de 36 ans, type caucasien) et ne sont décrits ici qu'à titre d'exemple. Des variations et différences dues aux systèmes de culture cellulaire utilisés et aux sources de cellules sont évidentes pour l'homme de 1'art.These results are of course relative to the cells used (36-year-old female donor, Caucasian type) and are described here only by way of example. Variations and differences due to the cell culture systems used and the cell sources are obvious to those skilled in the art.
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FEUILLE DE REMPLACEMENT BIBLIOGRAPHIE:REPLACEMENT SHEET BIBLIOGRAPHY:
Asselineau D, Beimard BA, Bailly C, Darmon M. Epidermal morphogenesis and induction of the 67Kd keratin polypeptide by culture of human keratinocytes et the liquid-air interface. Exp.Cell Res. 159:536-539; 1985Asselineau D, Beimard BA, Bailly C, Darmon M. Epidermal morphogenesis and induction of the 67Kd keratin polypeptide by culture of human keratinocytes and the liquid-air interface. Exp.Cell Res. 159: 536-539; 1985
Asselineau D, Bernard BA, Bailly C, Darmon M, Pruniéras M.Asselineau D, Bernard BA, Bailly C, Darmon M, Pruniéras M.
Human epider is reconstructed by culture: is it "normal"? J.Invest.Dermatol. 86:181-186; 1986Human epider is reconstructed by culture: is it "normal"? J.Invest.Dermatol. 86: 181-186; 1986
Asselineau D, Bernard BA, Bailly C, Darmon M. Retinoic acid improves epidermal morphogenesis. Developmental Biology 133:322-325; 1989Asselineau D, Bernard BA, Bailly C, Darmon M. Retinoic acid improves epidermal morphogenesis. Developmental Biology 133: 322-325; 1989
Bell E, Sher S, Hull B, Merrill C, Rosen S, Chamson A, Asselineau D, Dubertret L, Coulomb B, Lapierre C, Nusgens B, Neveux Y. The reconstitution of living skin. J.Invest.Dermatol.81:2s-10s; 1983Bell E, Sher S, Hull B, Merrill C, Rosen S, Chamson A, Asselineau D, Dubertret L, Coulomb B, Lapierre C, Nusgens B, Neveux Y. The reconstitution of living skin. J.Invest.Dermatol.81: 2s-10s; 1983
Bernstam LI, Vaughan FL, Bernstein IA. Stratified cornified primary cultures of human keratinocytes grown on microporous membranes at the air-liquid interface. J. Dermatol. Sci. 1:173-181; 1990Bernstam LI, Vaughan FL, Bernstein IA. Stratified cornified primary cultures of human keratinocytes grown on microporous membranes at the air-liquid interface. J. Dermatol. Sci. 1: 173-181; 1990
Bertaux B, Morlière P, Moreno G, Courtalon A, Masse JM, Dubertret L. Growth of melanocytes in a skin équivalent model in vitro. Brit. J. Dermatol. 119:503-512; 1988Bertaux B, Morlière P, Moreno G, Courtalon A, Masse JM, Dubertret L. Growth of melanocytes in a skin equivalent model in vitro. Brit. J. Dermatol. 119: 503-512; 1988
Boyce ST, Ham RG. Calcium regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free sériai culture. J.Invest. Dermatol. 81:33s-40s; 1983Boyce ST, Ham RG. Calcium regulated differentiation of normal human epidermal keratinocytes in chemically defined clonal culture and serum-free serial culture. J. Invest. Dermatol. 81: 33s-40s; 1983
Chardon A, Cretois I, Hourseau C. Skin colour typology and suntanning pathways. Int. J. Cosmetic Sci. 13:191-208; 1991Chardon A, Cretois I, Hourseau C. Skin color typology and suntanning pathways. Int. J. Cosmetic Sci. 13: 191-208; 1991
Coulomb B, Saiag P, Bell E, Breitburd F, Lebreton C, Heslan M, Dubertret L. A new method for studying epidermalization in vitro. Brit. J. Dermatol. 114:91-101; 1986Coulomb B, Saiag P, Bell E, Breitburd F, Lebreton C, Heslan M, Dubertret L. A new method for studying epidermalization in vitro. Brit. J. Dermatol. 114: 91-101; 1986
Coulomb B, Lebreton C, Dubertret L. Influence of human dermal fibroblasts on epidermalization. J. Invest. Dermatol. 92:122-125; 1989Coulomb B, Lebreton C, Dubertret L. Influence of human dermal fibroblasts on epidermalization. J. Invest. Dermatol. 92: 122-125; 1989
De Luca M, D'Anna F, Bondanza S, Tito Franzi A, Cancedda R.De Luca M, D'Anna F, Bondanza S, Tito Franzi A, Cancedda R.
Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. J.Cell Biol. 107:1919-1926; 1988Human epithelial cells induce human melanocyte growth in vitro but only skin keratinocytes regulate its proper differentiation in the absence of dermis. J. Cell Biol. 107: 1919-1926; 1988
Haake AR, Scott GA. Physiologie distribution and differentiation of melanocytes in human fetal and neonatal skin équivalents. J. Invest. Dermatol. 96:71-77; 1991Haake AR, Scott GA. Physiology distribution and differentiation of melanocytes in human fetal and neonatal skin equivalents. J. Invest. Dermatol. 96: 71-77; 1991
Halaban R, Pσmerantz SH, Marshall S, Lambert DT, Lerner AB.Halaban R, Pσmerantz SH, Marshall S, Lambert DT, Lerner AB.
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- 16 -- 16 -
FEUILLE DE REMPLACEMENT Fusenig NE, Breit reutz D, Dzarlieva RT et al. Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo. J.Invest.Dermatol. 81:168s-175s; 1983REPLACEMENT SHEET Fusenig NE, Breit reutz D, Dzarlieva RT et al. Growth and differentiation characteristics of transformed keratinocytes from mouse and human skin in vitro and in vivo. J.Invest.Dermatol. 81: 168s-175s; 1983
Lenoir MC, Bernard BA, Pautrat G, Darmon M, Shroot B. Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro. Developmental Biology 130:610-620; 1988Lenoir MC, Bernard BA, Pautrat G, Darmon M, Shroot B. Outer root sheath cells of human hair follicle are able to regenerate a fully differentiated epidermis in vitro. Developmental Biology 130: 610-620; 1988
Libow LF, Scheîde S, DeLeo VA. Ultraviolet radiation acts as an independent mitogen for normal human melanocytes in culture. Pigment Cell Res. 1:397-401; 1988Libow LF, Scheîde S, DeLeo VA. Ultraviolet radiation acts as an independent mitogen for normal human melanocytes in culture. Pigment Cell Res. 1: 397-401; 1988
Lillie JH, MacCallum DK, Jepsen A. Fine Structure of stratified squamous epithelium subcultivated on collagen rafts. Exp.Cell Res. 125:153-165; 1980Lillie JH, MacCallum DK, Jepsen A. Fine Structure of stratified squamous epithelium subcultivated on collagen rafts. Exp.Cell Res. 125: 153-165; 1980
Lillie JH, MacCallum DK, Jepsen A. Growth of stratified squamous epithelium on reconstituted extracellular matrices: long-term culture. J.Invest.Dermatol. 90:100-109; 1988Lillie JH, MacCallum DK, Jepsen A. Growth of stratified squamous epithelium on reconstituted extracellular matrices: long-term culture. J.Invest.Dermatol. 90: 100-109; 1988
Pittelkow MR, Shipley GD. Serum-free culture of normal human melanocytes: growth kinetics and growth factor requirements. J. Cell. Physiol. 140:565-576; 1989Pittelkow MR, Shipley GD. Serum-free culture of normal human melanocytes: growth kinetics and growth factor requirements. J. Cell. Physiol. 140: 565-576; 1989
Pruniéras M, Régnier M, Fougère S, Woodley D. Keratinocytes synthesize basal-lamina proteins in culture. J.Invest.Dermatol. 81:74s-81s; 1983aPruniéras M, Régnier M, Fougère S, Woodley D. Keratinocytes synthesize basal-lamina proteins in culture. J.Invest.Dermatol. 81: 74s-81s; 1983a
Pruniéras M, Régnier M, Woodley D. Methods for cultivation of keratinocytes with an air-liquid interface. J. Invest.Dermatol. 81:28s-33s; 1983bPruniéras M, Régnier M, Woodley D. Methods for cultivation of keratinocytes with an air-liquid interface. J. Invest Dermatol. 81: 28s-33s; 1983b
Régnier M, Pruniéras M, Woodley D. Growth and differentiation of adult human epidermal cells on dermal substrates. Front Matrix Biol.9:4-35; 1981Régnier M, Pruniéras M, Woodley D. Growth and differentiation of adult human epidermal cells on dermal substrates. Front Matrix Biol. 9: 4-35; nineteen eighty one
Régnier M, Darmon M. Human epidermis reconstructed in vitro: a model to study keratinocyte differentiation and its modulation by retinoic acid. In Vitro Cell & Dev Biology 25:1000-1008; 1989Régnier M, Darmon M. Human epidermis reconstructed in vitro: a model to study keratinocyte differentiation and its modulation by retinoic acid. In Vitro Cell & Dev Biology 25: 1000-1008; 1989
Rheinwald JG, Green H. Sériai cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6:331-344; 1975Rheinwald JG, Green H. Sériai cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331-344; 1975
