Cultured Skin Cells Research Articles

In Vitro Models of Cutaneous Inflammation.

The skin plays an essential role in the transmission of Lyme borreliosis since it is the first interface between the Ixodes tick and the host during the inoculation of Borrelia burgdorferi sensu lato. A better understanding of the inflammatory reaction at this location is key to develop better strategies (e.g., vaccine and diagnosis) to fight this disease. In vitro cell culture of resident skin cells might constitute an approach to decipher the complex interplay between the tick, the pathogen, and the vertebrate host.

Brassica rapa hairy root extracts promote skin depigmentation by modulating melanin production and distribution.

Skin whitening products, used for ages by Asian people for cultural and esthetic purposes, are very popular nowadays in Western countries as well, where the need to inhibit skin spots after sun exposure has become not only a cosmetic but also a health-related issue. Thus, the development of effective and safe depigmenting agents derived from natural products gets continuous attention by cosmetic brands and consumers. The aim of this study was to determine the effects of two preparations, obtained from the hairy root cultures of the species Brassica rapa, on melanogenesis and the expression of the extracellular matrix proteins involved in a correct pigment distribution. The two preparations, obtained by water-ethanol extraction and by digestion of cell-wall glycoproteins of the root cells, were chemically characterized and tested on skin cell cultures and on human skin explants to investigate on their dermatological activities. Both the extracts were able to decrease melanin synthesis pathway in melanocytes and modulate the expression of genes involved in melanin distribution. One of the extracts was also effective in inducing the expression of laminin-5 and collagen IV, involved into the maintenance of tissue integrity. The two extracts, when tested together on human skin explants, demonstrated a good synergic hypopigmenting activity. Taken together, the results indicate that the extracts from B. rapa root cultures can be employed as cosmetic active ingredients in skin whitening products and as potential therapeutic agents for treating pigmentation disorders.

Platelet lysate as a serum replacement for skin cell culture on biomimetic PCL nanofibers

Platelets are a popular source of native growth factors for tissue engineering applications. The aim of the study was to verify the use of platelet lysate as a fetal bovine serum (FBS) replacement for skin cell culture. The cytokine content of the platelet lysate was characterized using the Bio-Plex system. The cells (fibroblasts, melanocytes, and keratinocytes) were cultured on PCL nanofibrous scaffolds to mimic their natural microenvironment. The cytokine content of the platelet lysate was determined, and to the cells, a medium containing platelet lysate or platelet lysate in combination with FBS was added. The results showed that 7% (v/v) platelet lysate was sufficient to supplement 10% (v/v) FBS in the culture of fibroblasts and keratinocytes. The combination of platelet lysate and FBS had a rather inhibitory effect on fibroblasts, in contrary to keratinocytes, where the effect was synergic. Platelet lysate did not sufficiently promote proliferation in melanocytes; however, the combination of FBS and platelet lysate yielded a better outcome and resulted in bipolar morphology of the cultured melanocytes. The data indicated that platelet lysate improved cell proliferation and metabolic activity and may be used as an additive to the cell culture media.

Investigation of the Daily Fluctuation of Angiopoietin 1 Gene Expression Using Cultured Skin Cells

Investigation of the Daily Fluctuation of Angiopoietin 1 Gene Expression Using Cultured Skin Cells

RIS-1/psoriasin expression in epithelial skin cells indicates their selective role in innate immunity and in inflammatory skin diseases including acne.

