An improved method of human keratinocyte culture from skin explants: cell expansion is linked to markers of activated progenitor cells

Abstract:  Human keratinocyte primary cultures are commonly established by tissue dissociation and often rely on feeder cell supports and culture medium that is not defined. Further, contamination by unwanted fibroblasts can be problematic. Here, we developed a skin explant method for growing primary keratinocytes that was rapid, simple, and reliably generated keratinocyte cultures free of fibroblast contamination. The process capitalized on the observation that fibroblasts migrate out of adult skin explants later than epidermal cells, allowing the early harvesting of keratinocytes by trypsinization. When grown subsequently in defined medium in the absence of feeder cells, the explant-derived cells grew rapidly and could be cultured for multiple passages. Immunofluorescence microscopy revealed that a high percentage of cells harvested from the explant outgrowths expressed K15, while very few expressed the differentiation marker K10. Cells that were stained while migrating out from explants strongly expressed markers associated with progenitor cells, including p63, K15 and CD133, and displayed intense K6 expression, indicative of activated keratinocytes in wound-healing epidermis. By replenishing the explants with fresh medium after harvesting, further epidermal outgrowths could be obtained, offering the possibility of greatly increased keratinocyte yields for clinical applications.     

Fluorescent silica colloids for study and visualization of skin care products

BACKGROUND: The efficacy of skin care products depends on the time and dynamics of their absorbance by the skin, and its spatial distribution on the skin. Regular scrape-based methods may depend on the operator and are destructive and invasive in nature. Here, we describe a novel method based on non-contact optical measurements to trace the location and dynamics of skin care products on the skin. METHODS: We use fluorescent silica colloidal particles of micron sizes at a rather small concentration as non-invasive tracers. As an example of skin care products, we use two base materials: either glycerin or vaseline. A mixture of each product with fluorescent particles is applied on human skin. The amount of fluorescence is monitored by means of a fluorescent spectrometer. The scraping method is used to compare with the spectroscopic measurements. RESULTS: Fluorescent tracers make the skin care product visible under UV light. This allows obtaining an optical image of the spatial distribution of the product on the skin. The quantitative data of fluorescence are well correlated with the scrape data. Comparison of the difference in the spectral and scraped mass data reveals the details of accumulation of the skin products in skin cracks and crevices. CONCLUSION: We described an efficient non-invasive benign method to quantify dynamics and to perform mapping of emollients and humectants on the skin.     

An improved method for measurement of change in skin roughness caused by cleansing products under mild application conditions

Background: The aim of this study was to develop a method for the evaluation of subtle change in skin roughness caused by cleansing products under mild application conditions using a non‐invasive three‐dimensional (3D) analysis system. Methods: A double‐blind comparative study of the modified soap chamber test was performed using two soap bars and a syndet bar. Skin changes were evaluated by visual scoring [mean cumulative irritation index (MCII)] and by bioengineering measurements [transepidermal water loss (TEWL), skin capacitance, and skin surface roughness]. Results: MCII of the syndet bar was statistically higher than that of one soap bar, and TEWL increase after application of the syndet bar was statistically higher than that of both soap bars. Skin capacitance decreased significantly only after application of the syndet bar. The change in the average roughness of the skin surface was significantly greater after the application of the syndet bar than with classic soap bars. Conclusion: A simple, fast, and objective evaluation of skin surface topography was performed using a modified soap chamber test and a non‐invasive 3D analysis system. The results suggest that measurement of skin roughness using a non‐invasive 3D analysis system might be a good method for the evaluation of a subtle change caused by cleansing products under mild application conditions.     

Expression of stratum corneum chymotryptic enzyme in relation to other markers of epidermal differentiation in a skin explant model

The serine proteinase stratum corneum chymotryptic enzyme (SCCE) has been proposed to be involved in the degradation of intercellular cohesive structures in cornified squamous epithelia in the process of desquamation. Since SCCE is expressed late in epidermal differentiation and is found at all sites where there is a formation of cornified epithelia it also serves as a marker for terminal epidermal differentiation. Earlier studies have shown that the link between expression and the formation of cornified cells may be stronger for SCCE than for other well characterized markers of epidermal differentiation. In an attempt to further elucidate the regulation of SCCE expression we have in this study compared the expression of SCCE with the expression of keratin 10, filaggrin and involucrin in an in vitro model with skin explants cultured for various periods of time on de-epidermized dermis at the liquid-air interface. The markers were analysed by means of immunohistochemistry. We found that the expression of SCCE preceded the expression of keratin 10 and filaggrin. In contrast to involucrin, which was expressed by all suprabasal keratinocytes, SCCE was expressed only by high suprabasal cells. Our results indicate that the expression of SCCE may be regulated in a way that differs from the regulation of the expression of keratin 10, filaggrin and involucrin.     

