Acne is the disease manifesting from a multifactorial process involving the pilose- baceous appendages, and although the exact mechanism is unknown, four biologi- cal mechanisms are believed to play critical roles in the formation of a lesion:
1. increased sebum production,
2. aberrant keratinocyte proliferation and differentiation,
3. enhanced bacterial growth and metabolism, and
4. irritation and inflammation.
Acne is unique to humans, and although animals can be induced to form come- dones, there is no representative in vivo model aside from humans, which incorpor- ates the above-mentioned factors. Furthermore, of the four types of pilosebaceous appendages described by Kligman (1), only the sebaceous pilosebaceous unit, which contains a large sebaceous gland and a small vellus hair, has the propensity to develop acne. Even so, acne is still a remarkably rare event, with only a small per- centage of follicles being diseased at any single time.
A detailed structural analysis of the follicular plug, or comedone, that can develop into an acne lesion reveals a compacted agglomeration of desquamated follicular keratinocytes, bacteria, sebaceous gland products/remnants, and hair fibers. Finally, the static condition of the comedone allows for a favorable growth environ- ment for bacteria, such as Proprionibacterium acnes and Staphylococcus epidermidis that can lead to inflammation.
Although no in vivo or in vitro model exists to examine the entire complexity of lesion formation, several in vitro models are available to evaluate technologies targeting specific biological mechanisms occurring in acne. This review is aimed at disclosing the types of in vitro models available to evaluate technologies that may alleviate or prevent acne lesion formation.
EVALUATION OF TECHNOLOGIES TARGETING THE SEBACEOUS GLAND
The sebaceous gland of the skin is the part of the pilosebaceous unit, which secretes a unique lipid mixture of triglycerides, wax esters, and squalene during pro- grammed differentiation (2).
The sebaceous gland consists solely of one epithelial cell type, the sebocyte. Sebocyte migration out of the gland’s basal layer and full differentiation requires approximately two weeks, during which time the cells increase in volume 100- to
150-fold and become lipid-filled (3 – 6). Lipid secretion into the follicular duct
then occurs via a holocrine mechanism, and hence destroying the sebocyte. There- fore, the absolute rate and amount of sebum secretion is dependent upon sebaceous gland density and size, metabolic activity of the sebocytes, and lipogenic capacity of the ductal reservoir.
The movement of sebum through the follicular canal and onto the epidermal surface allows for interaction with bacterial and epidermal lipases that can degrade the triglyceride fraction partially or completely to free fatty acids (7 – 9).
Most of the literature documenting sebaceous gland physiology has resulted from the use of human volunteer subjects or animal models. Several in vitro models have been developed for further understanding sebocyte physiology, which are based on two technologies: the in situ culture of the human sebaceous gland and the in vitro culture of sebocytes in a monolayer with and without fibroblast feeder support.
Sebaceous Gland Organ Culture Maintenance
Culture of the entire sebaceous gland offers several advantages in comparison to sebocyte monolayer culture. For example, as the three-dimensional architecture is maintained, proliferation and lipid synthesis can be simultaneously examined in the basal and differentiated cell layers, respectively. An additional advantage over animal models lies in the fact that delivery of potential sebum suppressive compounds to the gland is not hindered, so absolute biological efficacy can be evaluated.
Sebaceous glands can be isolated by dissection of human skin (surgical waste, donor, cadaver, etc.) by a variety of methods including microdissection (10) and shearing (11). Once obtained, the glands are placed directly in growth medium (or can be floated by placement upon nitrocellulose filters) and incubated at 378C in the presence of carbon dioxide (12). Studies using intact sebaceous glands with and without the attached dermal/epidermal components have been utilized to measure lipogenesis rates in subjects with acne (13) and the effect of substrates on lipid rates and patterns (14 – 16). Acetate was determined to be the most pre- ferred substrate resulting in maximal incorporation into newly formed lipids. Pappas et al. (17) reported that the sebaceous gland selectively utilizes the 16:0 fatty acid for incorporation into wax esters and elongated fatty acids such as
18:0. As a control, acetate was incorporated into all of the cellular and secreted lipids.
