Human keratinocytes grown at a gas-permeable interface in vitro stratify correctly to generate engineered human epidermis.
Vaughan FeisstLisa Y. Y. ZhouChun‐Jen Jennifer ChenEloise WilliamsElliott DunnInken KelchSarah MeidingerJohn HuntAlex du RandH C RobinsonSaem ParkEvert Jan LoefJulian LoftsHilary M. SheppardMichelle LockeP. Rod Dunbar
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Abstract:
One of the key functions of human skin is to provide a barrier, protecting the body from the surrounding environment and maintaining homeostasis of the internal environment. A mature, stratified epidermis is critical to achieve skin barrier function and is particularly important when producing skin grafts in vitro for wound treatment. For decades epidermal stratification has been achieved in vitro by culturing keratinocytes at an air-liquid interface, triggering proliferating basal keratinocytes to differentiate and form all epidermal layers. We show here that culturing keratinocytes at a gas-permeable interface can induce epidermal stratification equivalent to an air-liquid interface. Culturing skin grafts at a gas-permeable interface has a number of advantages over the traditional air-liquid interface method including: less time input from highly skilled personnel, with consequent cost savings; fewer manipulations, with concomitant reduced risk of cell culture contamination; increased scalability of skin graft size; and improved potential for automation. These advantages confer significant benefits to the use of cell culture devices with gas-permeable interfaces to grow human skin for the treatment of major burns and other skin injuries.Keywords:
Epidermis (zoology)
Human skin
Sunscreen skin penetration and safety assessment should be considered together in order to ensure that in vitro cytotoxicity studies examine relevant doses of these organic chemical UV filters to which viable epidermal cells are realistically exposed. In this study, we sought to determine whether sufficient topically applied sunscreens penetrated into human viable epidermis to put the local keratinocyte cell populations at risk of toxicity. The penetration and retention of five commonly used sunscreen agents (avobenzone, octinoxate, octocrylene, oxybenzone and padimate O) in human skin was evaluated after application in mineral oil to isolated human epidermal membranes. Sunscreen concentration–human keratinocyte culture response curves were then defined using changes in cell morphology and proliferation (DNA synthesis using radiolabelled thymidine uptake studies) as evidence of sunscreens causing toxicity. Following 24 h of human epidermal exposure to sunscreens, detectable amounts of all sunscreens were present in the stratum corneum and viable epidermis, with epidermal penetration most evident with oxybenzone. The concentrations of each sunscreen found in human viable epidermis after topical application, adjusting for skin partitioning and binding effects, were at least 5-fold lower, based on levels detected in viable epidermal cells, than those appearing to cause toxicity in cultured human keratinocytes. It is concluded that the human viable epidermal levels of sunscreens are too low to cause any significant toxicity to the underlying human keratinocytes.
Human skin
Epidermis (zoology)
Penetration (warfare)
HaCaT
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We used the technique of autoradiography after incorporation of tritiated thymidine (3H-TdR) to evaluate keratinocyte proliferation in basal, epibasal, and other epidermal layers in 30 diseases affecting the epidermis. The number and proportion of 3H-TdR-labeled keratinocytes were counted in the different layers of the epidermis. Significant correlations were found between the proliferative indices of the different epidermal layers. Such links indicate that the epidermis responds in a rather stereotyped way to various pathological conditions. There exists some regulation in the distribution, number, and proportion of 3H-TdR-labeled keratinocytes in the various layers of the epidermis.
