Effects of Serum-Free Culture at the Air–Liquid Interface in a Human Tissue-Engineered Skin Substitute

Abstract

Previous studies have reported that well-defined culture conditions can improve keratinocytes terminal differentiation and reproducibility. The aim of our study was to compare skin substitutes cultured in a complete medium with those cultured in a serum-free medium at the air-liquid interface to optimize the self-assembly method. Skin substitutes, cultured in a serum-free medium over 7, 14, and 21 days, were compared with others cultured in a complete medium (5% serum) over the complete culture period. Masson's Trichrome staining showed that the substitutes cultured in a serum-free medium generated a well-developed and differentiated epidermis. Immunolabeling analyses between the substitutes cultured without serum and those cultured in complete serum showed similar expression of epidermal differentiation markers, dermo-epidermal junction, and dermal extracellular matrix components. On the basis of our Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) results, the skin substitutes cultured in serum-free condition over 21 days of culture at the air-liquid interface showed lower frequencies of the CH(2) symmetric mode of vibrations, which means a better lipid organization of the stratum corneum. No significant difference in hydrocortisone penetration was observed between serum-free medium substitutes and the controls. Results demonstrate that the absence of serum does not compromise the characteristics of the skin substitutes observed in this study.

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“…We demonstrated that our human skin equivalents mimic many aspects of human skin, namely, the morphology and expression of early and late differentiation markers [41]. Nevertheless, as reported by other groups, these skin substitutes showed a decreased permeability barrier compared with human skin when performing diffusion studies [46].…”

“…Thus, it allows for the production of skin substitutes exempt of exogenous material [39,40]. Their epidermal layer is fully differentiated with a well-defined stratum corneum, which shows a good barrier function [41]. In developing topical drug formulations and assessing their toxic effects, we often need to perform in vitro diffusion experiments through skin.…”

Keratinocytes Propagated in Serum-Free, Feeder-Free Culture Conditions Fail to Form Stratified Epidermis in a Reconstituted Skin Model

“…Moreover, evidence suggests that fibroblasts also provide key factors needed for differentiation; keratinocytes form fully stratified skin in serum-free media when seeded onto fibroblast-coated DEDs or on collagen gels embedded with supporting fibroblasts - an alternative skin equivalent model [13], [33], [34]. However, our work shows that fibroblasts feeder support is not absolutely required; keratinocytes that have never been co-cultured with fibroblasts or exposed to fibroblast-conditioned media will form stratified skin in serum-supplemented media.…”

Biokinetics and xenobiotic bioavailability

“…Acceptable intervals for periodic testing to confirm the genetic, phenotypic and immunological stability of the cell culture are highly case-dependent (Blázquez-Prunera et al, 2017 [43] ) (Daily et al, 2017 [44] ; Meza-Zepeda et al, 2008 [45] ). Therefore, this aspect should be included in the specific test system SOP(s).…”

“…Thus, in an in vitro system, the freely dissolved concentration of the test item in the medium or in the cell (being as close to the target as possible) is the central parameter. Some processes (Groothuis et al, 2015 [42] ; Heringa et al, 2006 [43] ); depicted schematically in , result in a freely dissolved concentration that is not the same as the nominal concentration (i.e., the added concentration).…”