Effects of retinoic acid on adult human epidermis in whole skin organ culture

Summary Adult human skin was cultured in wholeskin organ culture under chemically defined conditions. Retinoic acid was added to the culture at final concentrations of 5×10^-7 and 5×10^-6 M. Both concentrations elicited cell death in the upper epidermal layers and prevented the terminal differentiation of the cells to mature corneocytes. The inhibition of terminal differentiation was not permanent, as the corneocytes produced later during the culture showed no signs of inhibition. The upper vital cells in epidermis cultured with retinoic acid were very flattened and contained reduced amounts of cytoskeleton components. Fine, granular material not present in normal epidermis was oberved in both the intercellular spaces and the intracytoplasmic vesicles of retinoid-treated epidermis. The present results indicate that the response of normal human skin to retinoic-acid treatment involves the same kind of modulation of the epidermal structure previously described in embryonic avian and diseased human skin.     

Towards the development of a simplified long-term organ culture method for human scalp skin and its appendages under serum-free conditions

Organ culture of human scalp skin is usually performed with serum-containing medium, which limits its analytical usefulness. Here we report that intact human scalp skin can be grown at the air/liquid interface in supplemented, serum-free William's E medium for more than 2 weeks. Active hair shaft growth was visible until day 16 and was significantly enhanced compared with minimum essential medium (MEM) + 10% fetal bovine serum (FBS). Moreover, William's E medium protected better against cell death than MEM + 10% FBS before day 12. Using quantitative immunochemistry, proliferating (Ki-67+) cells could still be observed in the epithelium of hair follicles even on day 17 of serum-free skin organ culture. The number of apoptotic (TUNEL+) cells in the skin epithelium rose steadily after day 5. Giemsa stains revealed mature skin mast cells even after 13 days in culture. The percentage of surviving hair follicles (mostly with catagen- or telogen-like morphology) gradually increased over time displaying mostly catagen hair follicles after 17 days of culture. Although epidermis and hair follicle epithelium showed increasing atrophy and degeneration, and their pigmentation decreased gradually over time, some long-term-surviving epithelial islands were found in association with remnants of follicular structures as late as on day 88. These preliminary data suggest that a very simple serum-free organ culture method allows prolonged human skin and hair follicle survival as well as some limited hair follicle cycling in intact skin for more than 2 weeks under well-defined experimental conditions. This pragmatic assay invites multiple uses, and may become a valuable tool for both skin and hair research.     

Effects of skin care education for care staff at elderly care facilities on skin conditions of the residents

Asteatosis is common in elderly people due to a decrease in the moisture content of the epidermal stratum corneum through a loss of skin barrier function caused by aging. Because itching often accompanies asteatosis, this condition may cause a decrease in quality of life. Care staff in elderly care facilities have many opportunities to provide care for residents. In this study, we examined how educational training on skin care changed the thoughts and actions of care staff in these facilities and how these changes impacted the skin conditions of residents. The subjects for the training were all care staff in facilities because these staff work most closely with facility residents. We performed skin care training for the subjects and investigated changes in the skin conditions of the residents before and after the training. The training promoted the understanding of skin care among the care staff and improved the skin symptoms of residents with asteatosis. However, there were no changes in the severity of itchiness based on a verbal rating scale and in interviews of residents. This study showed that skin care training for the care staff in facilities is effective to improve skin conditions of residents. In addition, it was suggested that a full grasp of the residents’ skin symptoms based upon an interview on itching alone was difficult, and thus there is a need to observe skin conditions directly.     

An improved and rapid method to construct skin equivalents from human hair follicles and fibroblasts

To produce sufficient amounts of high quality skin equivalents (SE), either allogenic for dermatopharmacological and dermatotoxicological studies or autologous for transplantation purposes, we established a rapid, easy and cost effective three-dimensional SE model on the basis of human dermal fibroblasts, collagen and freshly plucked hair follicles. Acidic liquid collagen was polymerized with sodium hydroxide in the presence of fibroblasts to form a dermal equivalent (DE) resembling normal human dermis. At 24 h later, freshly plucked hair follicles were implanted into the surface of these DEs after cutting their bulbs off. Another 48 h later, the surface of the SEs was lifted to the air-liquid interface. Fourteen days after implantation, outgrowing keratinocytes from the outer root sheath of the hair follicles completely covered the surface of the SE and built a fully developed, multi-layered and cornified epidermis. Histology and immunofluorescence studies with specific antibodies directed against components of keratinocytes, fibroblasts, cell-adhesion molecules, different extracellular matrix and basement membrane proteins revealed the similarity of our three-dimensional SEs to the in vivo situation in normal human skin. Using autologous cell sources and cell culture media enriched with serum from the respective cell donor, it will be possible to use these SEs for autologous transplantation, thereby reducing the risk of transplant rejection.     

