Keywords

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FormalPara Core Messages

  • Seborrhea and acne are exclusively human disorders and sebaceous gland differentiation is species-specific, thus posing the need for human in vitro models.

  • Human sebaceous gland cell lines (SZ95 as well as SEB-1, Seb-E6E7) have been used in monolayer cultures as models to study specific functions involved in development, growth, and differentiation of sebaceous gland cells.

  • Maintenance of sebaceous gland cells in certain culture conditions has helped to investigate the physiology of the sebaceous gland, including its changes in acne.

  • Sebocyte culture models have provided insight in the mechanism of action of acne treatments, including retinoids, anti-androgens, and PPAR ligand antagonists.

  • More complex culture systems, including three-dimensional models are under development.

  • Sebocyte culture models provide new chances for further research on biologically active ingredients, new pharmaceutical and cosmetic drugs for antiaging, and acne treatment.

1 Introduction

Experimental sebaceous gland models are essential for a better understanding of the pathophysiology of human skin disorders involving the sebaceous gland, such as sebostasis, seborrhea, and acne, for thorough research and development of cosmetics and drugs, and for investigation of drug pharmacokinetics. So, the need for an established model for studies of sebocyte differentiation and for pharmacologic assays has led into considerable advances in this field (Table 6.1).

Table 6.1 Reported sebaceous gland experimental models
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Several attempts were made to cultivate animal [1, 2] or human sebaceous gland cells, using mechanical dissociation of isolated sebaceous glands or enzymatic separation of sebocytes from skin sections with a keratotome [3, 4]. However, seborrhea and acne are exclusively human diseases and sebaceous gland differentiation is species-specific, and no animal model was found to be predictive in assessing antiacne drug effects in humans. So, basic research on human sebaceous gland function and control requires human in vitro models.

Initial studies have been reported on experimental models carried out on whole human skin plugs, either incubated in vitro [5–7] or grafted on to nude mice [8]. However, it was not until the isolation of viable human sebaceous glands and pilosebaceous units [9, 10] and the establishment of the human sebocyte culture model in vitro [11] that fundamental research on human sebocyte activity and its regulation begun [12].

Human sebaceous gland experimental models have shed light on a plethora of functions of the sebaceous gland, highlighting its role in skin homeostasis [13]. Apart from acne, sebaceous glands are involved in embryology, development, and differentiation; in skin protection; and in inflammation and immunity and display complex endocrine properties (Table 6.2) [13]. Also, sebocytes, despite their programming for terminal differentiation, preserve characteristics of stem-like cells, as they present a remarkable potential of dual differentiation. The interactions between β-catenin and Sonic hedgehog promote proliferation of progenitors of the hair lineages, while Indian hedgehog stimulates proliferation of sebocytes precursors [14]. Overexpression of myc stimulates sebocyte differentiation, whereas overexpression of β-catenin stimulates interfollicular epidermal differentiation in vitro [15].

Table 6.2 Complex sebaceous gland functions identified by research in experimental sebocyte culture models
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2 Human Experimental Models of the Pilosebaceous Unit

Karasek and Charlton [3, 4] first described the cultivation of human sebocytes in collagen after enzymatic dissociation of isolated sebaceous glands and in monolayers after enzymatic digestion of sebaceous-gland-rich dermal slices obtained with a Castroviejo keratotome. Cells obtained by the first technique exhibited a significant loss of sebocyte characteristics in vitro. The latter method was modified later and further developed by Doran et al. [16] who removed the top 0.4-mm facial skin section containing the epidermis and some of the dermis and used the second 0.4-mm dermal section as the source of human sebocytes.

