The present invention relates to a model of reconstructed sebocyte epithelium.

Oily/dry skin disorders are often due to a malfunction of the pilosebaceous unit, including an abnormal production of sebum. Identifying solutions for these disorders entails designing biological models that are compatible with the screening of chemical or biological compounds, in particular in vitro models of sebocyte epithelium.

However, because of their in vivo function, primary sebocytes tend in vitro to differentiate rapidly through the accumulation of lipid droplets and finally degenerate, which renders the culture and the amplification thereof, although indispensable for obtaining a quantity of biological material that is sufficient for preparing models of reconstructed sebocyte epithelium, very difficult in vitro and therefore limits the use thereof in the framework of the screening of agents.

The most commonly used technique in literature to circumvent this problem of sebocyte differentiation is the immortalization thereof or the generation of cell lines. As such, the cell line SZ95 was developed in Zouboulis et at. (1999) J. Investig. Dermatol. 113:101 1 -1020, the cell line SEB-1 was developed in Thiboutot et al. (2003) J. Investig. Dermatol. 120:905-914 and the cell line SEB0662 was developed in Barrault et al. (2012) Exp. Dermatol. 21 :314-316. These are the cell lines that have been used until now for producing cell models.

However, immortalized sebocytes are genetically modified cells, which can turn out to be physiologically rather far from primary sebocytes. For example, such immortalized sebocytes partially lost their potential for differentiation.

Moreover, most of the work of modeling the sebaceous function has been carried out until now using cell models cultivated in 2 dimensions, in particular using immortalized cell lines SZ95 and SEB-1 . Currently, very few studies have shown the production of cell models in 3 dimensions. Mention can however be made of the article Guy et al. (1996) J. Investig. Dermatol. 106:454-460 which describes models of survival glands, the article Yoshida et al. (2013) Biochem. Biophys. Res. Com. 438:640-646 which describes micro- tissues manufactured with immortalized sebocytes and the articles Barrault et al. (2012) Exp. Dermatol. 21 :314-316 and Lo Celso et al. (2008) Stem Cells 26:1241 -1252 which describe reconstructed epitheliums from immortalized sebocytes. However, these three types of models have substantial disadvantages. Indeed, in the survival gland model, the preparation of models is highly limited by the supply of survival glands and the models obtained have a large intra- and inter-donor variability. For the two other types of models, the use of cell lines and therefore of genetically modified cells is not very physiological.

There is therefore a substantial need for new methods for preparing in vitro models of reconstructed sebocyte epithelium, implementing cells that are as close as possible to cells of the sebaceous gland in vivo, while still being available in a sufficient quantity and which makes it possible to produce a 3D model with differentiated sebocytes loaded with non-polar lipids of which the lipid metabolism is as close as possible to that of the sebaceous gland in vivo.

There is also a need to obtain an in vitro model of reconstructed sebocyte epithelium of which the lifetime is sufficient for studying over time the modulation of its lipid synthesis, and therefore for screening new molecules.

The present invention results from the combination by the inventors of innovative technical solutions:

- the amplification of non-immortalized primary sebocytes by culture in a specific culture medium comprising 7 factors allowing for the growth of sebocytes and a ROCK inhibitor, in such a way as to obtain a sufficient stock of sebocytes without requiring the immortalization thereof, which makes it possible to retain their potential for differentiation, - the culture of a model of reconstructed sebocyte epithelium in 3 dimensions on an extracellular matrix support with a culture phase at the air-liquid interface, and the adding of an inducing mixture in order to optimize the differentiation in mature sebocytes loaded with non-polar lipids, this mixture comprising linoleic acid as a precursor and a PPAR agonist, in a culture medium comprising only 3 factors allowing for the growth and/or the differentiation of the sebocytes, of which EGF at a high concentration, which makes it possible to push the sebocytes to differentiate themselves and produce a mixture of non-polar lipids approaching the composition of the sebum and obtaining a model that can be cultivated up to 17 days, allowing for a usage window that is longer than the models of the state of the art. The present invention therefore has for object a method, preferably in vitro, for amplifying primary sebocytes, said method comprising a step of culture, in the presence of feeder cells, of primary sebocytes, preferably human non-immortalized primary sebocytes, in a "G7F-sebo2D" culture medium comprising:

(i) adenine hydrochloride,

(ii) epidermal growth factor (EGF),

(iii) hydrocortisone, (iv) insulin,

(v) isoproterenol,

(vi) transferrin, and

(vii) triiodothyronine,

said culture medium further comprising a ROCK inhibitor.

Another object of the invention relates to a method, preferably in vitro, for preparing a model of reconstructed sebocyte epithelium, said method comprising the steps consisting in:

a) seeding, on a biomaterial for reconstructed epidermis (BPER) optionally covered with extracellular matrix protein, non-immortalized primary sebocytes in suspension in a "G7F-ERS" culture medium comprising:

(i) adenine hydrochloride,

(ii) epidermal growth factor (EGF),

(iii) hydrocortisone,

(iv) insulin,

(v) isoproterenol,

(vi) transferrin, and

(vii) triiodothyronine;

b) cultivating the sebocytes seeded in step a) in immersion on said BPER in G7F- ERS medium for 1 to 4 days;

c) cultivating the sebocytes obtained in step b) at the air - liquid interface on said BPER above a "G3F-ERS" culture medium comprising:

(I) epidermal growth factor (EGF),

(II) hydrocortisone, and

(III) insulin,

said G3F-ERS medium further comprising linoleic acid and a PPAR agonist, for a period that is suitable for allowing the differentiation of the sebocytes and lipid synthesis,

said model of reconstructed sebocyte epithelium being obtained at the end of step c).

The present invention also concerns a model of reconstructed sebocyte epithelium likely to be obtained by the preparation method according to the invention.

The present invention finally has for object a method, preferably in vitro, for screening a candidate compound for treating oily and/or glistening skin, comprising the putting into contact of said candidate compound with the model of reconstructed sebocyte epithelium according to the invention. Detailed description of the invention

Sebocytes

Preferably, the sebocytes used and/or amplified in the context of the invention are non-immortalized primary sebocytes.