Estimating dilutions for patch testing skin care products: A practical method

When patch testing, it is helpful to patch test with the patient's own topical products. However, the thickness (viscosity) of the product often prevents easy measurement for dilution. A method of estimating volume: volume dilutions that requires minimal investment in supplies is presented. This method is only applicable to personal products and is not suitable for industrial or household chemicals.     

Xenobiotic metabolizing enzymes in human skin and SkinEthic reconstructed human skin models

Skin metabolism is becoming a major consideration in the development of new cosmetic ingredients, skin being the first organ exposed to them. In order to replace limited samples of Excised human skin (EHS), in vitro engineered human skins have been developed. 3D models are daily used to develop and evaluate new cosmetic ingredients and have to be characterized and compared with EHS in terms of metabolic capabilities. This work presents the determination of apparent catalytic parameters (apparent Vmax, Km and the ratio Vmax/Km) in 3D models compared with EHS for cytochrome P450 dependent monooxygenase isoforms involved in drug metabolism, esterases, alcohol dehydrogenases, aldehyde dehydrogenases, peroxidases, glutathione S‐transferases, N‐acetyl transferases, uridinyl diphosphate glucuronyl transferases and sulfotransferases. Results show that all these enzymes involved in the metabolism of xenobiotics are expressed and functional in the EHS and 3D models. Also, the Vmax/Km ratios (estimating the intrinsic metabolic clearances) show that the metabolic abilities are the most often comparable between the skin models and EHS. These results indicate that the 3D models can substitute themselves for EHS to select cosmetic ingredients on the basis of their metabolism, efficacy or/and safety.     

An advanced mouse model for human skin wound healing

Here, we report a model for studying wound repair based on skin regenerated from human tissue culture‐expanded cells. The reconstituted skin (hRSK) responds to injury similar to that of intact human skin, and its constituent cells contribute to the healing process. As we have demonstrated that hRSK composed of GFP‐labelled cells also heals “normally,” we believe this model will be useful in analysing the wound repair process using genetically modified human cells.     

An improved method for in vitro perfusion of human skin

We have developed a procedure for establishing a long-living supravital skin preparation. Our model has made in vitro perfusion of human cutis, fatty tissue and lymph nodes possible. We describe the method of preparation, the technical construction and preliminary results of our experiments.     

The linear excisional wound: an improved model for human ex vivo wound epithelialization studies

Background/purpose: Wound healing is a complex process that involves multiple intercellular and intracellular processes and extracellular interactions. Explanted human skin has been used as a model for the re‐epithelialization phase of human wound healing. The currently used standard technique uses a circular punch biopsy tool to make the initial wound. Despite its wide use, the geometry of round wounds makes it difficult to measure them reliably. Methods: Our group has designed a linear wounding tool, and compared the variability in ex vivo human linear and circular wounds. Results: An F test for differences in variances demonstrated that the linear wounds provided a population of wound size measurements that was 50% less variable than that obtained from a group of matched circular wounds. This reduction in variability would provide substantial advantages for the linear wound technique over the circular wound punch technique, by reducing the sample sizes required for comparative studies of factors that alter healing. Conclusion: This linear wounding tool thus provides a method for wounding that is standardized, provides minimal error in wound gap measurements, and is easily reproducible. We demonstrate its utility in an ex vivo model for the controlled investigation of human skin wounds.     

S1 guideline on occupational skin products: protective creams,skin cleansers,skin care products (ICD 10: L23, L24) – short version

Job‐related hand dermatitis heads up the list of reported occupational diseases. So‐called skin products – understood to mean protective creams, skin cleansers and skin care products – are used for the primary and secondary prevention of job‐ related hand dermatitis. In the interests of evidence‐based medicine, the only preventive measures and/or occupational skin products that should be used are those whose potential uses and efficacy are underpinned by scientific research. To this end, the Arbeitsgemeinschaft für Berufs‐ und Umweltdermatologie e.V. (Working Group for Occupational and Environmental Dermatology, ABD) of the DDG (German Dermatological Society) and the Deutsche Gesellschaft für Arbeits‐ und Umweltmedizin (German Society for Occupational and Environmental Medicine, DGAUM) have summed up the latest scientific findings and recommendations in the updated guideline. The benefit of the combined application of protective creams and skin care products in the primary and secondary prevention of work‐related contact dermatitis has been widely confirmed by recent clinical‐epidemiological studies. The guideline clearly explains the necessity of demonstrating the efficacy of protective creams and cleansing products by means of in vivo methods in the sense of repetitive applications. Transferable standardised testing systems designed to examine the irritation potential and thus the compatibility of occupational skin cleansers and the reduction of irritation by protective skin creams have now been developed and validated by multicentre studies for skin protection creams and cleansers. The status of the current assessment of the safety of occupational skin products is also summarised.     