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The technique of obtaining human skin with dermis and epidermis reconstructed from cells isolated from patients can enable autologous skin grafting on patients with few donor sites. It also enables in vitro trials on chemicals and drugs. The objective of this work was to demonstrate a method for obtaining human skin composed of associated dermis and epidermis, reconstructed in vitro.Experimental laboratory study, in the Skin Cell Culture Laboratory of Faculdade de Ciências Médicas, Universidade Estadual de Campinas.Cells from human fibroblast cultures are injected into bovine collagen type I matrix and kept immersed in specific culturing medium for fibroblasts. This enables human dermis reconstruction in vitro. On this, by culturing human keratinocytes and melanocytes, differentiated epidermis is formed, leading to the creation of human skin composed of associated dermis and epidermis, reconstructed in vitro.We showed that human skin composed of associated dermis and epidermis can be successfully reconstructed in vitro. It is histologically formed in the same way as human skin in vivo. Collagen tissue can be identified in the dermis, with cells and extracellular matrix organized in parallel to multilayer epidermis.It is possible to obtain completely differentiated human skin composed of associated dermis and epidermis, reconstructed in vitro, from injection of human fibroblasts into bovine collagen type I matrix and culturing of human keratinocytes and melanocytes on this matrix.
Epidermis (zoology)
Human skin
Skin equivalent
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Human skin
Dermal fibroblast
Neutral red
Viability assay
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The expression of gene K51 in the cells of human normal epidermis and epithelial skin tumors was investigated using in situ hybridization method with radioactive probe. The K51 gene transcripts were detected in the epidermis, sebaceous and sweat glands of human embryo and adult skin. The level of gene expression was higher in the stratum granulosum than in the basal layer of the skin. K51 gene expression was also found in the basal cell and metatypical carcinomas, with the level of expression lower than in the neighbouring epidermis and higher than in the surrounding skin stromal cells. Thus, K51 gene is expressed in the skin epidermis of human embryo and adults but the level of its activity is dramatically decreased in the cells of skin epithelial tumors. This potentially may be important as a diagnostic test.
Stratum granulosum
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Human skin
Basal (medicine)
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Exposure to the sun affects the skin and may eventually result in UV-induced skin damage. It is generally known that hyaluronan (HA) is one of the main structural and functional components of the skin. However, UV-related changes in the HA metabolism in the skin have not yet been elucidated. Using qRT-PCR, confocal microscopy and LC-MS/MS we compared the naturally sun-exposed (SE), sun-protected, experimentally repeatedly UVA + UVB-exposed and acutely (once) UVA + UVB irradiated skin of Caucasian women. The epidermis was harvested by means of suction blistering 24 h after the acute irradiation. In addition, the epidermis was compared with a UV-irradiated in vitro reconstituted 3D epidermis (EpiDerm) and an in vitro 2D culture of normal human keratinocytes (NHEK). The amount of HA was found to be statistically significantly enhanced in the acutely irradiated epidermis. The acute UV evinced the upregulation of HA synthases (HAS2 and HAS3), hyaluronidases (HYAL2 and HYAL3), Cluster of differentiation 44 (CD44), and Cell Migration Inducing Proteins (CEMIP and CEMIP2), while only certain changes were recapitulated in the 3D epidermis. For the first time, we demonstrated the enhanced gene and protein expression of CEMIP and CEMIP2 following UV irradiation in the human epidermis. The data suggest that the HA metabolism is affected by UV in the irradiated epidermis and that the response can be modulated by the underlying dermis.
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Human skin
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Epidermis, the surface layer of human skin, is formed by cells called keratinocytes. They divide (making more cells) in the deeper parts of the skin and gradually move towards the surface. Keratinocytes eventually form a layer of dead cells on the skin's surface, and this layer is the main protective barrier against things that might damage or irritate the skin, such as chemicals and germs. A viscous substance called hyaluronan surrounds keratinocytes in the lower, living cell layers. All kinds of injuries to the epidermis, like wounding, infection, and sunburn, trigger a strong inflammatory response by keratinocytes. This response includes increased division of keratinocytes, and their movement to help the injury site recover, as well as increased hyaluronan production and turnover. In many skin diseases the keratinocyte injury‐response is excessive, or even completely unfounded, and harmful. Therefore, factors and treatments that calm down this reaction are potential medicines for some skin diseases. This study from Kuopio, Finland, studied hyaluronan metabolism (i.e. breakdown) in a special model developed in the laboratory, in which keratinocytes separate to produce a normal‐looking epidermis. This model allowed the researchers to demonstrate that vitamin C suppresses both production and degradation of hyaluronan in the epidermis. Since rapid hyaluronan metabolism is an indicator of inflammation, vitamin C – which is an antioxidant ‐ might have a use for suppressing this reaction in keratinocytes. This study calls for more work on the vitamin's mechanism of action, and possible roles in clinical practice.