The term "non-immortalized primary sebocytes" here means sebocytes that have not been immortalized, regardless of the immortalization technique used, or that do not come from cell lines of immortalized sebocytes. In particular, the sebocytes used and/or amplified in the context of the invention are not sebocytes of the cell line SZ95 (described in Zouboulis et al. (1999) J. Investig. Dermatol. 113:101 1 -1020), or sebocytes of the cell line SEB-1 (described in Thiboutot et al. (2003) J. Investig. Dermatol. 120:905-914), or sebocytes of the cell line SEB0662 (described in Barrault et al. (2012) Exp. Dermatol. 21 :314-316).

Using non-immortalized primary sebocytes is particularly advantageous as it makes it possible retain the potential for differentiation of the sebocytes, and in particular their capacity to synthesize a sebum equivalent in vitro that has a composition close to that of sebum in vivo, including in particular most of the major families of non-polar lipids present in the sebum in vivo.

The primary sebocytes used in the context of the invention are preferably human sebocytes.

These primary sebocytes can be obtained by any technique well known to those skilled in the art. The inventors have however developed a particular technique for obtaining these sebocytes from sebaceous glands coming preferably from surgical waste.

As such in a particular embodiment, the primary sebocytes used in the context of the invention are primary sebocytes, preferably non-immortalized primary human sebocytes, coming from sebaceous glands coming for example from surgical waste, such as lifting waste.

Preferably, the primary sebocytes used in the context of the invention are obtained by a method of extraction comprising the following steps:

1 ) recovering a piece of skin, such as a piece of lifting, and optionally removing the hair from the surface of the skin,

2) optionally cutting the piece of skin into pieces of about 0.5 cm ² in surface area, and treating them with antibiotics, for example in a culture medium such as the DMEM medium, preferably for 15 to 30 min, typically for 20 min, 3) covering the pieces of skin with dispase, preferably with dispase II, and incubating them for a suitable duration so that the dermis can then be separated from the epidermis via microdissection, for example by incubating them for one night at 4°C, or for 2 h at 37°C,

4) rinsing the pieces of skin and placing them in a "G7F-sebo2D" culture medium as defined hereinbelow,

5) separating the epidermis from the dermis of the piece of skin by microdissection and storing the sampled epidermis,

6) sampling the sebaceous glands of the hair follicles present under the epidermis and depositing them in the "G7F-sebo2D" culture medium as defined hereinbelow,

7) incubating the sampled sebaceous glands in trypsin, preferably for 5 to 10 min, then inhibiting the trypsin, for example by adding "G7F-sebo2D" culture medium as defined hereinbelow,

8) centrifuging the trypsinized sebaceous glands, preferably at 190 g, resuspending them and seeding them on feeder cells seeded beforehand, for example on irradiated fibroblasts such as 3T3i fibroblasts, in the "G7F-sebo2D" culture medium as defined hereinbelow further comprising a ROCK inhibitor as defined hereinbelow, preferably the Y-27632 compound, typically at 10 μΜ. Method of amplification

As indicated hereinabove, the inventors have developed a method that makes it possible to amplify non-immortalized primary sebocytes, in such a way as to retain their potential for differentiation during the preparation of the in vitro model of reconstructed sebocyte epithelium.

This method involves the use of a ROCK inhibitor in the presence of feeder cells in a culture medium comprising 7 factors enabling the growth of the sebocytes. This use makes it possible to retain the proliferative power of the primary sebocytes by reversibly blocking their differentiation, and thereby, to form substantial stocks of primary sebocytes.

The present invention thus relates to a method, preferably in vitro, for amplifying primary sebocytes, as defined in the "Sebocytes" section hereinabove, comprising a step of culture, in the presence of feeder cells, of primary sebocytes, as defined in the "Sebocytes" section hereinabove, preferably non-immortalized primary sebocytes, in particular non-immortalized primary human sebocytes, in a "G7F-sebo2D" culture medium comprising:

(i) adenine hydrochloride,

(ii) epidermal growth factor (EGF), (iii) hydrocortisone,

(iv) insulin,

(v) isoproterenol,

(vi) transferrin, and

(vii) triiodothyronine,

said culture medium further comprising a ROCK inhibitor.

The term "amplification" here means the proliferation or multiplication of cells.

Preferably, the cultivated primary sebocytes are non-immortalized primary human sebocytes, preferably coming from sebaceous glands coming for example from surgical waste, such as lifting waste. In a particular embodiment, the cultivated primary sebocytes come from the method of extraction defined in the "Sebocytes" section hereinabove.

Thus, in a particular embodiment, the method of amplification according to the invention comprises, before the step of culture, a step of extracting sebocytes, in particular from surgical waste, implementing the method of extraction defined in the "Sebocytes" section hereinabove.

The term "feeder cell" here means cells used in culture with other types of cells and which assist in the growth thereof. Feeder cells have their growth stopped, for example by irradiation, but are viable and form a substrate whereon the other cells can grow. In a particular embodiment, the feeder cells are fibroblasts, such as fibroblasts from fibroblast lines, for example 3T3 fibroblasts, and most preferably irradiated 3T3 fibroblasts, also called 3T3i.

The "G7F-sebo2D" culture medium used in the method of amplification according to the invention is a nutrient medium comprising a base medium and the 7 following factors that allow for the growth of the sebocytes:

(i) adenine hydrochloride,

(ii) epidermal growth factor (EGF),

(iii) hydrocortisone,

(iv) insulin,

(v) isoproterenol,

(vi) transferrin, and

(vii) triiodothyronine,

The base medium can in particular be a DMEM medium, DMEM/F12, MEM, RPMI medium, William's medium, KGM medium, dermalife-K medium, keratinocyte-SFM medium, or Glasgow medium. Preferably, the base medium is DMEM/F12 medium. Preferably, the base medium further contains L-glutamine, preferably at a concentration of 0.5 to 20 mM, more preferably at a concentration of 2 to 10 mM, still preferably at a concentration of 4 to 5 mM. Preferably, the base medium further comprises fetal calf serum, preferably at a concentration from 5 to 20%, and more preferably at a concentration of 10%.