Reorganization of hair follicles in human skin organ culture induced by cultured human follicle-derived cells

Studies of human hair follicle (HF) induction by follicle-derived cells have been limited due to a lack of suitable test systems. In this study, we established a skin organ culture system which supports HF formation by follicle-derived cells. Long-term skin organ cultures were set up from human retroauricular skin specimens and maintained in culture for up to 8 weeks. In vitro expanded human HF-derived cells from the dermal papilla (DP) and the outer root sheath (ORS) were injected together into the skin specimens and evaluated for their ability to induce reorganization of HFs. Macroscopic analysis of the cultured skin specimens demonstrated the growth of velus-like hair after 4 weeks in culture. Histologic evaluation of the cultured skin specimens after 8 weeks of culture revealed multiple miniaturized HFs with sebaceous glands. In addition, cell clusters of various differentiation stages could be demonstrated in serial sections of the cultured skin specimens. Labeling of HF-derived cells with the fluorescence dye CFDA-1 prior to injection suggested a de novo reorganization of HFs out of the injected cells. In conclusion, the study demonstrated HF formation by HF-derived cells in an in vitro skin organ culture model.     

Similar UV responses are seen in a skin organ culture as in human skin in vivo

Ultraviolet radiation (UVR) plays an important role in the development of non-melanoma skin cancer. Most tumors develop in chronically sun-exposed skin, most often in cosmetically sensitive locations, where in vivo experiments may be difficult to perform. In this study, we describe a skin organ culture model with preserved normal morphology and intact response to UVR. Skin explants from chronically sun-exposed and non-sun-exposed skin were irradiated with artificial UVA+UVB with and without topical sunscreen. UV-induced DNA damage, epidermal p53 response and repair kinetics were analyzed using immunohistochemistry. Four hours after UV-irradiation epidermal keratinocytes showed a strong immunoreactivity for thymine-dimers. Gradual repair during an incubation time resulted in few residual thymine-dimers after 48 h. Repair appeared to be more efficient in chronically sun-exposed skin compared with non-sun-exposed skin. There was also an accumulation of p53 protein in epidermal keratinocytes, peaking at 4-24 h after irradiation. Large interindividual differences with respect to formation and repair of thymine-dimers as well as induction and duration of the p53 response were observed. Skin explants treated with topical sunscreen prior to UV-irradiation showed a clear reduction of thymine-dimers and p53 expression. The epidermal UV-responses and repair kinetics in organ-cultured skin were similar to what was found in vivo. Our data suggest that organ-cultured skin provides a valuable tool for studies of UV-induced epidermal responses in chronically sun-exposed skin.     

Prolonged viability of human organotypic skin explant in culture method (hOSEC)

BACKGROUND:

Currently, the cosmetic industry is overwhelmed in keeping up with the safety assessment of the increasing number of new products entering the market. To meet such demand, research centers have explored alternative methods to animal testing and also the large number of volunteers necessary for preclinical and clinical tests.

OBJECTIVES:

This work describes the human skin ex-vivo model (hOSEC: Human Organotypic Skin Explant Culture) as an alternative to test the effectiveness of cosmetics and demonstrate its viability through cutaneous keratinocytes'' proliferative capacity up to 75 days in culture.

METHODS:

The skin explants obtained from surgeries were cultured in CO₂-humid incubator. After 1, 7, 30 and 75 days in culture, skin fragments were harvested for analysis with histomorphological exam (HE staining) on all days of follow-up and immunohistochemistry for Ck5/6, Ck10 and Ki-67 only on the 75th day.

RESULTS:

On the 7th day, the epidermis was perfect in the dermoepidermal junction, showing the viability of the model. On the 30th day, the epidermis was thicker, with fewer layers on the stratum corneum, although the cutaneous structure was unaltered. On the 75th day, the skin became thinner but the dermoepidermal junctions were preserved and epidermal proliferation was maintained. After the 75th day on culture, the skin was similar to normal skin, expressing keratinocytes with Ck5/6 on supra-basal layers; Ck10 on differentiated layers; and viability could be assessed by the positivity of basal cells by Ki-67.

CONCLUSION:

The hOSEC model seems a good alternative to animal testing; it can be used as a preclinical test analogous to clinical human skin test with similar effectiveness and viability proven by immunohistological analyses.     

日常护肤习惯与玫瑰痤疮发病风险研究

目的:探讨日常护肤习惯与玫瑰痤疮发病的相关性.方法:对我院面部皮炎专诊的玫瑰痤疮患者和非玫瑰痤疮患者进行日常护肤习惯的问卷调查,采用卡方检验比较两组在性别、年龄、护肤步骤数、洁面次数、是否每天使用面膜和酸类产品等因素的差异,采用二元Logistic回归方法分析玫瑰痤疮发生的独立危险因素.结果:收到有效问卷487份,其中...