J. Cosmet. Sci., 68, 183–194 (March/April 2017) 183 Evaluation of molecules or extracts modulating seborrhea and its consequences, using normal human culture of sebocytes and keratinocytes, skin explants models and in vivo methods: a case study PHILIPPE MONDON, ROBERTO DAL TOSO, CAROLINE RINGENBACH, LAURENT LAVAISSIÈRE, EMMANUEL DORIDOT, ÉMILIE OUVRAT, SANDRA BRAHIMI, SEDERMA, 78612 Le Perray-en-Yvelines, France (M.P., R.C., L.L., D.E., O.E., B.S.), IRB, 36077 Altavilla Vicentina, Italy (D.T.R.) Accepted for publication August 31, 2016. Synopsis Skin produces sebum through sebocytes. Hyper-seborrhea creates conditions for the development of infl amed cutaneous alterations through bacteria colonization triggering dead cell accumulation and pro-infl ammatory mediator release. Study of sebum production, its modulation, and its consequences requires complementary in vitro models in order to evaluate the effect of molecules on cell metabolisms. Clinical studies need to be performed to confi rm in vitro results. Effects of phenylpropanoids, obtained by elicitation and purifi cation from plant cell culture of Syringa vulgaris (CCSV), were studied on sebocytes, keratinocytes, and explants, all derived from normal human skins. Normal human sebocytes (NHSs) expressed markers such as cytokeratin-7, cytokeratin-4, and perilipin-2 (PLIN-2) (1) the latter being colocalized with lipid droplets. Lipid droplets clearly appeared and their size increased rapidly when lipogenic agents were used. NHS, normal human keratinocytes (NHK), and explants reacted to presence of bacterial fragments which trigger pre-infl ammatory mediator release. CCSV reduced lipid storage and release of pre-infl ammatory mediators in NHS, NHK and explants. CCSV also reduced P. acnes growth and triggered beta-defensin-2 and cathelicidin synthesis by NHS, two natural antimicrobial peptides. On volunteers, sebum production, infl amed blemishes, and retentional lesions were signifi cantly reduced after 1 month treatment with CCSV. INTRODUCTION Sebaceous glands are found in the skin of almost all mammals and are present all over the human body except palms of the hands and soles of the feet. The glands are numerous on the face, where 400–900 glands/cm² can be found ears, scalp, and upper part of the trunk are also rich in glands. Their main function is to produce sebum, a mixture of relatively Address all correspondence to Pascaline Criton at pascaline.criton@sederma.fr. Presented at the 28th Congress of IFSCC, October 27–30, 2014, Paris, France.

JOURNAL OF COSMETIC SCIENCE 184 neutral lipids which are synthesized de novo by sebocytes. Immature sebocytes are located at the periphery of sebaceous glands and are fl attened cells without lipids. When sebocytes progress to the center of the gland, they mature and increase in size due to lipid synthesis. Ultimately, as the cells differentiate, they disintegrate and release their lipid content into the infundibulum. Human sebum contains cholesterol (1.5–2.5%), cholesterol esters (3–6%), squalene (12–20%), free fatty acids (15–30%), triglycerides (30–50%), and wax esters (26–30%) (2). Overproduction of sebum is a common scheme for teenagers all around the world how- ever, it is also widely distributed in the general population whatever the age, the gender, and the geographical zone. It makes the skin look oily and shiny and renders people less attractive in addition, it creates conditions for the development of infl amed cutaneous alterations (3). Balance of lipids, such as linoleic and arachidonic acids, is usually modi- fi ed these acids act directly or indirectly on lipid storage increase through Peroxisome Proliferator Activated Receptors (PPARs) and perilipin expression. They also trigger pro- duction of infl ammatory mediators such as interleukins (IL-6 and IL-8) and prostaglandin E2 (PGE-2) (4, 5). These mediators, among others, are key players in the increase of lipid synthesis by sebocytes, in the proliferation of microorganism such as Propionibacterium acnes, in keratinocyte hyper-proliferation, and in macrophage invasion of nearby tissues. The lack of an ideal animal model comparable to human sebaceous glands was surmounted by the development of experimental models such as SEB-1, SEB-E6E7, and SZ95 cell lines all immortalized by SV40 (6,7) or using extracted human sebaceous glands. Growth of primary normal human sebocytes (NHSs) in monolayer was achieved by various teams however, these models could not be multiplied because cells loose quickly their features. So, this model remains a challenge in order to evaluate molecules with a nontransformed cell. This paper relates use of NHS to study modulators of lipid storage and variation of pro- infl ammatory mediator synthesis within these cells. Among products that were evalu- ated, a plant cell culture extract of Syringa vulgaris (CCSV) showed signifi cant effects on both lipid storage and on bacterial-induced pro-infl ammatory mediator release. Comple- mentary tests performed on NHK, macrophages, and skin explants confi rmed the interest of CCSV for reduction of PGE-2, IL-6, and IL-8 induced by lipopolysaccharide (LPS) or P. acnes. Moreover, CCSV was evaluated on volunteers with greasy skin, where it reduced sebum synthesis and consequences of over lipid production (skin blemishes, hyperkeratinization). MATERIALS AND METHODS CELL CULTURE, SKIN EXPLANTS, AND REAGENTS NHS. Mycoplasmas-free NHS cells, derived from facial sebaceous gland (woman, 26 years) and obtained in compliance with the procedures and international standards for donation and collection of human tissue used for cell isolation were used. They were amplifi ed and sub- cultured in appropriate and defi ned cell culture medium containing 2–10% of fetal calf serum (FCS, Gibco®,Thermo Fisher Scientifi c Inc., Waltham, MA). Various cell medium were tested for NHS growth and lipogenesis assays, such as Dulbecco’s Modifi ed Essential Medium (DMEM), Dulbecco’s Modifi ed Essential Medium and Ham’s F-12 Medium (DMEM-F12), Keratinocyte Serum-Free Medium (KSFM), Medium 154 (M154) completed with antibiotics