The "G7F-sebo2D" culture medium may further comprise antibiotics.

The "G7F-sebo2D" culture medium comprises the following 7 factors that allow for sebocyte growth:

(i) adenine hydrochloride, preferably at a concentration from 1 x10 ^~4 M to 2x10 ^"4 M, in particular at a concentration from 1 .4x10 ^"4 to 1 .9x10 ^"4 M, more particularly from 1 .5x10 ^"4 to 1 .8x10 ^"4 M or from 1 .6x10 ^"4 to 1 .7x10 ^"4 M,

(ii) epidermal growth factor (EGF), for example recombinant human EGF, preferably at a concentration from 5 to 25 ng/ml, in particular at a concentration from 7 to 20 ng/ml, more particularly from 8 to 15 ng/ml, and more particularly from 9 to 12 ng/ml or from 10 to 1 1 ng/ml,

(iii) hydrocortisone, for example hydrocortisone 21 -hemisuccinate sodium, preferably at a concentration from 0.1 to 1 μg/ml, in particular at a concentration from 0.2 to 0.9 μg/ml, more particularly from 0.3 to 0.8 μg/ml, from 0.4 to 0.7 μg/ml or from 0.5 to 0.6 μg/m\,

(iv) insulin, for example recombinant human insulin, preferably at a concentration from 1 to 10 μg/ml, in particular at a concentration from 2 to 9 μg/ml, more particularly from 3 to 8 μg/ml, from 4 to 7 μg/ml or from 5 to 6 μg/ml,

(v) isoproterenol (also called isoprenaline), for example (-)-isoproterenol hydrochloride, preferably at a concentration from 5x10 ^"7 to 5x10 ^"6 M, in particular at a concentration from 6x10 ^"7 to 4x10 ^"6 M, more particularly from 7x10 ^"7 to 3x10 ^"6 M, from 8x10 ^"7 to 2x10 ^"6 M or from 9x10 ^"7 to 1 x10 ^"6 M,

(vi) transferrin (also called siderophilin), preferably human transferrin, for example human apo-transferrin, preferably at a concentration from 1 to 10 μg/ml, in particular at a concentration from 2 to 9 μg/ml, more particularly from 3 to 8 μg/ml, from 4 to 7 μg/ml or from 5 to 6 μg/ml, and

(vii) triiodothyronine (also called 3,3',5-triiodo-L-thyronine or T ₃ ), for example 3, 3', 5- triiodo-L-thyronine sodium, preferably at a concentration from 1 x10 ^~9 to 5x10 ^"9 M, in particular at a concentration from 1 .5x10 ^"9 to 4x10 ^"9 M, more particularly from 1 .75x10 ^"9 to 3x10 ^"9 M or from 2x10 ^"9 to 2.5x10 ^"9 M.

In a particular embodiment, the "G7F-sebo2D" culture medium comprises:

(i) from 1 x10 ^~4 to 2x10 ^"4 M of adenine hydrochloride,

(ii) from 5 to 25 ng/ml of EGF, (iii) from 0.1 to 1 μg/ml of hydrocortisone,

(iv) from 1 to 10 μg/ml of insulin,

(v) from 5x10 ^"7 to 5x10 ^"6 M of isoproterenol,

(vi) from 1 to 10 μg/ml of transferrin, preferably human transferrin, and

(vii) from 1 x10 ^"9 to 5x10 ^"9 M of triiodothyronine.

In a particular embodiment, the "G7F-sebo2D" culture medium comprises:

(i) from 1 .5x10 ^"4 to 1 .8x10 ^"4 M of adenine hydrochloride,

(ii) from 9 to 12 ng/ml of EGF,

(iii) from 0.4 to 0.7 μg/ml of hydrocortisone,

(iv) from 4 to 7 μg/ml of insulin,

(v) from 8x10 ^"7 to 2x10 ^"6 M of isoproterenol,

(vi) from 4 to 7 μg/ml of transferrin, preferably human transferrin, and

(vii) from 1 .75x10 ^"9 to 3x10 ^"9 M of triiodothyronine.

In the method of amplification according to the invention, the "G7F-sebo2D" culture medium further comprises a ROCK inhibitor.

The term "ROCK inhibitor" here means a protein, a nucleic acid, a small molecule, an antibody or another agent that prevents the expression of ROCK or decreases the activity of ROCK such as its kinase activity. ROCK inhibitors are well known to those skilled in the art and include the compound Y-27632 and the compounds H-1 152, Y- 30141 , Wf-536, HA-1077, hydroxyl-HA-1077, GSK269962A and SB-772077-B.

Preferably, the ROCK inhibitor is the compound Y-27632.

The ROCK inhibitor, in particular the compound Y-27632, is preferably added to the "G7F-sebo2D" culture medium at a concentration from 5 to 15 μΜ, preferably from 6 to 14 μΜ, more preferably from 7 to 13 μΜ, from 8 to 12 μΜ, from 9 to 1 1 μΜ, and most preferably at a concentration of 10 μΜ.

The culture medium is preferably changed regularly during the culture of the sebocytes, for example every 1 , 2 or 3 days.

The primary sebocytes thereby amplified can be trypsinized and re-seeded as soon as a high level of confluence is obtained, for example a level of confluence of 70%.