ABSTRACTObjective: RIS-1/psoriasin/S100A7 is an epithelial antimicrobial peptide, whose expression is upregulated in inflammatory skin diseases and is induced by retinoids. Its molecular expression was investigated in skin cell cultures and in skin specimens to better understand its role in inflammatory procedures of the pilosebaceous unit. Methods: rtPCR and northern blotting of RIS-1/psoriasin and the retinoid-metabolizing genes CYP26AI and CRABP-II were performed in cells cultures (keratinocytes, sebocytes, fibroblasts, endothelial cells, melanocytes, lymphocytes and prostate cells; native and treated with retinoids) and in situ hybridization in normal and inflamed skin (acne, psoriasis). Results: a) RIS-1/psoriasin is expressed in keratinocytes and fibroblasts in vitro and in keratinocytes of the stratum granulosum in vivo. Retinoids in vitro and inflammatory conditions in vivo increase the levels of RIS-1/psoriasin in keratinocytes (both), sebocytes (inflammation only) and fibroblasts (retinoids). Sebocytes and fibroblasts are the metabolically most active skin cells, since they can upregulate the expression of CRABP-II and CYP26AI, genes responsible for retinoid metabolism. Inflammation modifies the compartmentation of RIS-1/psoriasin in sebaceous glands and the follicular root sheaths. Conclusion: The present data indicate that anti-inflammatory treatment targeting the epithelial compartments of the skin, including such with antibacterial peptides, may be promising for inflammatory skin diseases.

Galdieria sulphuraria Relieves Oily and Seborrheic Skin By Inhibiting the 5-α Reductase Expression in Skin Cells and Reducing Sebum Production In Vivo

The sebum has important protective functions for the skin, although its overproduction may lead to undesired aesthetic problems and even more serious skin diseases. Sebum overproduction is caused by the androgen hormone testosterone, which is regulated by the enzyme 5-α Reductase, and can be associated with an over-proliferation of parasitic microbes that cause serious infections linked to skin inflammatory reactions. From the extremophile microalga Galdieria sulphuraria, we obtained a water-soluble extract and we found that it was capable of inhibiting the enzyme 5-α Reductase, inducing the expression of the β-defensins, which represents the first defense response mechanism triggered by the skin cells to fight against undesired microbes, and stimulating the wound healing process in skin cell cultures. The microalga extract was finally employed in clinical tests on human volunteers and it was confirmed its capacity to regulate sebum production. The results suggested that the Galdieria extract had interesting potentialities as active ingredient in cosmetic and dermatological formulas, in particular in those addressed to oily and seborrheic skins.

Optimized method of dispersion of titanium dioxide nanoparticles for evaluation of safety aspects in cosmetics

Nanoparticles agglomerate when in contact with biological solutions, depending on the solutions’ nature. The agglomeration state will directly influence cellular response, since free nanoparticles are prone to interact with cells and get absorbed into them. In sunscreens, titanium dioxide nanoparticles (TiO2-NPs) form mainly aggregates between 30 and 150 nm. Until now, no toxicological study with skin cells has reached this range of size distribution. Therefore, in order to reliably evaluate their safety, it is essential to prepare suspensions with reproducibility, irrespective of the biological solution used, representing the above particle size distribution range of NPs (30–150 nm) found on sunscreens. Thus, the aim of this study was to develop a unique protocol of TiO2 dispersion, combining these features after dilution in different skin cell culture media, for in vitro tests. This new protocol was based on physicochemical characteristics of TiO2, which led to the choice of the optimal pH condition for ultrasonication. The next step consisted of stabilization of protein capping with acidified bovine serum albumin, followed by an adjustment of pH to 7.0. At each step, the solutions were analyzed by dynamic light scattering and transmission electron microscopy. The final concentration of NPs was determined by inductively coupled plasma-optical emission spectroscopy. Finally, when diluted in dulbecco’s modified eagle medium, melanocytes growth medium, or keratinocytes growth medium, TiO2–NPs displayed a highly reproducible size distribution, within the desired size range and without significant differences among the media. Together, these results demonstrate the consistency achieved by this new methodology and its suitability for in vitro tests involving skin cell cultures.

Juglone ameliorates skin wound healing by promoting skin cell migration through Rac1/Cdc42/PAK pathway.