Rosdy M, Clauss L-C. Terminal epidermal differentiation of human keratinocytes grown in chemically defined médium on inert filter substrates at the air-liquid interface. J. Invest. Dermatol. 95:409-414; 1990Rosdy M, Clauss L-C. Terminal epidermal differentiation of human keratinocytes grown in chemically defined médium on inert filter substrates at the air-liquid interface. J. Invest. Dermatol. 95: 409-414; 1990
Scott GA, Haake AR. Keratinocytes regulate melanocyte number in human fetal and neonatal skin équivalents. J. Invest. Dermatol. 97: 776-781; 1991Scott GA, Haake AR. Keratinocytes regulate melanocyte number in human fetal and neonatal skin equivalents. J. Invest. Dermatol. 97: 776-781; 1991
- 17 - FEUILLE DE REMPLACEMENT Tomita Y, Shibahara S, Takeda A, Okinaga S, Matsunaga J, Tagami H. The monoclonal antibodies TMH-1 and TMH-2 specifically bind to a protein encoded at the murine b-locus, not to the authentic tyrosinase encoded at the c-locus. J. Invest. Dermatol. 96:500-504; 1991- 17 - REPLACEMENT SHEET Tomita Y, Shibahara S, Takeda A, Okinaga S, Matsunaga J, Tagami H. The monoclonal antibodies TMH-1 and TMH-2 specifically bind to a protein encoded at the murine b-locus, not to the authentic tyrosinase encoded at the c -locus. J. Invest. Dermatol. 96: 500-504; 1991
Topol BM, Haimes HB, Dubertret L, Bell E. Transfer of melanosomes in a skin équivalent model in vitro. J. Invest. Dermatol. 87:642-647; 1986Topol BM, Haimes HB, Dubertret L, Bell E. Transfer of melanosomes in a skin equivalent model in vitro. J. Invest. Dermatol. 87: 642-647; 1986
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Vaughan FL, Gray RH, Bernstein IA. Growth and differentiation of primary rat keratinocytes on synthetic membranes. In Vitro Cell.& Develop.Biology 22:141-149; 1986Vaughan FL, Gray RH, Bernstein IA. Growth and differentiation of primary rat keratinocytes on synthetic membranes. In Vitro Cell. & Develop.Biology 22: 141-149; 1986
Yaar M, Grossman K, Eller M, Gilchrest BA. Evidence for nerve growth factor-mediated paracrine effects in human epidermis. J. Cell Biol. 115:821-828; 1991Yaar M, Grossman K, Eller M, Gilchrest BA. Evidence for nerve growth factor-mediated paracrine effects in human epidermis. J. Cell Biol. 115: 821-828; 1991
- 18 - FEUILLEDE REMPLACEMENT - 18 - REPLACEMENT SHEET

Claims

REVENDICATIONS:CLAIMS:
1- Test de bronzage caractérisé par le fait qu'on utilise des épidermes reconstitués humains par culture cellulaire qui bronzent sous l'action d'une stimulation physique et/ou chimique.1- Tanning test characterized by the fact that human skin reconstituted by cell culture is used which tans under the action of physical and / or chemical stimulation.
2- Epidermes humains reconstitués par culture cellulaire en milieu défini et exposés à l'interface air/milieu caractérisés par le fait que l'irradiation par des rayons ultraviolets et/ou la présence d'un produit chimique spécifique dans le milieu de culture stimule la production de grains de mélanine par les melanocytes présents, ainsi que leur transfert dans les keratinocytes.2- Human epidermis reconstituted by cell culture in a defined medium and exposed to the air / medium interface, characterized by the fact that irradiation with ultraviolet rays and / or the presence of a specific chemical in the culture medium stimulates the production of melanin grains by the melanocytes present, as well as their transfer into the keratinocytes.
3- Epidermes humains reconstitués par culture cellulaire en milieu défini et exposés à l'interface air/milieu selon la revendication 2, caractérisés par le fait qu'on ensemence un mélange précis de keratinocytes humains adultes normaux et de melanocytes humains adultes normaux cultivés auparavant séparément en immersion en milieu défini.3- Human epidermis reconstituted by cell culture in defined medium and exposed to the air / medium interface according to claim 2, characterized in that a precise mixture of normal adult human keratinocytes and normal adult human melanocytes previously cultivated is inoculated immersion in a defined environment.
4- Test de bronzage selon la revendication 1 caractérisé par le fait qu'on applique sur l'épiderme reconstitué décrit dans la revendication 3 le ou les produits à tester.4- Tanning test according to claim 1 characterized in that the product or products to be tested are applied to the reconstituted epidermis described in claim 3.