Method for preparing a model of reconstructed sebocyte epithelium. As indicated hereinabove, the inventors have furthermore developed a method for preparing a model of reconstructed sebocyte epithelium that implements non-immortalized primary sebocytes that makes it possible to obtain a three-dimensional model that is physiologically more pertinent with respect to the models described until now. Another object of the invention thus relates to a method, preferably in vitro, for preparing a model of reconstructed sebocyte epithelium that comprises the steps consisting in:

a) seeding, on a biomaterial for reconstructed epidermis (BPER) optionally covered with extracellular matrix protein, non-immortalized primary sebocytes in suspension in a "G7F-ERS" culture medium comprising:

(i) adenine hydrochloride,

(ii) epidermal growth factor (EGF),

(iii) hydrocortisone,

(iv) insulin,

(v) isoproterenol,

(vi) transferrin, and

(vii) triiodothyronine;

b) cultivating the sebocytes seeded in step a) in immersion on said BPER in G7F- ERS medium for 1 to 4 days;

c) cultivating the sebocytes obtained in step b) at the air - liquid interface on said BPER above a "G3F-ERS" culture medium comprising:

(I) epidermal growth factor (EGF),

(II) hydrocortisone, and

(III) insulin,

said G3F-ERS medium further comprising linoleic acid and a PPAR agonist, for a period that is suitable for allowing the differentiation of the sebocytes and lipid synthesis,

said model of reconstructed sebocyte epithelium being obtained at the end of step c).

The term "biomaterial for reconstructed epidermis" or "BPER" here means a dermal equivalent, used as a support and integrating ingredients that are naturally present in the dermis. Preferably, the BPER is a matrix of collagen, in particular of type I and/or IV collagens.

Preferably, the BPER is covered with extracellular matrix protein.

Extracellular matrix proteins are well known to those skilled in the art and include collagen, such as type IV or type VII collagen, fibronectin and laminin. Preferably, the BPER is covered with fibronectin, in particular human fibronectin.

The non-immortalized primary sebocytes seeded in step a) are as defined in the "Sebocytes" section hereinabove. Preferably, these are non-immortalized primary human sebocytes, preferably pre-amplified by the method of amplification as defined in the "Method of amplification" section hereinabove but in the absence of ROCK inhibitor.

Thus, in a particular embodiment, the method of preparation according to the invention comprises, before the step a) of seeding, a step pre-a) of pre-amplifying the non-immortalized primary sebocytes, preferably implementing the method of amplification as defined in the "Method of amplification" section hereinabove but in the absence of a ROCK inhibitor, in other words in the "G7F-Sebo2D" culture medium, as defined in the "Method of amplification" section, that does not contain any ROCK inhibitor.

The "G7F-ERS" culture medium used in the method of preparation according to the invention is a nutrient medium comprising a base medium and the 7 following factors that allow for the growth of the sebocytes:

(i) adenine hydrochloride,

(ii) epidermal growth factor (EGF),

(iii) hydrocortisone,

(iv) insulin,

(v) isoproterenol,

(vi) transferrin, and

(vii) triiodothyronine.

The base medium can in particular be a DMEM medium, DMEM/F12, MEM, RPMI medium, William's medium, KGM medium, dermalife-K medium, keratinocyte-SFM medium or Glasgow medium. Preferably, the base medium is DMEM/F12 medium. Preferably, the base medium further contains L-glutamine, preferably at a concentration of 0.5 to 20 mM, more preferably at a concentration of 2 to 10 mM, more preferably at a concentration of 4 to 5 mM. Preferably, the base medium further comprises fetal calf serum, preferably at a concentration from 5 to 20%, and more preferably at a concentration of 10%.

The "G7F-ERS" culture medium may further comprise antibiotics, sodium pyruvate preferably at a concentration of 0.1 to 10 mM, non-essential amino acids such as those contained in the MEM Non-Essential Amino Acids Solution (100X) product commercialized by Gibco under the reference 1 1 140, in particular diluted 100 times.

The "G7F-ERS" culture medium comprises the following 7 factors that allow for sebocyte growth:

(i) adenine hydrochloride, preferably at a concentration from 1 x10 ^~4 M to 2x10 ^"4 M, in particular at a concentration from 1 .4x10 ^"4 to 1 .9x10 ^"4 M, more preferably from 1 .5x10 ^"4 to 1 .8x10 ^"4 M or from 1 .6x10 ^"4 to 1 .7x10 ^"4 M, (ii) epidermal growth factor (EGF), for example recombinant human EGF, preferably at a concentration from 30 to 75 ng/ml, in particular at a concentration from 35 to 70 ng/ml, more preferably from 40 to 65 ng/ml, 45 to 60 ng/ml or from 50 to 55 ng/ml,

(iii) hydrocortisone, for example hydrocortisone 21 -hemisuccinate sodium, preferably at a concentration from 0.1 to 1 μg/ml, in particular at a concentration from 0.2 to 0.9 μg/ml, more preferably from 0.3 to 0.8 μg/ml, from 0.4 to 0.7 μg/ml or from 0.5 to 0.6 Mg ml,

(iv) insulin, for example recombinant human insulin, preferably at a concentration from 1 to 10 μg/ml, in particular at a concentration from 2 to 9 μg/ml, more preferably from 3 to 8 μg/ml, from 4 to 7 μg/ml or from 5 to 6 μg/ml,

(v) isoproterenol (also called isoprenaline), for example (-)-isoproterenol hydrochloride, preferably at a concentration from 5x10 ^"7 to 5x10 ^"6 M, in particular at a concentration from 6x10 ^"7 to 4x10 ^"6 M, more preferably from 7x10 ^"7 to 3x10 ^"6 M, from 8x10 ^"7 to 2x10 ^"6 M or from 9x10 ^"7 to 1 x10 ^"6 M,

(vi) transferrin (also called siderophilin), preferably human transferrin, for example human apo-transferrin, preferably at a concentration from 1 to 10 μg/ml, in particular at a concentration from 2 to 9 μg/ml, more preferably from 3 to 8 μg/ml, from 4 to 7 μg/ml or from 5 to 6 μg/ml, and

(vii) triiodothyronine (also called 3,3',5-triiodo-L-thyronine or T ₃ ), for example 3, 3', 5- triiodo-L-thyronine sodium, preferably at a concentration from 1 x10 ^~9 to 5x10 ^"9 M, in particular at a concentration from 1 .5x10 ^"9 to 4x10 ^"9 M, more preferably from 1 .75x10 ^"9 to 3x10 ^"9 M or from 2x10 ^"9 to 2.5x10 ^"9 M.