Skin cell regeneration and wound healing are key processes in the recovery from skin injuries. Rapid cell migration and regeneration of skin cells lead to faster and better healing of wounded skin. In the present study, we aimed to investigate the wound healing potential of juglone, a naturally occurring Pin1 inhibitor found in walnuts. Cultured skin cells (NHDF and HaCaT) and hairless mice were treated with juglone after wound creation to examine its effects on cell migration and wound healing rate. The expressions of cell migration related proteins (Rac1, Cdc42, and α-PAK), collagen deposition, and angiogenesis were analyzed. Juglone treatment resulted in faster rate of growth and migration and recovered cell morphology, particularly at a concentration of 5 µM, in skin cells compared to the untreated group. In vivo experiments showed that mice treated with juglone showed faster wound healing rate with better skin morphology and collagen deposition than the vehicle group. Furthermore, juglone increased the activation and/or expression of Cdc42, Rac1, and α-pak in HaCaT cells, and resulted in enhanced angiogenesis in endothelial cells (HUVECs). Juglone also activated MAPKs signaling by activation of ERK, JNK, and p38 proteins. Taken together, these data suggest that juglone may be a potential candidate for wound healing and skin regeneration which ameliorates wound healing mainly by promoting skin cell migration through Rac1/Cdc42/PAK pathway.

Preparation of biodegradable gelatin/PVA porous scaffolds for skin regeneration

Porous scaffolds composed of gelatin/poly (vinyl alcohol), (Gel/PVA), were prepared using combination of freeze gelation and freeze drying methods. The effect of polymer concentration, gelatin/PVA ratio, and glutaraldehyde/gelatin ratio (GA/Gel) was investigated on morphology of pores, swelling ratio, biodegradation, and skin cell culture. At optimum preparation conditions the scaffolds had uniform pore size distributions showing high swelling ratio of 23.6. The scaffolds were of biodegradable nature and almost degraded in 28 days. Human dermal fibroblast cells (HDF) were cultured on the scaffolds and MTS assay was conducted to evaluate the influence of PVA on growth and proliferation of the cells.

337 The protective effect of the radiation-resistant microbes-derived exopolysaccharide on the radiation damage in cultured skin cells

337 The protective effect of the radiation-resistant microbes-derived exopolysaccharide on the radiation damage in cultured skin cells

417 Modulation of microRNAs in skin cell culture models for aging and environmental pollution

417 Modulation of microRNAs in skin cell culture models for aging and environmental pollution

Reassessing the Role of the Active TGF-β1 as a Biomarker in Systemic Sclerosis: Association of Serum Levels with Clinical Manifestations

Objective. To determine active TGF-β1 (aTGF-β1) levels in serum, skin, and peripheral blood mononuclear cell (PBMC) culture supernatants and to understand their associations with clinical parameters in systemic sclerosis (SSc) patients. Methods. We evaluated serum samples from 56 SSc patients and 24 healthy controls (HC). In 20 SSc patients, we quantified spontaneous or anti-CD3/CD28 stimulated production of aTGF-β1 by PBMC. The aTGF-β1 levels were measured by ELISA. Skin biopsies were obtained from 13 SSc patients and six HC, and TGFB1 expression was analyzed by RT-PCR. Results. TGF-β1 serum levels were significantly higher in SSc patients than in HC (p < 0.0001). Patients with increased TGF-β1 serum levels were more likely to have diffuse subset (p = 0.02), digital ulcers (p = 0.02), lung fibrosis (p < 0.0001), positive antitopoisomerase I (p = 0.03), and higher modified Rodnan score (p = 0.046). Most of our culture supernatant samples had undetectable levels of TGF-β1. No significant difference in TGFB1 expression was observed in the SSc skin compared with HC skin. Conclusion. Raised active TGF-β1 serum levels and their association with clinical manifestations in scleroderma patients suggest that this cytokine could be a marker of fibrotic and vascular involvement in SSc.

