FR2665175A1 - Epidermal equivalent, process for obtaining it, and its use - Google Patents

Abstract

Process for obtaining an epidermal equivalent in a defined medium, the corresponding epidermal equivalent, and its use for pharmaceutical and cosmetics tests. According to the process, cells isolated from a sample of human epidermis (predominantly keratinocytes) are cultured in a chemically defined medium: immediately after their isolation, or else after at least one passage of a conventional monolayer culture, the cells are deposited on substrates formed by chemically inert filters which are covered or otherwise with gels or molecules which promote cell attachment, and the cells are cultured in a defined medium until confluence. The cultures are then exposed to the air, the defined culture medium reaching the cells only through the substrate.

Description

STATE OF THE ART:
SKIN RECONSTITUTED IN VITRO
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. 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.

 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.

a) Normal keratinocyte cultures on a de-epidermis dermis
The culture system on de-epidermalized dermis has the advantage of keeping an intact basement membrane as attachment surface for keratinocytes while eliminating interference due to fibroblasts of the dermis, since the latter are "killed" by successive freezing (Régnier et al. , 1981; Pruniéras et al., 1983). After exposure to air, the expression of keratin 67 kD in this system demonstrates that the presence of living fibroblasts is not necessary to induce terminal differentiation. This does not exclude an influence of the acellular components of the dermis: the filtering action of this substrate, ie the retention of molecules with inhibitory activity of the vitamin A type from the culture medium containing serum, is mentioned (Régnier et al., 1986). The terminal differentiation of normal and transformed human keratinocytes in this system has been described in more detail (Régnier et al., 1988; 1989), however these results cannot be attributed to a stimulating action of dermal substrate differentiation, since the undifferentiated control cultures were conventional submerged cultures: indeed, it is possible that exposure to air allows complete epidermal differentiation; the real control to estimate the influence of the substrate should therefore have been a culture on an inert substrate, but also exposed to air.

b) Normal keratinocyte cultures on a dermis equivalent: "the Bell'1 system
The dermis equivalent is prepared by including living fibroblasts in a collagen matrix which is modified and contracted by these resident cells (Bell et al., 1979). Normal rat or human keratinocytes are cultured on the surface of these substrates which are then exposed to the air by means of stainless steel grids. These reconstituted skins were successfully grafted onto rats (Bell et al., 1983). These reconstructed skins, in the presence of 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., 1986a).

Recently the system has been improved by adjusting the concentration of retinoic acid to 10 'M, thanks to the use of a delipidized serum: the expression of the different markers then strongly resembles the normal situation in vivo = orthokeratosis (Asselineau et al. ., 1989).

 The 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 agreement with the current observation that the hair roots which remain in a wound after a trauma are capable of regenerating a normally differentiated interfollicular epidermis. Other researchers cultivate keratinocytes on these same Bell lattices, but by incorporating a biopsy of human skin (Coulomb et al., 1986; 1989).

c) Other systems
They consist of inoculating mouse or rat epidermal cells on collagen films or gels, nylon or nitrocellulose filters covered with collagen or equivalent of the basement membrane deposited by endothelial cells from the cornea of the ox, then from expose these crops to air for 14 to 21, or even 65 days (Fusenig et al., 1983; Bernstam et al., 1986; Vaughan et al., 1986; Lillie et colt., 1980; 1988).

One 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 these experiments were carried out in the presence of serum in the culture medium, considered necessary for growth and complete differentiation. However, to be able to significantly assess if and how the components of the dermis influence epidermal proliferation and differentiation, the necessary culture system comprises a substrate: chemically inert without fibroblasts or collagen; Similarly, to determine the influence of serum factors (which, in vivo, migrate through the basement membrane), the use of a chemically defined culture medium becomes an essential research tool. In such minimal culture conditions ( neither dermal nor serum factors), obtaining a viable and moreover well-differentiated epidermis was considered improbable, the keratinocytes being assumed not to be self-sufficient, nor for the synthesis of a basement membrane (Bohnert et al., 1986), nor for their complete terminal differentiation (Fusenig et al., 1983;
Lillie et al., 1980; 1988).

