An improved human skin explant culture method for testing and assessing personal care products.

BACKGROUND: Cultured human skin models have been widely used in the evaluation of dermato-cosmetic products as alternatives to animal testing and expensive clinical testing. The most common in vitro skin culture approach is to maintain skin biopsies in an airlifted condition at the interface of the supporting culture medium and the air phase. This type of ex vivo skin explant culture is not, however, adequate for the testing of cleansing products, such as shampoos and body washes. One major deficiency is that cleansing products would not remain confined on top of the epidermis and have a high chance of running off toward the dermal side, thus compromising the experimental procedure and data interpretation. MATERIALS AND METHODS: Here, we describe an improved ex vivo method for culturing full-thickness human skin for the effective testing and evaluation of skin care products by topical application. RESULTS: This newly developed ex vivo human skin culture method has the ability to maintain healthy skin tissues for up to 14 days in culture. Importantly, the model provides a quick and safe way to evaluate skin care products at different time points after single or repetitive topical applications using a combined regimen of leave-on and wash-off. We found that the results obtained using the new skin culture method are reproducible and consistent with the data collected from clinical testing. CONCLUSION: Our new ex vivo skin explant method offers a highly efficient and cost-effective system for the evaluation and testing of a variety of personal care products and new formulations.

Quantitative morphometric analysis of intrinsic and extrinsic skin ageing in individuals with Fitzpatrick skin types II-III.

Skin ageing is an intricate physiological process affected by intrinsic and extrinsic factors. There is a demand to understand how the skin changes with age and photoexposure in individuals with Fitzpatrick skin types I-III due to accelerated photoageing and the risk of cutaneous malignancies. To assess the structural impact of intrinsic and extrinsic ageing, we analysed 14 skin parameters from the photoprotected buttock and photoexposed dorsal forearm of young and ageing females with Fitzpatrick skin types II-III (n = 20) using histomorphic techniques. Whilst the minimum viable epidermis (Emin ) remained constant (Q > 0.05), the maximum viable epidermis (Emax ) was decreased by both age and photoexposure (Q ≤ 0.05), which suggests that differences in epidermal thickness are attributed to changes in the dermal-epidermal junction (DEJ). Changes in Emax were not affected by epidermal cell proliferation. For the first time, we investigated the basal keratinocyte morphology with age and photoexposure. Basal keratinocytes had an increased cell size, cellular height and a more columnar phenotype in photoexposed sites of young and ageing individuals (Q ≤ 0.05), however no significant differences were observed with age. Some of the most striking changes were observed in the DEJ, and a decrease in the interdigitation index was observed with both age and photoexposure (Q ≤ 0.001), accompanied by a decreased height of rête ridges and dermal papilla. Interestingly, young photoexposed skin was comparable to ageing skin across many parameters, and we hypothesise that this is due to accelerated photoageing. This study highlights the importance of skin care education and photoprotection from an early age.

Tissue engineering strategies to bioengineer the ageing skin phenotype in vitro.

Human skin ageing is a complex and heterogeneous process, which is influenced by genetically determined intrinsic factors and accelerated by cumulative exposure to extrinsic stressors. In the current world ageing demographic, there is a requirement for a bioengineered ageing skin model, to further the understanding of the intricate molecular mechanisms of skin ageing, and provide a distinct and biologically relevant platform for testing actives and formulations. There have been many recent advances in the development of skin models that recapitulate aspects of the ageing phenotype in vitro. This review encompasses the features of skin ageing, the molecular mechanisms that drive the ageing phenotype, and tissue engineering strategies that have been utilised to bioengineer ageing skin in vitro.

The microfollicle: a model of the human hair follicle for in vitro studies.