The first successful human sebocyte culture was introduced in 1989 by Xia et al. [11] with the in vitro subcultivation of human sebaceous gland-derived cells. Intact sebaceous glands were isolated from full-thickness human skin after incubation in dispase and deoxyribonuclease. Human sebocyte monolayer cultures as outgrowths from the periphery of sebaceous gland organ cultures were obtained. The ducts of the glands were removed; the isolated gland lobules were seeded on a 3 T3-cell feeder layer in Dulbecco’s modified Eagle’s medium and Ham’s F 12 medium (3:1) supplemented with fetal calf serum (10 %), l-glutamine, antibiotics, epidermal growth factor (10 ng/ml), hydrocortisone (0.4 μg/ml), and cholera toxin (10-9 M), and were then cultivated in a CO2 incubator at 37° C. After 2–3 weeks, cell outgrowths resulted from the periphery of the gland lobules, and dispersed cells were subcultured thrice with or without 3 T3-cell feeder layer. The cultured cells preserved in vitro morphologic characteristics and differentiation patterns comparable to those described for normal human sebocytes in vivo with a high rate of viable cells. Their labeling pattern with monoclonal antibodies showed close similarities to the pattern of keratinocytes in vivo and in vitro. In their cytoplasm oil red- and nile red-stained droplets were detected, and the observed density and distribution evidenced in vitro lipogenesis. This technique demonstrated the growth of cells originating from intact human sebaceous glands and their long-term differentiation into lipid-producing cells in vitro [11]. Also, sebocyte cultures could be obtained not only from sebaceous-gland-rich skin areas but also from other areas of the human skin [17]. Disadvantages of the method included the fact that the exact separation of glands from the skin was time consuming and required skillful preparation and that the rather low number of proliferating cells in the intact sebaceous gland lobules did not provide optimal conditions for their in vitro growth.

Over the years, modifications of the technique of Xia and al [11]. have improved the culture of human sebocytes in vitro. Zouboulis et al. [18] modified the culture medium including 2 % human serum, 8 % fetal calf serum, and omitting hydrocortisone. Lee [19] treated sebaceous glands with collagenase before cultivating them in serum-free William’s E medium supplemented with 10 μg/ml insulin, 10 μg/ml transferring, 10 μg/ml hydrocortisone, 10 ng/ml epidermal growth factor, 10 ng/ml sodium selenite, 2 nmol/l l-glutamine, and penicillin/streptomycin [19]. Primary sebocyte cultures could also be obtained by omitting the 3 T3 fibroblast layer, and secondary cultures could be grown in a medium supplemented with delipidized serum and serum-free keratinocyte basal medium. [20–22]

Fujie et al. [23] cultured primary sebocytes after the technique of Xia et al. [11] for at least three passages in serum-free keratinocyte growth medium without a feeder cell layer. Keratinocyte growth factor, shown to be a mitogen for primary cultures of mammary epithelium alone or combined with epidermal growth factor and/or bovine serum albumin, was found to significantly improve yield rates and proliferation of human sebocytes [24–26].

Human sebocytes however, are predestined to differentiate by accumulating neutral fat droplets until they burst and die. Therefore, adequate cell amounts for large-scale experiments could only be obtained from multiple donors, whereas prolonged experiments were hindered by the short life spans of the cells, as normal human sebocytes could only be grown for 3–6 passages.

These drawbacks were overcome by the generation of an immortalized sebocyte cell line (SZ95) by Zouboulis et al. [27] by transfecting cultured human facial sebocytes from a 87-year old woman with the Simian virus 40 large T antigen. The SZ95 sebaceous gland cell line is nowadays protected by national and international patents as well as priority submissions [28]. SZ95 sebocytes exhibit similar morphologic, phenotypic, and functional characteristics of normal human sebocytes. Several studies have shown that SZ95 sebocytes retain major characteristics of normal human sebocytes, such as progressing differentiation with increasing cell volume and lipid synthesis, expression of markers of sebaceous lineage and terminal sebocyte differentiation, such as keratin 7 and epidermal membrane antigen (EMA), respectively [18], and can subsequently undergo apoptosis [29]. SZ95 sebocytes also express characteristic organ- and function-specific proteins of human sebaceous glands and exhibit expected biological responses to androgens and retinoids [27, 29].