Most of the reconstituted skin systems have been developed either to produce whole skin grafts or to establish models of complete terminal epidermal differentiation. Naturally, the dermis had been presented as an ideal in vitro substrate (Pruniéras et al., 1983). Dermal or pseudo-dermal matrices also made it possible to easily expose epidermal cultures to the air (Bell et al., 1983; Régnier et al., 1984). The culture medium arrived through the permeable substrate. From all these experiments, the researchers believed they could deduce the need for a mesenchymal substrate and serum to be able to obtain complete epidermal differentiation in vitro (Bernfield & Banerjee, 1982;
Holbrook & Hennings, 1983; Bohnert et al., 1986).

THE INVENTION:
Complete terminal epidermal differentiation in culture in the absence of dermal and serum factors
Terminal epidermal differentiation of human keratinocytes cultured in a chemically defined medium on substrates constituted by inert filters at the air-liquid interface
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 for 14 days. Vertical sections doubly stained 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. Under our environmental conditions, the use of de-epidermalized dermes as a substrate did not make it possible to obtain a differentiation of the keratinocytes better than that obtained on filters of cellulose acetate or polycarbonate.

 Obtaining a well differentiated epidermis in vitro, cultivated under simple and defined conditions, should allow the development of a standard system for.

the study of differentiation, reepidermization, keratinocyte cytotoxicity, epidermal permeation as well as transepidermal drug passage.

 The cultures described here clearly show that normal human keratinocytes are capable of producing a completely differentiated epidermis in the total absence of mesenchymal factors. In addition, the absence of basement membrane on the substrate has no influence on the quality of expression of the various markers of the basement membrane.

 The fact that such cultures are feasible does not make it possible to deduce that the human epidermis needs neither plasma factors, nor dermal supply to live and organize. However, the influence of such factors, active at very low concentrations (pM or nM), can only be clearly determined using the use of such a chemically defined system.

 (1) To elucidate the mechanisms of regulation of epidermal differentiation, (2) to develop an experimental model of the epidermis, usable for tests of cytotoxicity, cytocompatibility and (3) permeability to pharmaceutical or cosmetic products, it seems It is clear that a defined and "minimum" system of reconstructed skin capable of evolving towards terminal differentiation is very useful if not essential. In addition, this system may help understand the regulation of human epidermal differentiation and homeostasis.

 In addition, eliminating the serum makes it possible to design and interpret experiments that are difficult or impossible to carry out in its presence. In a defined medium it is possible to study the effect of hormones or pharmacological or cosmetic agents in the absence of unidentified components which can bind, inactivate, antagonize or mimic the action of the agent studied. Likewise, experiments concerning the release of cellular products, such as the factors secreted by differentiated cell lines, are much easier to carry out and to interpret in the absence of serum proteins.

METHOD 1: Methods of culturing epidermal cells
in defined medium on inert filters
Cell suspensions mainly containing normal human keratinocytes are obtained by the dermis / epidermis separation method in 0.25% of trypsin (Green et al., 1979) from samples of human skin. The optional preliminary primary cultures can be carried out according to the Green method (Rheinwald & Green, 1975, 1977), or else directly in a defined medium, such as MCDB 153 (Boyce, 1983; Tsao, 1982) for example.

Before reaching confluence, the cells of these cultures are harvested using gentle trypsinization and counted. Several subculture cycles can thus be carried out in order to increase the total number of cells available for the final culture which is exposed to air.

From a cell suspension obtained directly from a skin sample, or from subcultures, the selected substrates are sown at a cell density varying from 104 to 106 cells per cm2. The substrates used can be conventional filters made of cellulose acetate (Millipore PIHA), polycarbonate (Nucleopore Nucell or Costar Transwell), nylon, silicone or natural polymers such as collagen, elastin, agarose, etc. These substrates can be covered, before sowing by the cells, with solutions comprising molecules or factors promoting or not promoting cell attachment and / or growth. The substrate used must above all be permeable to the nutritive molecules of the defined medium, so that 'they can cross it to reach the cells of the first cell layer when exposed to crop air.