Access to complex in vitro models that recapitulate the unique markers and cell-cell interactions of the hair follicle is rather limited. Creation of scalable, affordable, and relevant in vitro systems which can provide predictive screens of cosmetic ingredients and therapeutic actives for hair health would be highly valued. In this study, we explore the features of the microfollicle, a human hair follicle organoid model based on the spatio-temporally defined co-culture of primary cells. The microfollicle provides a 3D differentiation platform for outer root sheath keratinocytes, dermal papilla fibroblasts, and melanocytes, via epidermal-mesenchymal-neuroectodermal cross-talk. For assay applications, microfollicle cultures were adapted to 96-well plates suitable for medium-throughput testing up to 21 days, and characterized for their spatial and lineage markers. The microfollicles showed hair-specific keratin expression in both early and late stages of cultivation. The gene expression profile of microfollicles was also compared with human clinical biopsy samples in response to the benchmark hair-growth compound, minoxidil. The gene expression changes in microfollicles showed up to 75% overlap with the corresponding gene expression signature observed in the clinical study. Based on our results, the cultivation of the microfollicle appears to be a practical tool for generating testable insights for hair follicle development and offers a complex model for pre-clinical substance testing.

Engineering a Multilayered Skin Equivalent: The Importance of Endogenous Extracellular Matrix Maturation to Provide Robustness and Reproducibility.

Human skin equivalents (HSEs) are a valuable tool for both academic and industrial laboratories to further the understanding of skin physiology and associated diseases. Over the last few decades, there have been many advances in the development of HSEs that successfully recapitulate the structure of human skin in vitro; however a main limitation is variability due to the use of complex protocols and exogenous extracellular matrix (ECM) proteins. We have developed a robust and unique full-thickness skin equivalent that is highly reproducible due to the use of a consistent scaffold, commercially available cells, and defined low-serum media. The Alvetex® scaffold technology allows fibroblasts to produce their own endogenous ECM proteins within the scaffold, which alleviates the need for exogenous collagen, and supports the differentiation and stratification of the epidermis. Our full-thickness skin equivalent is generated using a detailed step-by-step protocol, which sequentially forms the multilayered structure of human skin in vitro. This model can be adapted for many downstream applications such as disease modeling and testing of active compounds for cosmetics.

Bioengineering the microanatomy of human skin.

Recreating the structure of human tissues in the laboratory is valuable for fundamental research, testing interventions, and reducing the use of animals. Critical to the use of such technology is the ability to produce tissue models that accurately reproduce the microanatomy of the native tissue. Current artificial cell-based skin systems lack thorough characterisation, are not representative of human skin, and can show variation. In this study, we have developed a novel full thickness model of human skin comprised of epidermal and dermal compartments. Using an inert porous scaffold, we created a dermal construct using human fibroblasts that secrete their own extracellular matrix proteins, which avoids the use of animal-derived materials. The dermal construct acts as a foundation upon which epidermal keratinocytes were seeded and differentiated into a stratified keratinised epithelium. In-depth morphological analyses of the model demonstrated very close similarities with native human skin. Extensive immunostaining and electron microscopy analysis revealed ultrastructural details such as keratohyalin granules and lamellar bodies within the stratum granulosum, specialised junctional complexes, and the presence of a basal lamina. These features reflect the functional characteristics and barrier properties of the skin equivalent. Robustness and reproducibility of in vitro models are important attributes in experimental practice, and we demonstrate the consistency of the skin construct between different users. In summary, a new model of full thickness human skin has been developed that possesses microanatomical features reminiscent of native tissue. This skin model platform will be of significant interest to scientists researching the structure and function of human skin.

Quantitative proteogenomic profiling of epidermal barrier formation in vitro.

BACKGROUND: The barrier function of the epidermis is integral to personal well-being, and defects in the skin barrier are associated with several widespread diseases. Currently there is a limited understanding of system-level proteomic changes during epidermal stratification and barrier establishment. OBJECTIVE: Here we report the quantitative proteogenomic profile of an in vitro reconstituted epidermis at three time points of development in order to characterize protein changes during stratification. METHODS: The proteome was measured using data-dependent "shotgun" mass spectrometry and quantified with statistically validated label-free proteomic methods for 20 replicates at each of three time points during the course of epidermal development. RESULTS: Over 3600 proteins were identified in the reconstituted epidermis, with more than 1200 of these changing in abundance over the time course. We also collected and discuss matched transcriptomic data for the three time points, allowing alignment of this new dataset with previously published characterization of the reconstituted epidermis system. CONCLUSION: These results represent the most comprehensive epidermal-specific proteome to date, and therefore reveal several aspects of barrier formation and skin composition. The limited correlation between transcript and protein abundance underscores the importance of proteomic analysis in developing a full understanding of epidermal maturation.