During prolonged incubation at the air/liquid interface, newly formed sebo- cytes tend to differentiate similarly as interfollicular keratinocytes taking on a flat- tened phenotype with minimal lipid synthesis occurring. Through a thorough investigation of factors impeding differentiation, Guy et al. (18,19) reported that many of the common growth medium components, such as phenol red, HEPES buffer, epidermal growth factor (EGF), and serum, reduced overall lipogenesis. In the absence of phenol red (which is known to have estrogen-like properties) and EGF, the intact sebaceous gland can be maintained for up to two weeks without sig- nificant necrosis (Fig. 1). DNA, protein synthesis, and lipogenesis rates remained consistent over the maintenance period.
In the presence of estradiol (600 pM) and 13-cis-retinoic acid (1 mM), lipogen- esis rates were decreased on average by 36% and 48%, respectively (Fig. 1). Histo- logically, a thickening of the undifferentiated cell layer accompanied by luminal keratinization was evident.
FIGURE 1 Organ culture of the human sebaceous gland. Sebaceous glands can be maintained in culture over seven days with no discernable loss in proliferation or lipid synthesis. (A) Freshly isolated human sebaceous gland. (B) Sebaceous gland maintained in culture for seven days. (C) As in (B), but supplemented with 600 pM 17-b-estradiol. Rates of cell division are comparable to control but lipid synthesis is decreased on average by 41%. (D) As in (B), but supplemented with 1 mM
13-cis-retinoic acid. Rates of cell division and lipid synthesis are decreased by 36% and 48%,
respectively. Source: From Ref. 19.
Surprisingly, testosterone and dihydrotestosterone (DHT) had no effect on enhancing lipogenesis in the cultured glands, possibly because the glands were already at maximal lipogenesis rates. This finding implied that androgens are not necessary for the short-term stimulation of glandular function or, alternatively, that the growth conditions for sebaceous gland maintenance have been fully optimized.
Additionally, peroxisome proliferator activated receptors (PPARs) have been shown to be important in the regulation of lipid metabolism in adipose and liver cells. PPARs are nuclear hormone receptors and sebocytes express the subtypes a, b, and g that appear to play important roles in proliferation and differentiation. Downie et al. (20,21) investigated the role of PPAR and additional nuclear hormone receptor ligands on sebaceous gland proliferation and differentiation.
PPARa, PPARb, PPARg, retinoid-X-activated receptor a, liver-X-activated receptor a, and pregnane-X-receptor, but not farnesoid-X-activated receptors, were all detected in fresh and seven-day maintained glands. The PPAR ligands arachidonic acid, BRL-49653, clofibrate, prostaglandin J2, eicosatetraynoic acid (EYTA), leukotriene B4, linoleic acid, linolenic acid, oleic acid, and WY-14643 all inhibited lipid synthesis, whereas bezafibrate, juvenile hormone III, and farnesol were ineffective. With the exception of EYTA at 10 mM, no effects on gland prolifer- ation were evident (20,21).
The sebaceous gland in situ model is also applicable for the culture of sebac- eous glands isolated from animals. Toyoda and Morohashi (22) performed organ
culture on sebaceous glands obtained from mice to evaluate the effects of neuropep- tides (calcitonin gene-related peptide, substance P, vasoactive intestinal polypep- tide, and neuropeptide Y) in addition to nerve growth factor. Sebaceous glands treated with substance P resulted in accelerated lipid synthesis over the control glands by increasing the rate of proliferation and differentiation postulating that stress could participate in the pathogenesis of acne.