Calendula officinalis Protection Against Cytotoxicity Effects of Personal Care Products on HaCaT Human Skin Cells

Background: Human skin is normally exposed to ionizing and UV radiations and on occasions it may also be subjected to beauty products or drugs that the host uses; all of which can generate reactive oxygen species (ROS). Objective: In this study, Calendula officinalis extract, which contains antioxidant compounds, was examined for its protective effects against the products that generate ROS- induced cytotoxic activity towards human skin cells. Methodology: The protection against cytotoxicity by Calendula officinalis extract was investigated in vitro using a dose-response on HaCaT human skin cells. The proprietary aqueous calendula extract C from biodynamically grown plant was examined. Protection against cytotoxicity was measured via the methyl tetrazolium cytotoxicity (MTT) assay. Cells were exposed to the calendula extracts for 48h before being exposed to personal care products consisting of two head lice treatments -Lice Breaker (Permethrin 1% w/w) and Organix Pyrethrum treatment (4 g/L Pyrethrins, 16 g/LPiperonyl Butoxide) and two beauty products -Nivea Visage Q10 plus Anti-Wrinkle face moisturizer + TiO2; and Nivea Visage Q10 Plus Anti-Wrinkle face moisturizer (without TiO2) for 1h. Results: The effect of different concentrations of calendula extract on HaCaT human skin cells in vitro was explored. Doses of 1% (v/v) [1.4 mg dry weight equiv/ml] or less showed no toxicity. Cells were also exposed to the calendula extract for 48 hours before being exposed to the four personal care products for 1h. using the MTT cytotoxicity assay; it was observed that extract of Calendula officinalis gave time-dependent and concentrationdependent protection against harmful products that induce cell killing. Pre-incubation with the calendula extract for 48 hours significantly increased survival relative to the population without the extract by 30% and 47.4%, respectively, following treatment with personal care products. Conclusion: This study demonstrates that calendula extract contains bioactive and free radical scavenging compounds that significantly protect against personal care products that generate oxidative stress cytotoxicity in a human skin cell culture model.

Electrospun polyvinyl alcohol-polyvinyl pyrrolidone nanofibrous membranes for interactive wound dressing application

Cross-linked polyvinyl alcohol (PVA) and polyvinyl pyrrolidone (PVP) composite nanofibrous membranes have been prepared by electrospinning. Mechanical properties of the membranes improved significantly with PVP addition. PVP improved hydrophilicity and sustainable degradation of the membranes. Biocompatibility of the membranes was assessed by in vitro culture of native skin cells (L929 fibroblast and HaCaT keratinocytes). Tests showed sustained release of the antibiotic ciprofloxacin hydrochloride monohydrate by the membranes. Further, zone of inhibition study against Staphylococcus aureus growth demonstrated protective action against external pathogenic microbes. These studies show these simple PVA–PVP nanofibrous membranes are promising interactive antibiotic-eluting wound dressing materials.

Mechanical Stretch on Human Skin Equivalents Increases the Epidermal Thickness and Develops the Basement Membrane.

All previous reports concerning the effect of stretch on cultured skin cells dealt with experiments on epidermal keratinocytes or dermal fibroblasts alone. The aim of the present study was to develop a system that allows application of stretch stimuli to human skin equivalents (HSEs), prepared by coculturing of these two types of cells. In addition, this study aimed to analyze the effect of a stretch on keratinization of the epidermis and on the basement membrane. HSEs were prepared in a gutter-like structure created with a porous silicone sheet in a silicone chamber. After 5-day stimulation with stretching, HSEs were analyzed histologically and immunohistologically. Stretch-stimulated HSEs had a thicker epidermal layer and expressed significantly greater levels of laminin 5 and collagen IV/VII in the basal layer compared with HSEs not subjected to stretch stimulation. Transmission electron microscopy revealed that the structure of the basement membrane was more developed in HSEs subjected to stretching. Our model may be relevant for extrapolating the effect of a stretch on the skin in a state similar to an in vivo system. This experimental system may be useful for analysis of the effects of stretch stimuli on skin properties and wound healing and is also expected to be applicable to an in vitro model of a hypertrophic scar in the future.

Short peptides stimulate cell regeneration in skin during aging

The search for new safe and efficient low-molecular weight substances stimulating skin regeneration is an important objective of geriatric cosmetology. The present study addressed the effects of the peptides LK and AEDG at concentrations of 0.05–2.00 ng/mL on the proliferation of organotypic tissue cultures of skin from young and old animals. The application of peptides enhanced fibroblast proliferation in skin cell cultures from young and old animals by 29–45%. The effect on aging skin was observed in a narrower concentration range; in addition, it was less pronounced than the effect on skin cell cultures obtained from young animals. These data reveal the potential of LK and AEDG as components of cosmetic products that restore the structure of aging skin.

