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Characterization of microfluidic human epidermal keratinocyte culture.

ABSTRACT: Human epidermal keratinocytes (HEK) are skin cells of primary importance in maintaining the body's defensive barrier and are used in vitro to assess the irritation potential and toxicity of chemical compounds. Microfluidic systems hold promise for high throughput irritant and toxicity assays, but HEK growth kinetics have yet to be characterized within microscale culture chambers. This research demonstrates HEK patterning on microscale patches of Type I collagen within microfluidic channels and maintenance of these cells under constant medium perfusion for 72 h. HEK were shown to maintain 93.0%-99.6% viability at 72 h under medium perfusion ranging from 0.025-0.4 mul min(-1). HEK maintained this viability while approximately 100% confluent-a level not possible in 96 well plates. Microscale HEK cultures offer the ability to precisely examine the morphology, behavior and viability of individual cells which may open the door to new discoveries in toxicological screening methods and wound healing techniques.

SUBMITTER: O'Neill AT

PROVIDER: S-EPMC2553630 | biostudies-other | 2008 Mar

REPOSITORIES: biostudies-other

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Biologically relevant laminin as chemically defined and fully human platform for human epidermal keratinocyte culture.

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Dynamic Transcriptional and Epigenetic Regulation of Human Epidermal Keratinocyte Differentiation.

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Gene Expression Profile Comparison of Human Epidermal Keratinocyte Cell Culture Models Following Sulfur Mustard Exposure

Project description:Normal human epidermal keratinocytes (NHEKs) and HaCaTs have been widely used as cell culture models to study the effects of cutaneous sulfur mustard (HD) exposure. While these cell lines are similar, one key difference is that NHEKs are primary cells, whereas HaCaT cells are a transformed line with a mutation in the p53 gene. However, the impact of this mutation on the response of HaCaT cells to HD is unclear. Thus, gene expression profiling was performed to compare the transcriptional responses of NHEKs and HaCaTs after HD exposure. Cells were exposed to 0, 25, and 200 µM HD, harvested at 1 h and 8 h post-exposure, and processed for microarray analysis. Principal component analysis of the microarray data suggested profile differences based on cell type, but both cell types respond similarly to HD with regard to dose and time. To further analyze the expression profiles at various doses, the dataset was filtered by dose, and an analysis of variance was performed using cell type as the factor; the pathways most significantly different between these cell types were identified. At all three doses, the p53 and N-glycan degradation pathways were significantly different between NHEKs and HaCaTs. Interestingly, p53 responsive genes showed differences and similarities across cell types, which may provide insight into the role of p53 in HD toxicity. The inflammatory pathways expected to respond to HD exposure were not significantly different between cell types, suggesting that both NHEKs and HaCaTs are appropriate models to study the inflammatory effects of HD.

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Identification and characterization of small molecule inducers of epidermal keratinocyte differentiation

Project description:An essential function of the human epidermis is the maintenance of a protective barrier against the environment. As a consequence, keratinocytes, which make up this layer of the skin, undergo an elaborate process of self-renewal, terminal differentiation and cell death. Misregulation of these processes can lead to several human diseases including psoriasis and basal cell and squamous cell carcinomas. To identify novel regulators of keratinocyte differentiation, a cell-based screen of small molecule libraries was carried out for molecules that induce terminal differentiation of normal human epidermal keratinocytes (NHEKs). One class of molecules was identified, the 2-(3,4,5-trimethoxy-phenylamino)-pyrrolo[2,3-d]pyrimidines, which were shown to induce differentiation of epidermal progenitor cells to terminally differentiated keratinocytes. These molecules serve as useful mechanistic probes of the cellular differentiation programs that regulate the formation and homeostasis of the epidermis, and may lead to novel therapeutic approaches for the treatment of skin hyperproliferative disorders. Keywords: time course of compound treatment

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Amniotic Fluid primary human keratinocyte culture

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2021-08-25 | GSE182704 | GEO

Amniotic Fluid primary human keratinocyte culture

Project description:Amniotic Fluid primary human keratinocyte culture

| PRJNA757450 | ENA

Mechanism of interleukin-1? transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line.

Project description:S100A9 is a calcium-binding protein and subunit of antimicrobial calprotectin complex (S100A8/A9). Produced by neutrophils, monocytes/macrophages and keratinocytes, S100A9 expression increases in response to inflammation. For example, IL-1? produced by epithelial cells acts autonomously on the same cells to induce the expression of S100A8/A9 and cellular differentiation. Whereas it is well known that IL-1? and members of the IL-10 family of cytokines upregulate S100A8 and S100A9 in several cell lineages, the pathway and mechanism of IL-1?-dependent transcriptional control of S100A9 in epithelial cells are not established. Modeled using human epidermal keratinocytes (HaCaT cells), IL-1? stimulated the phosphorylation of p38 MAPK and induced S100A9 expression, which was blocked by IL-1 receptor antagonist, RNAi suppression of p38, or a p38 MAPK inhibitor. Transcription of S100A9 in HaCaT cells depended on nucleotides -94 to -53 in the upstream promoter region, based upon the use of deletion constructs and luciferase reporter activity. Within the responsive promoter region, IL-1? increased the binding activity of CCAAT/enhancer binding protein ? (C/EBP?). Mutated C/EBP? binding sequences or C/EBP?-specific siRNA inhibited the S100A9 transcriptional response. Hence, IL-1? is strongly suggested to increase S100A9 expression in a human epidermal keratinocyte cell line by signaling through the IL-1 receptor and p38 MAPK, increasing C/EBP?-dependent transcriptional activity.

| S-EPMC3719387 | biostudies-literature

Histamine suppresses epidermal keratinocyte differentiation and impairs skin barrier function in a human skin model.

Project description:BackgroundDefects in keratinocyte differentiation and skin barrier are important features of inflammatory skin diseases like atopic dermatitis. Mast cells and their main mediator histamine are abundant in inflamed skin and thus may contribute to disease pathogenesis.MethodsHuman primary keratinocytes were cultured under differentiation-promoting conditions in the presence and absence of histamine, histamine receptor agonists and antagonists. The expression of differentiation-associated genes and epidermal junction proteins was quantified by real-time PCR, Western blot, and immunofluorescence labeling. The barrier function of human skin models was tested by the application of biotin as tracer molecule.ResultsThe addition of histamine to human keratinocyte cultures and organotypic skin models reduced the expression of the differentiation-associated proteins keratin 1/10, filaggrin, and loricrin by 80-95%. Moreover, the addition of histamine to skin models resulted in the loss of the granular layer and thinning of the epidermis and stratum corneum by 50%. The histamine receptor H1R agonist, 2-pyridylethylamine, suppressed keratinocyte differentiation to the same extent as did histamine. Correspondingly, cetirizine, an antagonist of H1R, virtually abrogated the effect of histamine. The expression of tight junction proteins zona occludens-1, occludin, claudin-1, and claudin-4, as well as that of desmosomal junction proteins corneodesmosin and desmoglein-1, was down-regulated by histamine. The tracer molecule biotin readily penetrated the tight junction barrier of skin cultures grown in the presence of histamine, while their diffusion was completely blocked in nontreated controls.ConclusionsOur findings suggest a new mechanism by which mast cell activation and histamine release contribute to skin barrier defects in inflammatory skin diseases.

| S-EPMC3555427 | biostudies-literature

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