Characterization of Reconstructed Human Epidermis in a Chemically-Defined, Animal Origin-Free Cell Culture

Abstract

The Reconstructed Human Epidermis (RHE) model derived from epidermal keratinocytes offers an ethical and scientific alternative to animal experimentation, particularly in cutaneous toxicology and dermatological research, where the elimination of animal cruelty is of paramount importance. Thus, we compared commercially available chemically defined animal origin-free (cdAOF) supplements, designed for regenerative medicine, to the widely utilized supplement (human keratinocyte growth supplement), which contains growth factors and bovine pituitary extract. Herein we present the extended characterization of RHE derived from newborn, adult, and immortalized N/telomerase reverse transcriptase keratinocytes under cdAOF conditions. Culture of RHE in the cdAOF media produced histological features that were similar to that produced using human keratinocyte growth supplement, with the exception that the basal keratinocytes were less cylindrical. Additionally, immunolocalization of involucrin in the basal layer and increased mRNA expression of several inflammatory-proliferative markers were observed under cdAOF conditions. In RHEs cultured in cdAOF media, expression and immunolocalization of other expected markers of keratinization were similar, while monitoring of barrier function (transepithelial electrical resistance) revealed results that were statistically equal to, or lower than those observed in RHE cultured in human keratinocyte growth supplement. Our study indicates that reconstruction of RHE was accomplished under cdAOF culture conditions and that further refinement could promote an expanded use beyond regenerative medicine, for in vitro toxicology applications.

Keywords: Animal-free culture; Keratinocytes; Reconstructed human epidermis; Tissue engineering.

Figure 1

Microanatomy and barrier differences of the skin models reconstructed with HEK, HEK, or immortalized N/TERT keratinocytes, as cultured with HKGS, HKS, HKS + HKGE, or HFS supplement, respectively. (a) Histological sections of RHE stained with H&E. Scale bar = 50 μm. Pictures are representatives of 3 independent experiments. Magnification (×100). (b) TEER measurements performed on RHE (n = 3 independent experiments). Data represent mean ± SEM. Two-way ANOVA was performed, followed by Dunett`s tests. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. HEK, human epidermal keratinocytes; HFS, human fibroblast supplement; HKGE, human keratinocyte growth supplement; HKGS, Human keratinocyte growth supplement; HKS; high keratinocyte serum-free medium; RHE, reconstructed human epidermis; TEER, trans-epidermal electrical resistance; TERT, telomerase reverse transcriptase.

Figure 2

Proliferation of keratinocytes in RHE via Ki67 immunolocalization. (a) IHC staining for Ki67 revealed differences in proliferation marker intensity in skin models reconstructed with HEKn, HEKa, or N/TERT keratinocytes cultured with HKGS, HKS, HKS + HKGE, or HFS supplements (n = 3 independent experiments). Scale bar = 50 μm. Magnification (×100). (b) Quantification of proliferation marker Ki67 by double-blind test (n = 9). Quantification was made on three different areas of each skin model of every condition. Data represent mean ± SEM. Two-way ANOVA was performed, followed by Dunett`s tests. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. HEK, human epidermal keratinocytes; HFS, human fibroblast supplement; HKGE, human keratinocyte growth supplement; HKGS, Human keratinocyte growth supplement; HKS; high keratinocyte serum-free medium; RHE, reconstructed human epidermis; TERT, telomerase reverse transcriptase.

Figure 3

Immunostaining and localization of differentiation-related markers in RHE. (a) CK14. (b) IVL. (c) CK10 (d) LOR. (e) FLG. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Pictures are representatives of 3 independent experiments. CK, cytokeratin; IVL, involucrin; LOR, Loricrin; RHE, reconstructed human epidermis.

Figure 3

Immunostaining and localization of differentiation-related markers in RHE. (a) CK14. (b) IVL. (c) CK10 (d) LOR. (e) FLG. Nuclei were stained with DAPI (blue). Scale bar: 50 μm. Pictures are representatives of 3 independent experiments. CK, cytokeratin; IVL, involucrin; LOR, Loricrin; RHE, reconstructed human epidermis.

Figure 4

Transcriptional study of genes related to epidermal differentiation, inflammation, and proliferation. mRNA expression was assessed by RT-qPCR. Gene expression in RHE reconstructed of (a) HEKn, (b) HEKa, and (c) immortalized N/TERT keratinocytes. Data represent mean ± SEM (n = 3 independent experiments). A 2-way ANOVA test was performed, followed by Dunnett`s tests. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. HEK, human epidermal keratinocytes; RHE, reconstructed human epidermis; TERT, telomerase reverse transcriptase.