In a particular embodiment, the "G7F-ERS" culture medium comprises:

(i) from 1 x10 ^~4 to 2x10 ^"4 M of adenine hydrochloride,

(ii) from 30 to 75 ng/ml of EGF,

(iii) from 0.1 to 1 μg/ml of hydrocortisone,

(iv) from 1 to 10 μg/ml of insulin,

(v) from 5x10 ^"7 to 5x10 ^"6 M of isoproterenol,

(vi) from 1 to 10 μg/ml of transferrin, preferably human transferrin, and

(vii) from 1 x10 ^~9 to 5x10 ^"9 M of triiodothyronine.

In a particular embodiment, the "G7F-ERS" culture medium comprises:

(i) from 1 .5x10 ^"4 to 1 .8x10 ^"4 M of adenine hydrochloride,

(ii) from 45 to 65 ng/ml of EGF,

(iii) from 0.4 to 0.7 μg/ml of hydrocortisone,

(iv) from 4 to 7 μg/ml of insulin, (v) from 8x10 ^"7 to 2x10 ^"6 M of isoproterenol,

(vi) from 4 to 7 μg/ml of transferrin, preferably human transferrin, and

(vii) from 1 .75x10 ^"9 to 3x10 ^"9 M of triiodothyronine.

After the step of seeding, the sebocytes are advantageously cultivated in immersion in the G7F-ERS medium, as defined hereinabove, for 1 to 4 days, preferably for 3 days.

During this step b) of culture, the G7F-ERS culture medium can be replaced regularly, for example every day or every 2 days.

This step of culture allows for the proliferation of the sebocytes.

This step b) of culture in immersion is followed by a step c) of culture of sebocytes at the air - liquid interface on the BPER above a "G3F-ERS" culture medium comprising:

(I) epidermal growth factor (EGF),

(II) hydrocortisone, and

(III) insulin,

and further comprising linoleic acid and a PPAR agonist.

The "G3F-ERS" culture medium used in the method of preparation according to the invention is a nutrient medium comprising a base medium and the 3 following factors that allow for the growth and/or differentiation of the sebocytes:

(I) epidermal growth factor (EGF),

(II) hydrocortisone, and

(III) insulin.

The base medium can in particular be DMEM medium, DMEM/F12, MEM, RPMI medium, William's medium, KGM medium, dermalife-K medium, keratinocyte-SFM medium or Glasgow medium. Preferably, the base medium is DMEM/F12 medium. Preferably, the base medium further contains L-glutamine, preferably at a concentration of 0.5 to 20 mM, more preferably at a concentration of 2 to 10 mM, still preferably at a concentration of 4 to 5 mM. Preferably, the base medium further comprises fetal calf serum, preferably at a concentration from 5 to 20%, more preferably at a concentration of 10%.

The "G3F-ERS" culture medium comprises the following 3 factors that allow for sebocyte growth and/or differentiation:

(I) epidermal growth factor (EGF), for example recombinant human EGF, preferably at a concentration from 30 to 75 ng/ml, in particular at a concentration from 35 to 70 ng/ml, from 40 to 65 ng/ml, 45 to 60 ng/ml or from 50 to 55 ng/ml, (II) hydrocortisone, for example hydrocortisone 21 -hemisuccinate sodium, preferably at a concentration from 0.1 to 1 μg/ml, in particular at a concentration from 0.2 to 0.9 μg/ml, from 0.3 to 0.8 μg/ml, from 0.4 to 0.7 μg/ml or from 0.5 to 0.6 μg/ml, and

(III) insulin, for example recombinant human insulin, preferably at a concentration from 1 to 10 μg/ml, in particular at a concentration from 2 to 9 μg/ml, from 3 to 8 μg/ml, from 4 to 7 μg/ml or from 5 to 6 μ9ΛηΙ.

The "G3F-ERS" medium thus has the particularity of comprising only 3 factors that allow for sebocyte growth and/or differentiation. Preferably, the "G3F-ERS" medium is devoid of adenine hydrochloride, isoproterenol, human transferrin and/or triiodothyronine.

In a particular embodiment, the "G3F-ERS" culture medium comprises:

(I) from 30 to 75 ng/ml of EGF,

(II) from 0.1 to 1 μg/ml of hydrocortisone, and

(III) from 1 to 10 μg/ml of insulin.

In a particular embodiment, the "G3F-ERS" culture medium comprises:

(I) from 45 to 65 ng/ml of EGF,

(II) from 0.4 to 0.7 μg/ml of hydrocortisone, and

(III) from 4 to 7 μg/ml of insulin.

The "G3F-ERS" medium can furthermore comprise vitamin C, preferably at a concentration from 0.05 to 0.5 mg/ml, in particular at a concentration from 0.06 to 0.4 mg/ml, from 0.07 to 0.3 mg/ml, from 0.08 to 0.2 mg/ml or from 0.09 to 0.1 mg/ml.

The "G3F-ERS" medium can further comprise antibiotics, sodium pyruvate preferably at a concentration of 0.1 to 10 mM, non-essential amino acids such as those contained in the MEM Non-Essential Amino Acids Solution (100X) product commercialized by Gibco under the reference 1 1 140, in particular diluted 100 times.

The "G3F-ERS" medium as defined hereinabove further comprises linoleic acid and a PPAR agonist.

These additional compounds act in the method of preparation according to the invention as inducers of differentiation and of lipid synthesis.

In a particular embodiment, the "G3F-ERS" medium comprises several PPAR agonists, in particular at least two PPAR agonists, preferably at least three PPAR agonists.

The term "PPAR (peroxisome proliferator-activated receptor) agonist" or "PPAR inducer" here means a compound that activates the signaling pathway mediated by PPAR.

The term "PPAR" or "peroxisome proliferator-activated receptor" here means a protein of the superfamily of nuclear receptors that naturally binds lipids and that acts as a transcription factor of genes involved in particular in metabolism and adipogenesis. Three types of PPAR have been identified: alpha, gamma and delta (beta). Like all nuclear receptors, PPAR is structured in four separate regions. An N-terminal domain has a transactivating function independent of the ligand (AF-1 ). The DNA binding domain or DBD is constituted of two zinc fingers characteristic of the superfamily of nuclear receptors. PPAR has a hinge region preceding the ligand binding domain or LBD that has a transactivating function dependent of the ligand (AF-2).