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Human skin
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We examined 20 punch biopsies taken from five patients at varying intervals following CO2 laser-induced thermal injury. The regenerating epidermis was studied with monoclonal antibodies AE1 and AE3 (directed against low and high molecular weight keratins), and involucrin, a protein found within the cellular envelope of the most mature keratinocytes. Twenty-four hours following thermal damage, there was extensive spillage of keratins and involucrin into the papillary dermis and disarray of all constituents of the necrotic keratinocytes. Early ingrowth of basaloid keratinocytes weakly expressed AE1. By 1 week, keratinocytes expressed AE1 in varying intensities throughout the epidermis. AE3 was present in its normal distribution, staining all but the most basaloid keratinocytes. Involucrin stained cells deep within the epidermis. Six weeks following the initial injury, the staining pattern within the epidermis had returned to normal. Thus, it appears that the regenerating epidermis produces low molecular weight keratins in cells at all levels and forms premature cellular envelopes, perhaps as a protective measure, before expression of these constituents reverts to the normal pattern. These findings suggest that keratinocyte differentiation in wound-healing following laser-induced thermal injury is similar to that seen in other types of injury. Observed clinical differences may be attributable to differences in keratinocyte proliferative or migratory capabilities.
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In order to validate a model for predictive screening of dermatological drugs, we used a customized cDNA macro-array system containing 475 skin-related genes to analyze the gene expression patterns in human keratinocytes from different origins: (1) normal human epidermal keratinocyte mono-layer cultures, (2) the commercially available SkinEthic reconstituted human epidermis model, and (3) biopsies of normal human epidermis. Few markers of those that were detected significantly in keratinocyte mono-layers or in reconstituted epidermis were undetected or detected at very low level in the normal epidermis biopsies. A comparative expression of more than 100 markers could be evidenced in both normal epidermis and reconstituted epidermis samples; however, only 90% of these were detected in keratinocyte mono-layers: expression of several terminal differentiation markers, such as filaggrin, loricrin, and corneodesmosin were strongly detected in normal epidermis and reconstituted epidermis, but were not significantly expressed in keratinocyte mono-layers. Under the experimental conditions described herein, the reconstituted human epidermis model was found to significantly reproduce the gene expression profile of normal human epidermis. Using the same methodology, we then investigated the effects of all-trans retinoic acid, 9-cis retinoic acid, all-trans retinol and a commercialized tretinoin-containing cream (Retacnyl) on the gene expression profiles of reconstituted human epidermis. According to the nature and the length of the treatments, more than 40 genes were found significantly modified. Among the genes whose expression was decreased, we found cytokeratins 1, 10, 2E, and 6B, several cornified envelope precursors, integrins alpha 3, alpha 6, beta 1, beta 4, some components of desmosomes, of hemi-desmosomes and of the epidermal basement membrane. Transcriptional upregulation was observed for keratins 18 and 19, autocrine and paracrine growth factors such as HB-EGF, IGF 1, PDGF-A, calgranulins A and B, interleukin-1 alpha and the other IL-1-related markers, type II IL-1 receptor and type I IL-1-receptor antagonist. Our results confirm most of the known effects of retinoids on human epidermis, but also give new insights into their complex pharmacological activity on skin. The reconstituted human epidermis used proves to be a highly predictive model for efficacy evaluation of skin-targeted compounds, such as retinoids.
Epidermis (zoology)
Loricrin
Human skin
Corneocyte
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Reconstructed human epidermis models are used as epidermis alternatives in skin research studies.
Epidermis (zoology)
Human skin
Barrier function
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