Ex Vivo Live Full-Thickness Porcine Skin Model as a Versatile In Vitro Testing Method for Skin Barrier Research

Abstract

Since the European Union (EU) announced their animal testing ban in 2013, all animal experiments related to cosmetics have been prohibited, creating a demand for alternatives to animal experiments for skin studies. Here, we investigated whether an ex vivo live porcine skin model can be employed to study the safety and skin barrier-improving effects of hydroxyacids widely used in cosmetics for keratolytic peels. Glycolic acid (1-10%), salicylic acid (0.2-2%), and lactobionic acid (1.2-12%) were used as representative substances for α-hydroxyacid (AHA), β-hydroxyacid (BHA), and polyhydroxyacid (PHA), respectively. When hydroxyacids were applied at high concentrations on the porcine skin every other day for 6 days, tissue viability was reduced to 50-80%, suggesting that the toxicity of cosmetic ingredients can be evaluated with this model. Based on tissue viability, the treatment scheme was changed to a single exposure for 20 min. The protective effects of a single exposure of hydroxyacids on skin barrier function were evaluated by examining rhodamine permeability and epidermal structural components of barrier function using immunohistochemistry (IHC) and immunofluorescence (IF) staining. Lactobionic acid (PHAs) improved skin barrier function most compared to other AHAs and BHAs. Most importantly, trans-epidermal water loss (TEWL), an important functional marker of skin barrier function, could be measured with this model, which confirmed the significant skin barrier-protective effects of PHAs. Collectively, we demonstrated that the ex vivo live full-thickness porcine skin model can be an excellent alternative to animal experiments for skin studies on the safety and efficacy of cosmetic ingredients.

Keywords: ex vivo skin model; hydroxyacids; skin barrier; skin permeability; stratum corneum.

Figure 1

Classification of hydroxyacids used in this study (A) AHA (glycolic acid), (B) BHA (salicylic acid), and (C) PHA (lactobionic acid). AHA: α-hydroxyacid, BHA: β-hydroxyacid, PHA: polyhydroxyacid.

Figure 2

Damage to ex vivo porcine skin after repeated application of hydroxyacids at high concentrations Hydroxyacids were applied at high concentrations on the porcine skin every other day for six days: AHA, 5–10%; BHA, 1–2%; and PHA, 6–12%. (A) Tissue viability of the treated ex vivo porcine skin was determined using the WST-1 assay. Values are mean ± SE (n ≥ 3). Significant differences are denoted by * p < 0.05 and ** p < 0.01. (B) Porcine skin was processed and H&E stained. A representative microphotograph is shown. NC; negative control (30% ethanol).

Figure 2

Damage to ex vivo porcine skin after repeated application of hydroxyacids at high concentrations Hydroxyacids were applied at high concentrations on the porcine skin every other day for six days: AHA, 5–10%; BHA, 1–2%; and PHA, 6–12%. (A) Tissue viability of the treated ex vivo porcine skin was determined using the WST-1 assay. Values are mean ± SE (n ≥ 3). Significant differences are denoted by * p < 0.05 and ** p < 0.01. (B) Porcine skin was processed and H&E stained. A representative microphotograph is shown. NC; negative control (30% ethanol).

Figure 3

Effects of a single treatment with low-concentration hydroxyacids on ex vivo porcine skin Hydroxyacids were applied once for 20 min at low concentrations: AHA, 1–5%; BHA, 0.2–1%; and PHA, 1.2–6%. The ex vivo porcine skin was washed and incubated for six days. (A) Tissue viability of the treated ex vivo porcine skin was determined using the WST-1 assay. Values are mean ± SE (n ≥ 3). (B) Porcine skin was processed and H&E stained. A representative microphotograph is shown. NC; negative control (30% ethanol). The area in which the stratum corneum is well-organized compared to the negative control is indicated by black arrow heads.

Figure 4

Effect of hydroxyacids on the skin penetration of rhodamine B Permeability of rhodamine B was evaluated after 24-h incubation of treated tissue. The fluorescent images represent rhodamine B (red) and DAPI (blue) staining. D: Dermis, E: Epidermis.

Figure 5

Effect of hydroxyacids on skin barrier function. Measurement using a vapometer with a closed chamber. Measurements were consecutively repeated three times for each skin biopsy. The mean of the three measurements was used as a representative value. Significant differences are denoted by * p < 0.05 and ** p < 0.01.

Figure 6

Immunohistochemical staining of key protein components of the skin epidermis Immunohistochemical images of ex vivo porcine skin with antibodies against (A) K5, (B) K1, (C) K10, (D) LOR, (E) FLG, and (F) PCNA. K1: Keratin 1, K5: Keratin 5, K10: Keratin 10, LOR: loricrin, FLG: filaggrin, PCNA: proliferating cell nuclear antigen. The regions in which the expression of the specific protein is significantly higher than the negative control are indicated by red arrows. All scale bar represents 50 μm.

Figure 6

Immunohistochemical staining of key protein components of the skin epidermis Immunohistochemical images of ex vivo porcine skin with antibodies against (A) K5, (B) K1, (C) K10, (D) LOR, (E) FLG, and (F) PCNA. K1: Keratin 1, K5: Keratin 5, K10: Keratin 10, LOR: loricrin, FLG: filaggrin, PCNA: proliferating cell nuclear antigen. The regions in which the expression of the specific protein is significantly higher than the negative control are indicated by red arrows. All scale bar represents 50 μm.

Figure 7

Immunofluorescence staining of tight junction protein, claudin 1, in the skin epidermis. Immunofluorescence images of ex vivo porcine skin with antibodies against tight junction protein, CLDN1: claudin 1 is shown in red and DAPI is shown in blue.

Figure 8

Quantification of the cells expressing marker proteins in immunohistochemistry and immunofluorescence images. 3,3′-Diaminobenzidine (DAB)-positive pixels were quantified in the epidermis excluding the hair follicle region using Qupath software [16]. The DAB positive pixels were normalized with total stained pixel counts (hematoxylin + DAB) in IHC or DAPI positive pixels in IF to calculate the percentage of cells expressing the marker protein. (A) K1, (B) K5, (C) K10, (D) LOR, (E) FLG, (F) PCNA, and (G) Claudin 1. Data are presented as the mean ± SEM (n = 3–4 fields per a group). p < 0.05 was considered significant (Student’s t test vs NC; * p < 0.05; ** p < 0.01; *** p < 0.001).