The PPAR agonist used in the context of the invention can be a pan-PPAR agonist (i.e. an inducer of the three types of PPAR, alpha, gamma and beta/delta), a selective PPAR-alpha agonist and/or a selective PPAR-delta agonist and/or a selective PPAR- gamma agonist.

Examples of PPAR agonists are well known to those skilled in the art and include, as a pan-PPAR agonist, aleglitazar, muraglitazar, saroglitazar and tesaglitazar, as a selective PPAR-delta agonist, the compound GW501516 ({4-[({4-methyl-2-[4- (trifluoromethyl)phenyl]-1 ,3-thiazol-5-yl}methyl)sulfanyl]-2-methylphenoxy}acetic acid), and the compound GW0742 ([4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5- thiazolyl]methyl]thio]-2-methylphenoxy]-acetic acid) referenced as CAS [317318-84-6], as a PPAR-alpha agonist, derivatives of fibric acid such as aluminum cofibrate, beclobrate, bezafibrate, binifibrate, ciprofibrate, clinofibrate, clofibrate, clofibric acid, clofibride, dulofibrate, eniclobrate, ethofibrate, etofylline clofibrate, fenofibrate, fenofibric acid, gemfibrozil, nicofibrate, pirifibrate, ronifibrate, simfibrate and theofibrate.

Preferably, the PPAR agonist used in the context of the invention is the compound GW0742.

Advantageously, the "G3F-ERS" medium contains only one PPAR agonist, preferably the PPAR agonist is the compound GW0742.

In a particular embodiment, the "G3F-ERS" medium comprises from 1 x10 ^~7 to 2.5x10 ^"5 M of linoleic acid, preferably from 5x10 ^"7 to 2x10 ^"5 M, from 1 x10 ^~6 to 1 .5x10 ^"5 M, from 5x10 ^"6 to 1 x10 ^"5 M, or from 9x10 ^"6 to 1 x10 ^"5 M.

In another particular embodiment, the "G3F-ERS" medium comprises from 1 x10 ^~6 to 5x10 ^"6 M of a PPAR agonist, in particular of compound GW0742, preferably from 1 .5x10 ^"6 to 4x10 ^"6 M, from 2x10 ^"6 to 3.5x10 ^"6 M or from 2.5x10 ^"6 to 3x10 ^"6 M.

In a particular embodiment, the "G3F-ERS" medium comprises from 5x10 ^"6 to 2.5x10 ^"5 M of linoleic acid and from 1 x10 ^~6 to 5x10 ^"6 M of a PPAR agonist, said PPAR agonist being preferably the compound GW0742. In a particular embodiment, the "G3F-ERS" medium comprises from 8x10 ^"6 to 2x10 ^"5 M of linoleic acid and from 2x10 ^"6 to 5x10 ^"6 M of a PPAR agonist, said PPAR agonist being preferably the compound GW0742.

It is during this phase c) of culture at the air-liquid interface that the sebocytes differentiate and produce a mixture of neutral lipids (such as glycerides, cholesterol, esters of cholesterol and free fatty acids).

Thus, the step c) of culture at the air-liquid interface is implemented during a suitable duration for allowing the differentiation of sebocytes and lipid synthesis.

Preferably, the step c) of culture at the air-liquid interface is implemented for 5 to 25 days, preferably for 7 to 20 days, for 8 to 17 days or for 10 to 15 days.

In particular, the step c) of culture at the air-liquid interface can be continued once the sebocytes are differentiated, for example in order to use the obtained model in screening tests.

During the step c) of culture at the air-liquid interface, the G3F-ERS culture medium can be replaced regularly, partially or entirely, for example every day, every 2 days or every 3 days.

The model of reconstructed sebocyte epithelium is obtained at the end of step c). This model is different from the models of sebocyte epithelium of the state of the art by the fact that the sebocytes that form it are not immortalized and that its histology is very close to that of the in vivo sebaceous gland. In particular, almost all of the large families of non-polar lipids (glycerides, cholesterol, fatty acids, esters of cholesterol) present in the sebum are present in the model of reconstructed sebocyte epithelium obtained by the preparation method according to the invention

The present invention therefore also concerns a model of reconstructed sebocyte epithelium likely to be obtained by the preparation method according to the invention.

Uses

The inventors have shown that the lipid synthesis of the model of reconstructed sebocyte epithelium according to the invention could be modulated using compounds known for modulating this activity in vivo such as 13-cis-retinoic acid.

The model of reconstructed sebocyte epithelium according to the invention can therefore be used to identify compounds that modulate the lipid synthesis of sebocytes.

The present invention thus relates to a method, preferably in vitro, for screening compounds that modulate the lipid synthesis of sebocytes, comprising putting into contact a candidate compound with the model of reconstructed sebocyte epithelium according to the invention. The present invention also has for object a method, preferably in vitro, for screening compounds for treating oily and/or glistening skin, comprising putting into contact a candidate compound with the model of reconstructed sebocyte epithelium according to the invention.

The screening methods according to the invention can further comprise a step of analyzing lipids synthesized by the sebocytes of the model of reconstructed sebocyte epithelium in the presence of the candidate compound, for example by labeling neutral lipids, for example with BODIPY, Lipidtox or oil-red-o. The present invention shall be illustrated in more detail by the figures and example hereinbelow.

Brief description of the figures

Figure 1 : Scheme of extraction of primary human sebocytes obtained from a biopsy.

Figure 2: Observations under the microscope of sebocytes extracted from sebaceous glands after 6 days of culture on plastic. Part A: culture in G7F-sebo2D medium - Part B: culture in G7F-sebo2D medium in the presence of a ROCK inhibitor (Y27632).