In 2003, Thiboutot et al. [30] applied the transfection system administered by Zouboulis et al. [27] to develop a second immortalized human sebaceous gland cell line, termed SEB-1. SEB-1 was established from sebaceous glands of normal skin of the preauricular area of a 55-year-old male. Like SZ95 sebocytes, SEB-1 sebocytes also express characteristic sebaceous gland proteins and their cytoplasm-induced oil red O-positive lipid droplets. In gene array studies, genes characteristic for the sebaceous gland and such involved in lipid and steroid metabolism were expressed in SEB-1 sebocytes.

A third immortalized sebaceous gland cell line, Seb-E6E7, has been generated from adult human facial skin following a facelift procedure. Human sebocytes were immortalized by introduction of HPV16 E6 and E7 genes. Seb-E6E7 sebocytes were transduced by coculture with mitomycin C-treated packaging cells in the presence of 3 T3-J2 cells. Seb-E6E7 sebocytes, like SZ95 sebocytes, express both K7 and involucrin. In first experiments, Seb-E6E7 seem to respond to chemicals in a similar manner with SZ95 sebocytes despite their different transfection methods [15].

3 Acne Treatments Investigated in Experimental Sebocyte Culture Models

Cell culture models, especially the SZ95 sebaceous gland cell line, have advanced in excellent models to investigate new ingredients against seborrhea, acne, and aging skin.

Antiacne therapies investigated in vitro include retinoids, anti-androgens, and zileuton, a potent peroxisome proliferator-activated (PPAR)-α ligand antagonist [31, 32] (Table 6.3). Among these, the most investigated therapy in vitro is 13cis retinoic acid. Its mechanism of action has been elucidated by in vitro sebocyte research revealing that 13cis retinoic acid and all-trans retinoic acid inhibit the proliferation of cultured sebocytes in a dose- and time-dependent manner [22, 33, 34]. Marked decreases in wax esters, a slight decrease in squalene, and a relative increase in cholesterol level have been measured. It has been shown that 13cis retinoic acid undergoes intracellular isomerization to all-trans retinoic acid in human sebocytes, which then exerts its antiproliferative effect on sebocytes via binding to retinoic acid receptors (RAR) [35]. Also, 13cis retinoic acid causes cell cycle arrest and induced apoptosis in cultured sebocytes by a RAR independent mechanism [36] and may reduce the mRNA expression of pro-matrix metalloproteinase (MMP)-2, proMMP-9, proMMP-13, which are increased in acne [37].

Table 6.3 Mechanism of action of acne treatments investigated in in vitro sebocyte culture models
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Anti-androgens have been studied regarding their mechanism of action in acne. Anti-androgens, like spironolactone, inhibit the stimulatory effect of testosterone and 5α-dihydrotestosterone on sebocyte proliferation. They inhibit lipogenesis under the presence of peroxisome proliferator-activated receptor (PPAR) ligands [21]. Cyproterone acetate inhibits the activity of 3β-hydroxy-steroid dehydrogenase and blocks the androgen receptor [38].

Zileuton, the only known potent PPARα ligand antagonist, inhibits leukotriene B4 synthesis, thus reducing lipid synthesis [31, 32, 39]. Also, ectopeptidase inhibitors reduce proliferation and cytokine production in SZ95 sebocytes as well as the topical function of human peripheral T cells in vivo and in vitro [40].

Conclusions

Mammal sebocytes and sebocyte-like cells (human, mouse, hamster, rat) and human sebaceous gland cell lines (SZ95, SEB-1, Seb-E6E7) have been used in monolayer cultures as models to study specific functions involved in development, growth, and differentiation of sebaceous gland cells.

Maintenance of these cells in certain culture conditions has helped investigate the physiology of the sebaceous gland, including its changes in acne. Also, sebocyte culture models provide new chances for further research on biologically active ingredients, new pharmaceutical and cosmetic drugs for antiaging, and acne treatment. More complex culture systems, including three-dimensional models, are under development.