Modulation of stratum corneum lipid composition and organization of human skin equivalents by specific medium supplements.

Our in-house human skin equivalents contain all stratum corneum (SC) barrier lipid classes, but have a reduced level of free fatty acids (FAs), of which a part is mono-unsaturated. These differences lead to an altered SC lipid organization and thereby a reduced barrier function compared to human skin. In this study, we aimed to improve the SC FA composition and, consequently, the SC lipid organization of the Leiden epidermal model (LEM) by specific medium supplements. The standard FA mixture (consisting of palmitic, linoleic and arachidonic acids) supplemented to the medium was modified, by replacing protonated palmitic acid with deuterated palmitic acid or by the addition of deuterated arachidic acid to the mixture, to determine whether FAs are taken up from the medium and are incorporated into SC of LEM. Furthermore, supplementation of the total FA mixture or that of palmitic acid alone was increased four times to examine whether this improves the SC FA composition and lipid organization of LEM. The results demonstrate that the deuterated FAs are taken up into LEMs and are subsequently elongated and incorporated in their SC. However, a fourfold increase in palmitic acid supplementation does not change the SC FA composition or lipid organization of LEM. Increasing the concentration of the total FA mixture in the medium resulted in a decreased level of very long chain FAs and an increased level of mono-unsaturated FAs, which lead to deteriorated SC lipid properties. These results indicate that SC lipid properties can be modulated by specific medium supplements.

Nature versus nurture: does human skin maintain its stratum corneum lipid properties in vitro?

Human skin equivalents (HSEs) mimic human skin closely, but show differences in their stratum corneum (SC) lipid properties. The aim of this study was to determine whether isolation of primary cells, which is needed to generate HSEs, influence the SC lipid properties of HSEs. For this purpose, we expanded explants of intact full thickness human skin and isolated epidermal sheets in vitro. We investigated whether their outgrowths maintain barrier properties of human skin. The results reveal that the outgrowths and human skin have a similar morphology and expression of several differentiation markers, except for an increased expression of keratin 16 and involucrin. The outgrowths show a decreased SC fatty acid content compared with human skin. Additionally, SC lipids of the outgrowths have a predominantly hexagonal packing, whereas human skin has the dense orthorhombic packing. Furthermore, the outgrowths have lipid lamellae with a slightly reduced periodicity compared with human skin. These results demonstrate that the outgrowths do not maintain all properties observed in human skin, indicating that changes in properties of HSEs are not caused by isolation of primary cells, but by culture conditions.

Increased presence of monounsaturated fatty acids in the stratum corneum of human skin equivalents.

Previous results showed that our in-house human skin equivalents (HSEs) differ in their stratum corneum (SC) lipid organization compared with human SC. To elucidate the cause of the altered SC lipid organization in the HSEs, a recently developed liquid chromatography/mass spectrometry method was used to study the free fatty acid (FFA) and ceramide composition in detail. In addition, the SC lipid composition of the HSEs and human skin was examined quantitatively with high-performance thin-layer chromatography. Our results reveal that all our HSEs have an increased presence of monounsaturated FFAs compared with human SC. Moreover, the HSEs display the presence of ceramide species with a monounsaturated acyl chain, which are not detected in human SC. All HSEs also exhibit an altered expression of stearoyl-CoA desaturase, the enzyme that converts saturated FFAs to monounsaturated FFAs. Furthermore, the HSEs show the presence of 12 ceramide subclasses, similar to native human SC. However, the HSEs have increased levels of ceramides EOS and EOH and ceramide species with short total carbon chains and a reduced FFA level compared with human SC. The presence of unsaturated lipid chains in HSE offers new opportunities to mimic the lipid properties of human SC more closely.

Unraveling barrier properties of three different in-house human skin equivalents.

Human skin equivalents (HSEs) are three-dimensional culture models that are used as a model for native human skin. In this study the barrier properties of two novel HSEs, the fibroblast-derived matrix model (FDM) and the Leiden epidermal model (LEM), were compared with the full-thickness collagen model (FTM) and human skin. Since the main skin barrier is located in the lipid regions of the upper layer of the skin, the stratum corneum (SC), we investigated the epidermal morphology, expression of differentiation markers, SC permeability, lipid composition, and lipid organization of all HSEs and native human skin. Our results demonstrate that the barrier function of the FDM and LEM improved compared with that of the FTM, but all HSEs are more permeable than human skin. Further, the FDM and LEM have a relatively lower free fatty acid content than the FTM and human skin. Several similarities between the FDM, LEM and FTM were observed: (1) the morphology and the expression of the investigated differentiation markers were similar to those observed in native human skin, except for the observed expression of keratin 16 and premature expression of involucrin that were detected in all HSEs, (2) the lipids in the SC of all HSEs were arranged in lipid lamellae, similar to human skin, but show an increase in the number of lipid lamellae in the intercellular regions and (3) the SC lipids of all HSEs show a less densely packed lateral lipid organization compared with human SC. These findings indicate that the HSEs mimic many aspects of native human skin, but differ in their barrier properties.

Generation of human skin equivalents under submerged conditions-mimicking the in utero environment.

In this study we generated human skin equivalents (HSEs) under submerged conditions mimicking the aqueous in utero environment and investigated the morphology and differentiation process of the formed epidermis. Further, the skin barrier, which resides in the stratum corneum (SC), was characterized by its lipid content, hydration level, and natural moisturizing factor level. The submerged HSEs showed comparable tissue morphology and similar expression of several differentiation markers and SC lipid composition compared with HSEs grown at the air-liquid interface and native human skin. The SC of the submerged HSEs, however, contained more free water and less natural moisturizing factors compared with the air-exposed counterparts. These results show that the presented cell culture method can be utilized to generate HSEs under submerged conditions to study epidermal formation under aqueous conditions.