Sebocyte Monolayer Culture
Although the advantages of sebaceous gland organ culture are evident, the pro- cedure is time-consuming and dependent on continued sources of viable tissue for additional glands. Cell culture offers an alternative over organ culture mainten- ance, as large numbers of cells can be processed and frozen back allowing multiple experiments to be performed on a similar cell lineage. However, continued holo- crine destruction of sebocytes also necessitates a stem cell population required for maintenance. Stem cells are self-renewing and are capable of generating large numbers of differentiated progeny throughout an individual’s life span (23). Stem cells can also generate a transit amplifying population, which has a high prob- ability of postmitotic differentiation. Within the human sebaceous gland, transit amplifying cells have been detected (24,25). For a recent review on hair follicle stem cells, refer to Lavker et al. (26).
The in vivo data appeared consistent with early in vitro experiments that failed to yield viable sebocytes during extended culture conditions (27 – 29). This resulted in a similar but less stringent need as organ culture for ample biopsies to supply sufficient sebocytes for experimentation.
Several laboratories have successfully cultured human sebocytes in the pre- sence of fibroblast support and in serum-free conditions to examine the effects of serum, growth factors, and biopsy location (30 – 33). Sebocyte culture provides an excellent model to change local environmental growth conditions (i.e., hormones, vitamins, carbohydrates, etc.) and to evaluate lipogenesis inhibitors in a higher throughput mode than that which could normally be obtained through the use of organ culture maintenance. This can be accomplished either through extended culture to evaluate gross morphological changes (i.e., sebocyte size, keratin expression patterns, and lipid vacuole content) or through rapid evaluation using radiolabeled substrates. Tritiated thymidine will be incorporated into the cells as a marker for proliferation, whereas radiolabeled glucose and/or acetate can be used as tracers for lipid synthesis.
Two procedures are suitable for generating sebocyte monolayer cultures. In the explant outgrowth method, isolated sebaceous glands are attached to culture plates by limited drying followed by addition of growth medium. Fibroblast- feeder support can later be added, but this appears not to be necessary. After several days of incubation, proliferative basal sebocytes are seen as visible out- growths from the gland (Fig. 2).
Alternatively, a much more common approach is to digest sebaceous glands by limited trypsin proteolysis and plate the released cells in mass culture upon a fibroblast feeder layer. After several days, colonies of attached cells will begin to proliferate into small colonies that can be further cloned and/or passed.
During the proliferative stage, sebocytes are indistinguishable from keratino- cytes expressing keratins 5 and 14. Upon reaching confluence, the cells take on a sebocytic phenotype as they enlarge, express keratins 4, 7, and 13, and become
FIGURE 2 Primary culture of human sebocytes derived from explant growth. Human sebocytes can be propagated in monolayer culture by outgrowth of sebaceous glands through in vitro explant culture. (A, inset) Colony size at day 5. (B, main photograph) Colony size at day 10. Source: From Ref. 31.
lipid filled. Detection of an intracellular lipid droplet is evident by retention of lipo- philic dyes such as Nile red, oil red O, or Sudan black (Fig. 3). Sebocytes, in com- parison to cultured keratinocytes, synthesize and accumulate considerably more intracellular lipids at comparable stages of differentiation (Fig. 4), and analysis by thin layer chromatography (TLC) reveals similar classes of sebum-specific lipids as those made de novo (Fig. 5).
Although sebocytes can be cultured for an extensive passage number (34), there may be a tendency for sebaceous cells to de-differentiate to varying degrees, especially in regard to sebum specific lipids. The rate-limiting step in squa- lene synthesis shifts, as the cells tend to accumulate higher levels of cholesterol, which is then esterified into cholesterol esters. This de-differentiation appears to have been corrected by Zouboulis et al. (35), when freshly isolated cells were immortalized by transfection with a PBR-322 based plasmid containing the coding region of the simian virus-40 large T antigen. These sebocytes have been pas- saged over 50 times with no discernable loss of phenotype (Fig. 6).
Regardless of the culture conditions being utilized, sebocyte monolayer culture has been used successfully to answer many questions regarding cell metabolism and lipid synthesis, specifically the androgen metabolism and retinoid pathways.