Figure 3: A: Example of a histological section of an in vivo sebaceous gland adapted from Woldow et al. (2009) Dermatology Online Journal 15:14. B: Scheme representing an in vivo sebaceous gland adapted from Niemann et al. (2012) Seminars in Cell & Developmental Biology 23:928-936.

Figure 4: Left part: Examples of histological sections of the model of reconstructed sebocyte epithelium according to the invention at D13 colored with HES.

Right part: for comparison, example of a histological section of the EPISKIN epidermis model colored with HES.

Figure 5: Compilation of 3 studies of modulation of the synthesis of neutral lipids on the model of reconstructed sebocyte epithelium according to the invention carried out by BODIPY labeling and analyzing IMAGEJ images. 4 molecules evaluated: isotretinoin (= 13-cis-retinoic acid) at 1 μΜ (Iso), compound A at 50 μΜ (A), compound B at 20 μg/ml (B) and compound C at 10 μg/ml (C). Two controls were carried out: an non-treated control (NC) and a control treated with the solvent DMSO (DMSO). The bar graphs show the surface labeled with BODIPY standardized with respect to untreated controls, the percentage indicated above the bar graphs corresponding to the percentage of surfaces labeled with respect to the DMSO control.

* ^* p < 0.05 vs. DMSO, ^*** p < 0.005 vs. DMSO, Student's T test

Examples

Example 1 : Extraction of primary human sebocytes obtained from a biopsy

This example describes the extraction of primary human sebocytes carried out by using the extraction protocol according to the invention.

Pieces of lifting comprising hairs were used.

The medium in which the pieces of lifting were stored and the blood were eliminated.

The pieces of lifting were selected and placed in a Petri dish containing PBS. The surface of the skin was shaved using a scalpel in order to remove the hair.

The pieces were then cut into pieces of about 0.5 cm ² and placed in a bath of antibiotics (medium with 10% antibiotics) for 20 min.

3T3i irradiated fibroblasts were defrosted and seeded in the "G7F-sebo2D" medium comprising 10 μΜ of Y-27632.

The pieces of skin were covered with an aqueous solution of Dispase II at 2.4 U/ml, and incubated at 4°C overnight or 2h at 37°C.

The dispase was then removed, the pieces rinsed in PBS, and the epidermis was separated from the dermis by using microdissection tweezers. The dermis was eliminated. The sebaceous gland of each hair follicle found under the epidermis was removed and deposited into a well comprising "G7F-sebo2D" medium.

Composition of the G7F-sebo2D medium supplemented with antibiotics for a final volume of 500 ml:

Product Volume or final Reference concentration

DMEM Glutamax 333 ml Gibco n° 31966

F12 Glutamax 1 1 1 ml Gibco n° 31765

FetalClone II Serum 10% Hyclone n°SH30066.03

Antibiotics-antimycotics 1 % Gibco n° 15240-062

Adenine hydrochloride 1 .8x10 ^"4 M Sigma n° A-9795

EGF 10 ng/ml Chemicon inter ^nal ref. GF144 Hydrocortisone 0.4 μg/ml Sigma n° H-4881

Insulin 5 μ9/ηιΙ Sigma n° I-9278

Isoproterenol 10 ^-6 M Sigma n° I-6504

Human transferrin 5 μο/ηιΙ Sigma n° T-2252

Triiodothyronine 2x10 ^"9 M Sigma n° T-2752

The medium comprising the glands was then recovered and centrifuged 8 min at 190 g. The supernatant was eliminated and the glands were resuspended in trypsin. After 5 to 10 min of incubation, the tubes were vortexed in order to dissociate the glands. The trypsin was then inhibited by adding "G7F-sebo2D" medium supplemented with antibiotics.

The whole was then centrifuged for 8 min at 190 g and the pellet was resuspended.

The cells were counted with trypan blue then seeded on the fibroblasts in culture in "G7F-sebo2D" medium supplemented with antibiotics and comprising 10 μΜ of Y-27632.

This method of extraction is shown in Figure 1.

Example 2: Method for amplifying non-immortalized human sebocytes

This example describes the amplification of primary human sebocytes carried out by using the method of amplification according to the invention.

Composition of the G7F-sebo2D medium supplemented with antibiotics for a final volume of 500 ml:

10 μΜ of Y-27632 were added to the "G7F-sebo2D" medium supplemented with antibiotics. The day before, or 2 hours before the seeding of the sebocytes, 3T3i fibroblasts were seeded in G7F-sebo2D medium supplemented with antibiotics + Y27632, at a rate of 40,000 cells / cm ² .

The non-immortalized primary human sebocytes were put into suspension in G7F- sebo2D medium + Y27632, and seeded on the fibroblasts at a rate of 2,400 cells / cm ² .

The culture medium was changed every 48h.

After 5 days of culture, the cells arrived at confluence and about 5 million sebocytes could be recovered by trypsinization.

Figure 2 shows microscopic observations of sebocytes after 6 days of culture on plastic in G7F-sebo2D medium supplemented with antibiotics with and without Y27632.

Example 3: Method for preparing a model of reconstructed sebocyte epithelium.

This example describes the preparation of a model of reconstructed sebocyte epithelium from non-immortalized primary human sebocytes using the method of preparation according to the invention.

Preparation of BPERs

The BPERs comprising type I and type IV collagen were rinsed twice in PBS and once in DMEM/F12 Glutamax 3:1 base medium + antibiotics.

The BPERs were then coated with fibronectin the day before inoculation of sebocytes.

Amplification of sebocytes

The 3T3i fibroblasts and the sebocytes were either co-seeded, or seeded sequentially, the sebocytes then being seeded at least 2h after the fibroblasts.