Mouse hair cycle expression dynamics modeled as coupled mesenchymal and epithelial oscillators.

The hair cycle is a dynamic process where follicles repeatedly move through phases of growth, retraction, and relative quiescence. This process is an example of temporal and spatial biological complexity. Understanding of the hair cycle and its regulation would shed light on many other complex systems relevant to biological and medical research. Currently, a systematic characterization of gene expression and summarization within the context of a mathematical model is not yet available. Given the cyclic nature of the hair cycle, we felt it was important to consider a subset of genes with periodic expression. To this end, we combined several mathematical approaches with high-throughput, whole mouse skin, mRNA expression data to characterize aspects of the dynamics and the possible cell populations corresponding to potentially periodic patterns. In particular two gene clusters, demonstrating properties of out-of-phase synchronized expression, were identified. A mean field, phase coupled oscillator model was shown to quantitatively recapitulate the synchronization observed in the data. Furthermore, we found only one configuration of positive-negative coupling to be dynamically stable, which provided insight on general features of the regulation. Subsequent bifurcation analysis was able to identify and describe alternate states based on perturbation of system parameters. A 2-population mixture model and cell type enrichment was used to associate the two gene clusters to features of background mesenchymal populations and rapidly expanding follicular epithelial cells. Distinct timing and localization of expression was also shown by RNA and protein imaging for representative genes. Taken together, the evidence suggests that synchronization between expanding epithelial and background mesenchymal cells may be maintained, in part, by inhibitory regulation, and potential mediators of this regulation were identified. Furthermore, the model suggests that impairing this negative regulation will drive a bifurcation which may represent transition into a pathological state such as hair miniaturization.

Transcriptional profiling of epidermal barrier formation in vitro.

BACKGROUND: Barrier function is integral to the health of epithelial tissues. Currently, there is a broad need to develop and improve our knowledge with regard to barrier function for reversal of mild skin irritation and dryness. However, there are few in vitro models that incorporate modulations of both lipids and epidermal differentiation programs for pre-clinical testing to aid in the understanding of barrier health. OBJECTIVE: We have generated a reconstituted epidermis on a decellularized dermis (DED) and characterized its barrier properties relative to human epidermis in order to determine its utility for modeling barrier formation and repair. METHODS: We followed the process of epidermal differentiation and barrier formation through immunocytochemistry and transcriptional profiling. We examined barrier functionality through measurements of surface pH, lipid composition, stratum corneum water content, and the ability to demonstrate topical dose-dependent exclusion of surfactant. RESULTS: Transcriptional profiling of the epidermal model during its formation reveals temporal patterns of gene expression associated with processes regulating barrier function. The profiling is supported by gradual formation and maturation of a stratum corneum and expression of appropriate markers of epidermis development. The model displays a functional barrier and a water gradient between the stratum corneum and viable layers, as determined by confocal Raman spectroscopy. The stratum corneum layer displays a normal acidic pH and an appropriate composition of barrier lipids. CONCLUSION: The epidermal model demonstrates its utility as an investigative tool for barrier health and provides a window into the transcriptional regulation of multiple aspects of barrier formation.

Kazrin, a novel periplakin-interacting protein associated with desmosomes and the keratinocyte plasma membrane.

Periplakin forms part of the scaffold onto which the epidermal cornified envelope is assembled. The NH2-terminal 133 amino acids mediate association with the plasma membrane and bind a novel protein, kazrin. Kazrin is highly conserved and lacks homology to any known protein. There are four alternatively spliced transcripts, encoding three proteins with different NH2 termini. Kazrin is expressed in all layers of stratified squamous epithelia; it becomes membrane associated in the suprabasal layers, coincident with up-regulation of periplakin, and is incorporated into the cornified envelope of cultured keratinocytes. Kazrin colocalizes with periplakin and desmoplakin at desmosomes and with periplakin at the interdesmosomal plasma membrane, but its subcellular distribution is independent of periplakin. On transfection, all three kazrin isoforms have similar subcellular distributions. We conclude that kazrin is a novel component of desmosomes that associates with periplakin.