Fujie et al. (31) reported that the androgens testosterone and DHT stimulated the proliferation as well as the lipid synthesis of sebocytes in a dose-dependent manner with maximal effects occurring at 1029 and 10210 M, respectively. Zoubou- lis et al. (32) found that testosterone stimulated facial sebocyte proliferation by 50% at 1026 M and DHT at 1028 M. In the presence of the antiandrogen spironolactone, sebocyte proliferation was reduced by 50% at 1027 M and additionally, the stimu- latory effect seen with DHT was neutralized. Spironolactone has been successfully used as a treatment for acne (36,37), suggesting that the inhibitory effects of anti- androgens may be occurring at the cellular level.
The stimulatory effect seen with androgens in sebocyte monolayer culture is in contrast to the results seen in organ culture in which no effects were evident. This
FIGURE 3 The in vitro culture of human sebocytes. Human sebocytes can be cultured in vitro under a variety of conditions including the presence/absence of serum, phenol red, and fibroblast support to attain sebocyte-specific differentiation characteristics. (A) Preconfluent human sebocytes cultured in vitro in serum-free medium in the absence of fibroblast support. (B) Sebocytes cultured in phenol red-free and serum-free medium at seven days postconfluence. Note the accumulation of intracellular lipid droplets. (C) Sebocytes at seven days postconfluence stained with oil red O confirming the presence of intracellular lipids.
may be due to a dilution of inherent androgens, as sebocytes multiply to a greater extent in monolayer culture. However, it has been documented that sebocytes are able to synthesize testosterone from adrenal precursors in parallel to an inactivation process to maintain androgen homeostasis (38). Alternatively, the presence of ingre- dients in the growth medium such as phenol red, which has inhibitory activity via estrogen-like properties, may account for the stimulatory effects seen with androgens. It is suggested that all experiments with cultured sebocytes be performed using phenol red-free medium under reduced lighting conditions for optimal benefits.
FIGURE 4 Oil red O staining of sebocyte and keratinocyte cultures. Upon reaching confluence, sebocyte differentiation involves synthesis and accumulation of intracellular lipid droplets. (Left): Human sebocytes cultured in vitro for seven days postconfluence in serum-free/phenol red-free medium (60 mm2 area). Note the extensive oil red O staining within the dish confirming the presence of lipids. (Right): Similar aged culture of human keratinocytes stained with oil red O. Note the absence of significant intracellular lipids.
It is well-known that oral administration of 13-cis-retinoic acid reduces sebaceous gland size and lipid synthesis, whereas the all-trans and 9-cis isomers of retinoic acid are significantly less effective. Therefore, cultured sebocytes have also been utilized as a model to further understand retinoid responsiveness and potential mechanism of action.
Tsukada et al. (39) reported that isomerization of 13-cis-retinoic acid to all- trans retinoic acid occurs in SZ-95 sebocytes, subsequently providing the biological benefit. No isomerization was evident in HaCaT keratinocytes and the reverse
FIGURE 6 Culture of immortalized sebocytes. Human facial sebocytes can be immortalized to retain sebocyte-specific differentiation characteristics by transfection with simian virus-40 large T antigen. The resulting cell line (SZ-95) was further cloned and extensively passaged with no discernible loss of features. (A) Preconfluent cultures of human sebocytes prior to transfection. (B) First passage of SZ-95 immortalized sebocytes. (C) SZ-95 immortalized sebocytes at 50th passage. Note the appearance similar to sebocytes from first passage. Source: From Ref. 35.
conversion of all-trans to 13-cis-retinoic acid was not evident in sebocytes. Both
13-cis and all-trans retinoic acid suppressed mRNA expression of cytochrome P450-1A2, and in addition, 13-cis, all-trans, and 9-cis-retinoic acid reduced sebo- cyte proliferation on average by 40% at 1027 M. Retinoids also do not appear to effect the programmed cell death of human sebocytes (40).
In additional studies, Zouboulis et al. (41,42), using nontransfected sebocytes, reported that 13-cis-retinoic acid reduced radiolabeled acetate incorporation by
48%, whereas all-trans and acitretin only reduced incorporation by 38% and 27%, respectively.