The inoculum is done in defined medium (DMEM, Ham F-12,
MCDB 153, other synthesis media) capable of promoting cell growth over differentiation. When the cultures reach confluence (after 1 to 5 days, depending on the density of the inoculum and the proliferation of cells), their respective substrate is hoisted, using fine and sterile forceps, on a sterile support allowing the access of the culture medium to the entire underside of the substrate, so that the cultures are fed, from below and through the substrate, by the defined culture medium which is chosen to promote epidermal differentiation and to limit the proliferation of cells to that observed during the regeneration of a human epidermis in vivo.

Thus the medium defined MCDB 153 is then with a high concentration of calcium (approximately 1.2 mM), the medium F-12 of
Ham then no longer contains any growth factor, while the additives necessary to allow the correct formation of a compact horny layer are added to the chosen medium (lipids, steroids, vitamins). The cover of each culture dish must be placed in such a way as to prevent drops from the condensation of the culture medium from falling on the horny layers in formation, which would induce excessive hydration of the extracellular lipids, some of which are responsible for the relative impermeability of the epidermis (Madison, 1988).

After incubation in an unsaturated humidity atmosphere, between 40 and 95% relative humidity, preferably around 60%, for at least one week, with changes in the defined medium every three days, the cross sections of cultures fixed in paraffin or simply frozen reveal an architecture strongly resembling that of a human epidermis in vivo, with expression of all the classic epidermal differentiation markers, formation of a compact horny layer (at least ten cell layers), synthesis of typical lipids of the stratum corneum, but with the particularity of having a basal base following the contours of the substrate, that is to say rectilinear. see figures 1, 2 and 3.

METHOD 2: Analysis of the morphology and the compositions
lipid and protein of the epithelia formed
in culture: Cytocompatibility tests
The cultures described above are used to test the effects of exposure of a medical, pharmaceutical or cosmetic product to their surface on the being of the horny layers, of the overall architecture of the cultures and of the morphology of the cells. For this, classical histology and electron microscopy techniques are used (Rosdy, 1990a; Asselineau, 1989;
Madison, 1988; Régnier, 1988). Similarly for the preparation of protein or lipid extracts, conventional techniques are used (Rosdy, 1986, 1988; Asselineau, 1989; Ponec, 1989; Lampe, 1983). The proteins are then visualized by immunoelectrophoresis or simply by staining of gels electrophoresis, while the lipids typical of well-differentiated epidermis are detected by semi-quantitative two-dimensional thin layer chromatography (Brod, 1987; Ponec, 1989).

 The results are of course compared with those obtained with parallel control cultures (positive and negative) and at least two independent cultures are carried out per sample to be tested.

METHOD 3: Analysis of molecules released into the environment
defined by equivalent cells
epidermis: cytocompatibility tests
The fact of using a chemically defined medium to obtain the epidermis equivalents described above makes it possible to measure an additional parameter of great importance: the release of molecules (in small quantities) synthesized by the cells of the epidermis equivalent; indeed, the presence of serum in the culture systems of reconstructed skins prevented this type of parameter from being measured. Epidermal cells and more specifically keratinocytes synthesize in vivo and in vitro a wide range of molecules and factors having important biological regulatory functions (Akhurst, 1988; Bikle, 1986;
Coffey, 1987; Miyazaki, 1989; Nickoloff, 1989).

The analytical techniques used are very varied, because they closely depend on the type of molecules that we want to detect, or that we think we can detect: the presence (in the defined medium) of factors can be demonstrated by the techniques now classics of
HPLC, thin layer chromatography, rapid electrophoresis, immunoelectrophoresis (Westernblot), indirect immunofluorescence, by indirect tests of a known biological function of the molecule (growth test on fibroblast culture for example). These tests for the release of molecules by an epidermis equivalent are relatively complex to perform, but the information obtained on the mechanisms of cellular toxicity is very precious and interesting. Their obtaining is only possible thanks to the use of a medium. chemically defined for the duration of the cultures.

LEGEND OF THE FIGURES:
Figure 1: Culture of epidermal cells exposed to air
and in defined medium on acetate filter
cellulose (Millipore PIHA) (A) and on filter
polycarbonate (B) after 14 days incubation at
37 "C at 60 * humidity.

Figure 2: same, but in more detail. Note the
complete and normal epidermal differentiation.