An oil-soluble extract of Rubus idaeus cells enhances hydration and water homeostasis in skin cells.

Raspberry plants, belonging to the species of Rubus idaeus, are known for their excellent therapeutic properties as they are particularly rich in compounds with strong antioxidant activity, which promote health and well-being of human cells. Besides their high content of phenolic compounds, Rubus plants are rich in oil-soluble compounds, which are also primary components of the hydrolipidic film barrier of the skin. As plant cell cultures represented a valuable system to produce interesting compounds and ingredients for cosmetic applications, we developed liquid suspension cultures from Rubus idaeus leaves and used them to obtain an active ingredient aimed at improving hydration and moisturization capacity in the skin. Rubus idaeus cells, grown in the laboratory under sterile and controlled conditions as liquid suspension cultures, were processed to obtain an oil-soluble (liposoluble) extract, containing phenolic compounds and a wide range of fatty acids. The extract was tested on cultured keratinocytes and fibroblasts and then on the skin in vivo, to assess its cosmetic activities. When tested on skin cell cultures, the extract induced the genes responsible for skin hydration, such as aquaporin 3, filaggrin, involucrin and hyaluronic acid synthase, and stimulated the expression and the activity of the enzyme glucocerebrosidase, involved in ceramide production. Moreover, the liposoluble extract increased the synthesis of the extracellular matrix components in cultured fibroblasts and showed a remarkable skin-hydrating capacity when tested on human skin in vivo. Thanks to these activities, the Rubus idaeus liposoluble extract has several potential applications in skin care cosmetics: it can be used as hydrating and moisturizing ingredient in face and body lotions, and as anti-ageing product in face creams specifically designed to fight wrinkle formation.

In vitro growth, cytopathic effects and clearance of monolayers by clinical isolates of Balamuthia mandrillaris in human skin cell cultures

Balamuthia mandrillaris is a free-living ameba (FLA) that has been isolated or its DNA identified in soil, dust and water. It causes a fatal central nervous system infection in humans and animals. Although it is environmental as Acanthamoeba and Naegleria fowleri, the two other free-living amebae that also cause CNS infections in humans and other animals, Balamuthia does not feed on bacteria as the other FLA. In the laboratory, it can be grown on a variety of mammalian cell cultures. In this study we examined the ability of three different Balamuthia isolates to grow on several different human skin cell cultures including the WT/A keratinocyte cell cultures. A corneal isolate of Acanthamoeba castellanii was used for comparison.

Basal level of autophagy is increased in aging human skin fibroblasts in vitro, but not in old skin.

Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubule-associated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5-fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP.

Human Skin Models for Research Applications in Pharmacology and Toxicology: Introducing NativeSkin®, the “Missing Link” Bridging Cell Culture and/or Reconstructed Skin Models and Human Clinical Testing

The use of human skin models in pharmacology and toxicology has become a widely accepted approach for the evaluation of dermato-cosmetic products, both finished formulations and active ingredients. In particular, in vitro–reconstructed human skin models consisting on human keratinocytes and/or fibroblasts are used globally in both industrial and academic research laboratories, and regulatory bodies endorse the use of several of them as valid alternatives to animal testing requirements. Despite the recent progress that has been made in the reconstruction of more complex skin models containing other cell types, such models still have inherent limitations, as they are not an exact copy of human skin in vivo, and hence the predictability of the human in vivo response remains limited. Here we describe a new testing approach based on the use of NativeSkin®, a standardized ex vivo model of human skin, maintaining physiological skin biology, skin barrier function, and metabolism during 7 days of culture in tissue culture inserts. NativeSkin models are cultivated in quality-controlled and assured conditions, and demonstrate high reproducibility among donors. Where initial product testing is often performed on skin cell cultures and in vitro–reconstructed models, we propose the use of NativeSkin being the most complete skin model available, as highly predictive and cost-effective “last-line screen in laboratory conditions” prior to clinical evaluation in humans.