5- Test de bronzage selon la revendication 1 caractérisé par le fait qu'on ajoute dans le milieu de culture sousjacent au substrat de culture des épidermes reconstitués décrit dans5- Tanning test according to claim 1 characterized in that added in the culture medium underlying the culture substrate reconstituted epidermis described in
- 19 - FEUILLEDE REMPLACEMENT la revendication 3, le ou les produits à tester.- 19 - REPLACEMENT SHEET claim 3, the product or products to be tested.
6- Test de bronzage selon la revendication 1 caractérisé par le fait que le bronzage des épidermes reconstitués est mesuré par quantification des grains de mélanine sur une ou plusieurs coupes verticales colorées par analyse dimage assistée par ordinateur.6- Tanning test according to claim 1 characterized in that the tanning of the reconstructed epidermis is measured by quantification of the melanin grains on one or more colored vertical sections by computer-assisted image analysis.
7- Test de bronzage selon la revendication 1 caractérisé par le fait que le bronzage des épidermes reconstitués est mesuré par dosage biochimique de la mélanine et/ou de l'activité tyrosinase.7- Tanning test according to claim 1 characterized in that the tanning of the reconstructed epidermis is measured by biochemical determination of melanin and / or tyrosinase activity.
8- Test de bronzage selon la revendication 1 caractérisé par le fait que le bronzage des épidermes reconstitués est mesuré par marquage par immunofluorescence indirecte à l'aide d'anticorps reconnaissant des antigènes jouant un rôle dans la mélanogénèse et/ou le transfert des mélanosomes.8- Tanning test according to claim 1 characterized in that the tanning of the reconstituted epidermis is measured by labeling by indirect immunofluorescence using antibodies recognizing antigens playing a role in melanogenesis and / or the transfer of melanosomes.
9- Test de bronzage selon la revendication 1 caractérisé par le fait que le bronzage des épidermes reconstitués est mesuré par microscopie électronique.9- Tanning test according to claim 1 characterized in that the tanning of the reconstructed epidermis is measured by electron microscopy.
10- Test de bronzage selon la revendication 1 caractérisé par le fait que le bronzage des épidermes reconstitués est évalué par la mesure instrumentale de leur couleur (colorimétrie) .10- Tanning test according to claim 1 characterized in that the tanning of the reconstructed epidermis is evaluated by the instrumental measurement of their color (colorimetry).
- 20 - FEUILLE DE REMPLACEMENT - 20 - REPLACEMENT SHEET
PCT/FR1993/000997 1993-10-08 1993-10-08 In vitro human epidermal sun-tanning test WO1995010600A1 (en)

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FR2738914A1 (en) * 1995-09-20 1997-03-21 Biopredic Bio-assay for testing photo-toxicity
FR2792728A1 (en) * 1999-04-20 2000-10-27 Oreal Evaluating effects of compounds on epidermal lipogenesis, useful e.g. for testing skin care products, uses in vitro skin equivalent
FR2823217A1 (en) * 2001-04-09 2002-10-11 Oreal Producing suspension of individual cells from skin equivalent, useful for testing genotoxicity, comprises incubation with mixture of collagenases and protease
EP2103687A1 (en) 2008-03-17 2009-09-23 L'Oréal Functional pigmented skin equivalent
US8222031B2 (en) 2000-05-31 2012-07-17 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Three-dimensional skin model
EP4299719A1 (en) 2022-06-28 2024-01-03 Univerza v Mariboru A complex in vitro model of human skin, a process for preparation and use thereof

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Publication number Priority date Publication date Assignee Title
FR2738914A1 (en) * 1995-09-20 1997-03-21 Biopredic Bio-assay for testing photo-toxicity
FR2792728A1 (en) * 1999-04-20 2000-10-27 Oreal Evaluating effects of compounds on epidermal lipogenesis, useful e.g. for testing skin care products, uses in vitro skin equivalent
US8222031B2 (en) 2000-05-31 2012-07-17 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Three-dimensional skin model
FR2823217A1 (en) * 2001-04-09 2002-10-11 Oreal Producing suspension of individual cells from skin equivalent, useful for testing genotoxicity, comprises incubation with mixture of collagenases and protease
EP2103687A1 (en) 2008-03-17 2009-09-23 L'Oréal Functional pigmented skin equivalent
EP4299719A1 (en) 2022-06-28 2024-01-03 Univerza v Mariboru A complex in vitro model of human skin, a process for preparation and use thereof
LU502391B1 (en) 2022-06-28 2024-01-09 Univerza V Mariboru A complex in vitro model of human skin, a process for preparation and use thereof

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