Additional reports using immortalized sebocytes have also been documented to measure expression of steroidal enzymes (43), PPARs, and transcriptional factors (44), in addition to melanocortin receptors and the effects of select interleukins (ILs) (45). Finally, the patent databases are an excellent source of reviewing intellectual property on sebum suppressive technologies.
Rat Preputial Sebocyte Monolayer Culture
The rodent preputial gland has also been used as a model for the human sebaceous gland. These specialized glands open to the surface on either side of the urethral meatus, and secretions are involved in territorial marking and mating behavior (46).
Using techniques similar to that used with human sebaceous glands, rat prepu- tial glands can be isolated, digested, and cultured upon a fibroblast feeder layer to produce monolayer cultures (47,48) that can be manipulated and studied. Rosenfield (49) reported on the relationship of sebaceous cell stage with growth in culture in that
fully differentiated sebocytes are not capable of attachment and proliferation, whereas early differentiated cells are. Subsequent studies by Laurent et al. (48) docu- mented the requirement of serum and growth factors to enhance growth and that preputial sebocytes also expressed cytokeratin-4 and formed fewer cornified envelopes than keratinocytes. The effects of estrogen on preputial cell behavior (50) and characterization of the cultured cells (51,52) along with the mechanism of androgen action (53) and effects of PPARs (54) have also been reported.
Preputial cells differentiate in a similar process, but to an overall lesser extent than human sebocytes even in the presence of androgens (Fig. 7). Rosenfield et al. (55) discovered that preputial cells cultured in vitro lack the presence of PPAR ligands that will induce lipid droplet formation. The effect of PPAR ligands was found to be distinct and additive to the effect seen with androgens in increasing lipid droplet formation.
FIGURE 7 Differentiation of preputial sebocytes. Preputial sebocytes can be cultured in vitro with 3T3-fibroblast support. Differentiated sebocytes containing lipids are positively identified via staining with oil red O. (Top left): Oil red O staining of preputial sebocytes cultured in vitro for nine days in which 11% of the culture has differentiated into lipid-forming colonies. (Lower left): Oil red O staining of preputial sebocytes cultured in the presence of
1026 M dihydrotestosterone (DHT) in which 25% of the culture has differentiated into lipid-
forming colonies. (Top right): Oil red O staining of preputial sebocytes cultured in the presence of 1026 M peroxisome proliferator activated receptor g ligand BRL-49653 in which
66% of the culture has differentiated into lipid-forming colonies. (Lower right): Oil red O staining of preputial sebocytes cultured in the presence of 1026 M DHT and BRL-49653 in which 80% of the culture has differentiated into lipid-forming colonies. Abbreviation: DHT, dihydrotestosterone. Source: From Ref. 53.
Kim et al. (56), furthermore, investigated the effect of retinoids on prepu- tial cell growth and differentiation. All-trans retinoic acid and retinoic acid receptor agonists resulted in a decrease in cell number, colony size, and number of lipid-forming colonies. Kim concluded that retinoic acid receptors and retinoid-X-receptors play select roles in sebocyte proliferation and differentiation.
Other In Vitro Sebaceous-Cell-Based Models
Animal models such as the hamster flank organ and hamster ear have been devel- oped to study the effects of anti-acne and sebum-suppressive capabilities of com- pounds and formulations in vivo. Following application, the target areas are excised, weighed, and processed for histological examination. In other animal models, monitoring of sebum secretions follows intravenous or oral administration of sebum-suppressive agents.
The hamster auricle sebaceous gland has been successfully cultured and utilized as a model for human sebaceous glands. Glands are isolated and cells are grown on a 3T3 fibroblast-feeder layer using standard protocols. Ito et al. (57), Sato et al. (58), and Akimoto et al. (59) have reported that the amount of lipid produced increases following the peak of sebocyte proliferation and that androgens stimulate, whereas retinoic acid, EGF, and vitamin D3 sup- press overall activity.