The "G7F-sebo2D" culture medium supplemented with antibiotics was used:

Composition of the "G7F-sebo2D" medium supplemented with antibiotics for a final volume of 500 ml:

Product Volume or final Reference concentration

DMEM Glutamax 333 ml Gibco no. 31966

F12 Glutamax 1 1 1 ml Gibco no. 31765

FetalClone II Serum 10% Hyclone no. SH30066.03

Antibiotics-antimycotics 1 % Gibco no. 15240-062

Adenine hydrochloride 1 .8x10 ^"4 M Sigma no. A-9795 EGF 10 ng/ml Chemicon internal ref. GF144

Hydrocortisone 0.4 μg/ml Sigma no. H-4881

Insulin 5 μ9/ηιΙ Sigma no. I-9278

Isoproterenol 10 ^-6 M Sigma no. I-6504

Human transferrin 5 μο/ηιΙ Sigma no. T-2252

Triiodothyronine 2x10 ^"9 M Sigma no. T-2752

The flask comprising the cells was incubated at 37°C with 5% of C0 ₂ for 6 days, changing the medium every two days.

Seeding of the sebocvtes on BPER

"G7F-ERS" medium supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, fetal bovine serum and antibiotics, was deposited under BPER inserts.

Composition of the supplemented "G7F-ERS" medium for a final volume of 500 ml:

The amplified sebocytes were trypsinized, centrifuged at 190 g and resuspended in supplemented "G7F-ERS" medium in such a way as to obtain a suspension at 0.8.10 ⁶ cells/ml.

500 μΙ of cell suspension were deposited on each BPER (DO) and incubated at 37°C, 5% C0 ₂ .

Culture in immersion from DO to D3:

On D1 , the culture medium was renewed with "G7F-ERS" medium above and under each insert. Culture in emersion from D3 to D13:

On D3, D6, D8 and D10, the culture medium above and under the inserts was aspirated. "G3F-ERS" medium supplemented with L-glutamine, sodium pyruvate, nonessential amino acids, fetal bovine serum, antibiotics, vitamin C, linoleic acid and GW0742, was deposited under each insert.

Composition of the G3F-ERS medium for a final volume of 500 ml:

They were then incubated at 37°C, 5% C0 ₂ .

It is during this phase of culture in particular that the sebocytes differentiate and produce a mixture of neutral lipids (glycerides, cholesterol, esters of cholesterol, free fatty acids).

Figure 4 shows histological sections of the model obtained at the end of this protocol, compared to an Episkin epidermis model (referenced EPISKIN/L/13 by EPISKIN S.A.) and to an in vivo sebaceous gland (Figure 3).

Example 4: Study of the modulation of the synthesis of the neutral lipids by different molecules in a model of reconstructed sebocyte epithelium according to the invention

This example shows that the lipid synthesis of the model of reconstructed sebocyte epithelium according to the invention can be modulated by using compounds known to modulate this activity in vivo such as 13-cis-retinoic acid and that the model of reconstructed sebocyte epithelium according to the invention can therefore be used to identify compounds that modulate the lipid synthesis of sebocytes. Materials and methods

Labeling of lipids with BODIPY:

BODIPY® (493/503) is a fluorochrome that has a particular affinity for neutral lipids.

Products:

This labeling is carried out on sections of 10 μηι thick obtained from cryomicrotom sections of samples frozen at OCT (Optimal Cutting Temperature).

The following protocol was implemented:

- Take the blades for labeling out of the freezer, let them dry flat for about 30 min.

- Fixation: 4% formaldehyde bath 20 min at room temperature

- Allow to dry under the fume cupboard until there is no more formaldehyde

- Surround the sections with Dako pen (allow to dry well)

- PBS rinsing 5 min at +4°C

- Incubation in PBS + goat serum 10% + BSA 1 %: 100μΙ /section, 20 min at room temperature in a wet chamber, in the dark

- Preparation PBS + BSA 1 %: 10g BSA + 1 liter PBS histo

- Preparation PBS + BSA 1 %+- goat serum 10%: 1 ,ΟΟΟμΙ -Goat serum + 9,000μΙ PBS+BSA 1 %

- PBS rinsing 5 min at +4°C

- Bodipy Incubation 1 h at room temperature in a wet chamber, in the dark, 100μΙ /section

- Preparation of PBS +- goat serum 10%: 1 .5ml - Goat serum + 13.5ml PBS.

- Bodipy preparation at 25μg/ml (dilution to 1/40 of aliquots prepared beforehand at 1 mg/ml DMSO), 250μΙ Bodipy 1 mg/ml + 9750 μΙ PBS + -Goat serum.

- PBS rinsing 5min at +4°C

- Incubation with Hoechst 1/1000 5 min at room temperature in a wet chamber, in the dark, 100μΙ /section - Hoechst Preparation: 15μΙ Hoechst + 15 ml PBS

- 2 PBS rinsings 5 min at +4°C

- Mounting the coverglasses with Fluoromount G. Acquisition and analysis of the labeling:

The acquisition of the fluorescence images is carried out under standardized conditions (exposure time, white-black settings...) and with a magnification that makes it possible to visualize the epithelium over its entire thickness.

The images were acquired in fluorescence with a Zeiss Axioplan 2 imaging microscope combined with a Zeiss AxioCam MRm camera, and with the Zeiss ZEN lite image acquisition software. Images are recorded in a format that is compatible with the image analysis software and with the scale of the sizes.

Analysis of images is carried out with IMAGEJ: the labelling is thus selected, and the labeled surface is measured for each sample.

In order to have a more robust evaluation, this analysis is conducted over several fields per histological section, and over several histological sections per biological sample. For each sample the measurements are averaged, and for each test condition the average of the replicates per treatment condition is taken.

If an agent has been tested over several studies, the results can be standardized with respect to the untreated control in order to be able to express a significance over all of the tests conducted.

Results

Figure 4 shows the results obtained by labeling with BODIPY and image analysis with IMAGEJ. For each condition the surface results were obtained by compiling 3 different studies after standardizing values with respect to the untreated controls. All of the compounds tested being solubilized in DMSO, a solvent control was integrated into each study. Isotretinoin was used as a pharmaceutical reference or positive control for the functionality of the model, and made it possible to validate the studies. 3 cosmetic compounds were evaluated in parallel (compounds A, B and C).

Compounds A and B made it possible to decrease the synthesis of neutral lipids, contrary to compound C which did not show any activity in this test.