Figure 3: Culture of epidermal cells exposed to air
and in defined medium on acetate filter
cellulose (Millipore PIHA) after 14 days of incubation at
37 "C at 60% humidity. Marking of cuts
verticals of frozen cultures using
monoclonal antibodies recognizing
essential markers of differentiation
epidermal: keratins K1 and K10 (A), filaggrin
(B), membrane transglutaminase (C), and
involucrine, main precursor of the envelope
cornea (D).

BIBLIOGRAPHY
Akhurst RJ, Fee F, Balmain A. Localized production of
TGF-beta mRNA in tumor promoter-stimulated mouse epidermis.

Nature 331: 363-365; 1988
Asselineau D, Bernard 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.

Human epidermis 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; 1989
Bell E, Ivarssen B, Merrill C. Production of a tissue-like structure by contraction 0f collagen lattices by human fibroblasts of different proliferative potential in vitro.

Proc.Natl.Acad.Sci.USA 76: 1274-1278; 1979
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-îOs; 1983
Bernfield M, Banerjee SD. The turnover of basal lamina glycosaminoglycan correlates with epithelial morphogenesis.

Dev. Biol. 90: 291-305; 1982
Bernstam LI, Vaughan FL, Bernstein lA. Keratinocytes grown at the air-liquid interface. In Vitro Cellular & BR <
Developmental Biology 22: 695-705; 1986
Bikle DD, Nemanic MK, Gee E, Elias P. 1,25-Dihydroxyvitamin
D3 production by human keratinocytes.

J. Clin. Invest. 78: 557-566; 1986
Bohnert A, Hornung J, Mackenzie IC, Fusenig NE. Epithelial -mesenchymal interactions control basement membrane production and differentiation in cultured and transplanted mouse keratinocytes. Cell Tissue Res. 244: 413-429; 1986
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
Brod J, Justine P, Bernaud F, Thevenet E. Quantitative determination of skin epidermal lipids using thin-layer chromatography with flame ionization detection. In:
Instrumental High Performance Thin-Layer Chromatography (Eds .: Traitler H, Kaiser RE), Inst. for Chromatography, Bad
Durkheim-FRG, pp. 93-100; 1987
Coffey RJ, Derynck R, Wilcox JN, Bringman TS, Goustin AS,
Moses H, Pittelkow MR. Production and auto-induction of transforming growth factor-alpha in human keratinocytes.

Nature 328: 817-820; 1987
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; 1986
Coulomb B, Lebreton C, Dubertret L. Influence of human dermal fibroblasts on epidermalization. J.Invest.Dermatol.

92: 122-125; 1989
Fusenig NE, Breitkreutz 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
Green H, Kehinde O, Thomas J. Growth of cultured human epidermal cells into multiple epithelia suitable for grafting. Proc.Natl.Acad.Sci.USA 76; 11: 5665-5668; 1979
Holbrook KA, Hennings H. Phenotypic expression of epidermal cells in vitro: a review. J.Invest.Dermatol. 81 (suppl): lls-24s; 1983 Lampe- MA, Williams ML, Elias PM. Human epidermal lipids: characterization and modulations during differentiation.

J. Lipid Res. 24: 131-140; 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; 1988
Lillie 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; 1988
Madison KC, Swartzendruber DC, Wertz PW, Downing DT.

Lamellar granule extrusion and stratum corneum intercellar lamellae in murine keratinocyte cultures. J.Invest.Dermatol.

90: 110-116; 1988
Miyazaki K, Horio T. Growth inhibitors: molecular diversity and roles in cell proliferation. In Vitro Cell.Dev.Biol.

25.10: 866-872; 1989
Nickoloff BJ, Mitra RS. Inhibition of 1251-epidermal growth factor binding to cultured keratinocytes by antiproliferative molecules gamma interferon, cyclosporin A, and transforming growth factor-beta. J.Invest.Dermatol.

93: 799-803; 1989
Ponec M, Weerheim A, Kempenaar J, Elias PM, Williams-ML.

Differentiation of cultured human keratinocytes: effect of culture conditions on lipid composition of normal vs.

clever ones. In Vitro Cell Dev. Biol. 25: 689-696; 1989
Pruneras M, Régnier M, Fougere S, Woodley D. Keratinocytes synthesize basal-lamina proteins in culture.

