Development of new 3D human ex vivo models to study sebaceous gland lipid metabolism and modulations

Abstract

Objectives: Sebaceous glands maintain skin homeostasis by producing sebum. Low production can induce hair loss and fragile skin. Overproduction provokes seborrhoea and may lead to acne and inflammatory events. To better study sebaceous gland maintenance, sebocyte maturation, lipid production and ageing or inflammatory processes, we developed innovative 3D ex vivo models for human sebaceous glands.

Materials and methods: Culture conditions and analytical methods optimized on sebocyte monolayers were validated on extracted sebaceous glands and allowed the development of two 3D models: (a) "air-liquid" interface and (b) human fibronectin-coated "sandwich" method. Lipid production was assessed with microscopy, fluorometry or flow cytometry analysis after Nile Red staining. Specific lipids (particularly squalene and peroxidized squalene) were measured by Gas or liquid Chromatography and Mass spectrometry.

Results: This study allowed us to select appropriate conditions and design Seb4Gln culture medium inducing sebocyte proliferation and neutral lipid production. The "air-liquid" model was appropriate to induce sebocyte isolation. The "sandwich" model enabled sebaceous gland maintenance up to 42 days. A treatment with Insulin Growth Factor-1 allowed validation of the model as we succeeded in mimicking dynamic lipid overproduction.

Conclusion: Functional sebocyte maturation and physiological maintenance were preserved up to 6 weeks in our models. Associated with functional assays, they provide a powerful platform to mimic physiological skin lipid metabolism and to screen for active ingredients modulating sebum production.

Keywords: 3D model; sebaceous gland; sebocyte; sebum; skin lipid metabolism; squalene.

Figure 1

Protocol used for SG isolation and ex vivo culture. A, Skin sample with epidermis (Ep), dermis (De) and a single hair (black arrow). B, C, Progressively isolated SG (red arrow). D, Two SG culture protocols. D1, “Air‐Liquid” 3D model: SG cultivated in a fibronectin‐coated plate with only 600 µL culture medium; D2, “Sandwich” 3D model: SG cultivated between fibronectin‐coated plate bottom and coverglass. Scale bar 600 µm

Figure 2

Excitation and emission wavelengths used for lipid analysis compared to fluorescence excitation and emission spectra of Hoechst, Nile Red in triglycerides (neutral lipids) and in phospholipids (polar lipids). Dotted and solid line curves represent, respectively, excitation and emission spectra of Hoechst (blue), Nile Red in triglycerides (yellow) or phospholipids (red). Excitation and emission intensity (relative intensity) are expressed as a percentage of excitation or emission peak (=100%). Under each graph are represented wavelengths used in fluorometry (A) and in flow cytometry (B). Exc FL and EM FL, Excitation and Emission fluorescence wavelengths

Figure 3

Effect of different media on sebocyte starting P2 and lipid production. Seb7, Green and Seb5 media were tested as control. A, Sebocyte growth curves. Average of three biological replicates. Error bars represent sd B‐ Ratio (relative fluorescence) between Nile Red (Excitation 475 nm—Emission 530 nm) and Hoechst Signal (Excitation 356 nm—Emission 465 nm) obtained by fluorimetry and revealing total lipid production. C, Ratio between Nile Red (Excitation 520 nm—Emission 625 nm) and Hoechst Signal revealing neutral lipid production. D‐M, Pictures of sebocytes in brightfield (I‐M) or stained with DAPI and Nile Red (D‐H) after 5 (P5) (D‐G) or four subcultures (P4) (H‐M) in Seb7 (D, I), Seb4 (E, J), Seb4Gln (F, K), Seb5 (G, L) or Green (H, M) medium. In red, total lipid. In yellow, merge of total (red) and neutral lipid (green). In blue, nuclei (DAPI). Scale bar 100 µm

Figure 4

Lipid production by sebocytes and glutamine effect revealed by flow cytometry. A, Neutral lipid production by sebocytes grown 3 weeks with (Seb4Gln) or without glutamine (Seb4) revealed by FL1 channel after Nile Red staining. Green and Seb5 media were tested as control. B, Nile Red MFI obtained with the four different channels FL1, FL2, FL3 and FL4 in the same conditions. C, Dot plots showing Nile Red signal in FL1 channel (neutral lipids—C1, C2, C4, C5) or FL4 channel (polar lipids—C3, C6) for sebocytes grown 7 days with (Seb4Gln—C5, C6) or without glutamine (Seb4—C2, C3). Sebocytes grown in Seb5 (C1) and Green medium (C4) were used as control. NR‐dim and NR‐bright, sebocyte population with, respectively, low and high Nile Red signal. D, Nile Red MFI ratio between NR‐bright and NR‐dim populations after 7 days (Week1) or 21 days (Week3) of sebocyte culture in Seb4 and Seb4Gln media. One repetition was done each week

Figure 5

Staining of isolated sebocytes obtained with “air‐liquid” method (A) and SG cultivated during a long period with “sandwich” method (B). A, Isolated sebocytes growing in Seb4Gln, around a SG from an abdomen skin sample (A1), after 3 (A2‐A4) or 5 subcultures (A5). B, Fresh (B1‐B3) and ex vivo cultivated SG during 42 days with “sandwich” method (B4‐B6) observed before (B1) or after staining (B2‐B6) with MC5R (B2, B5—green) or Muc‐1 antibody (B3—Green, B6—red). B2: Brightfield control. Photos obtained with confocal microscope (B2, B3) or fluorescence microscope (A1‐A5, B1, B4‐B6). DAPI: 4′,6‐diamidino‐2‐phénylindole, K7: cytokeratin 7, K5: cytokeratin 5, Muc 1: Mucin‐1, MC5R: Melanocortin 5 Receptor, PPARγ: Peroxisome proliferator‐activated Receptor gamma. Scale bar 100 µm

Figure 6

Lipid quantification in SG after 6 days’ maintenance according to skin origin and culture media (Green or Seb4Gln media supplemented or not with IGF‐1)