J.Invest.Dermatol. 81: 74s-81s; 1983a
Pruneras M, Regnier 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; nineteen eighty one
Régnier M, Pautrat G, Pauly G, Pruniéras M. Natural substrates for the reconstruction of skin in vitro.

Brit.J.Dermatol. 111, Suppl. 27: 223-224; 1984
Régnier M, Schweizer J, Michel S, Bailly C, Pruniéras M.

Expression of high molecular weight (67K) keratin in human keratinocytes cultured on dead de-epidermized dermis.

Exp. Cell Research 165: 63-72; 1986
Régnier M, Desbas C, Bailly C, Darmon M. Differentiation of normal and tumoral human keratinocytes cultured on dermis: reconstruction of either normal or tumoral architecture. In
Vitro Cell & Dev Biology 24: 625-632; 1988
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; 1989
Rheinwald JG, Green H. Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells. Cell 6: 331-344; 1975
Rheinwald JG, Green H. Epidermal growth factor and the multiplication of cultured human epidermal keratinocytes.

Nature 265: 421-424; 1977
Rosdy M, Bernard BA, Schmidt R, Darmon M. Incomplete epidermal differentiation of A431 epidermoid carcinoma cells. In Vitro Cellular & Developmental Biology 22: 295-300; 1986
Rosdy M. Opposite effects of EGF on involucrin accumulation of A431 keratinocytes and a variant which is not growth-arrested by EGF. In Vitro Cell.Develop.Biology 24: 1127-1132; 1988
Rosdy M, Clauss LC. Cytocompatibility testing of wound dressings using normal human keratinocytes in culture.

J. Biomed, Materials Research 24 (3): 363-377; 1990a
Rosdy M, Clauss LC. Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air-liquid interface.

J.Invest.Dermatol. 95 (2): xxx-xxx; August 1990b
Tsao MC, Walthall BJ, Ham RG. Clonal growth of normal human epidermal keratinocytes in a defined medium. J. Cellular
Physiology 110: 219-229; 1982
Vaughan FL, Gray RH, Bernstein IA. Growth and differentiation of primary rat keratinocytes on synthetic membranes. In, Vitro Cell. & Develop.Biology 22: 141-149; 1986

Claims (10)

 at least 5 days.  e) the cultures thus exposed to air are incubated  (without any unknown components), and  d) using chemically defined culture medium  lower only through the substrate,  the medium reaches the layer cells  the air / culture medium interface, so that  after the confluence, the substrate is raised to  cell attachment,  or not a deposit of molecules favoring  permeable and chemically inert substrate, covered  b) these human epidermal cells are seeded on a  human epidermis sample,  directly or after at least one subculture, of a  a) a suspension of cells is prepared,  in which: CLAIMS: 1- Process for obtaining an equivalent of human epidermis, 2- A method according to claim 1, characterized by  that we use a defined medium whose  calcium concentration is greater than 1.15 mM. 3- A method according to claim 1, characterized by  using a defined medium that does not contain  no peptide growth factor. 4- A method according to claim 1, characterized by  the fact that filters are used as substrate  cellulose acetate, polycarbonate, silicone,  nylon, collagen or agarose. 5- Method according to claim 1, characterized by the  placing cell cultures  epidermal in an incubator that has an atmosphere  controlled from 40 to 95% humidity. 6- A method according to claim 1, characterized by  placing cell cultures  epidermal in an incubator that has an atmosphere  controlled by 60% (+/- 5%) humidity. 7- Equivalent to epidermis, characterized by the fact that it is  obtained by the method according to one of claims 1 to  6. 8- Use of the epidermis equivalent according to the  claim 7 for cytotoxicity tests on  cellular culture. 9- Use of the skin equivalent according to the  claim 7 for testing the toxicity or quality of  pharmaceutical or cosmetic products, or one or  many of their ingredients. 10- Use of the epidermis equivalent according to the  claim 7 for measuring speed and / or quality  penetration through the epidermis of products  pharmaceutical or cosmetic, or one or more of  their ingredients.