scholarly journals Isolation of Mouse Epidermal Keratinocytes and Their In Vitro Clonogenic Culture

10.3791/58701 ◽  
2019 ◽  
Author(s):  
Rebecca J. Morris ◽  
Nyssa Readio ◽  
Kelsey Boland ◽  
Kelly Johnson ◽  
Sonali Lad ◽  
...  
Keyword(s):  
Epidermal Keratinocytes ◽  
Mouse Epidermal Keratinocytes

Alterations of ultrastructure and elemental composition in cultured human epidermal keratinocytes after treatment with nitrofurazone and silver sulfadiazine

1987 ◽  
Vol 45 ◽  
pp. 676-677
Author(s):  
A. R. Crooker ◽  
M. C. Myers ◽  
T. L. Beard ◽  
E. S. Graham
Keyword(s):  
Electron Microscopy ◽  
Elemental Composition ◽  
Pyrolytic Carbon ◽  
Human Keratinocytes ◽  
Silver Sulfadiazine ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Culture Systems ◽  
Cytotoxicity Endpoints

Cell culture systems have become increasingly popular as a means of screening toxic agents and studying toxic mechanisms of drugs and other chemicals at the cellular and subcellular levels. These in vitro tests can be conducted rapidly in a broad range of relevant mammalian culture systems; a variety of biological and biochemical cytotoxicity endpoints can be examined. The following study utilized human keratinocytes to evaluate the relative cytotoxicities of nitrofurazone (NF) and silver sulfadiazine (SS), the active ingredients of FURACIN(R) Topical Cream and SILVADENE(R) Cream, respectively. These compounds are anti-infectives used in the treatment of burn patients. Cell ultrastructure and elemental composition were utilized as cytotoxicity endpoints.Normal Human Epidermal Keratinocytes (HK) were prepared from the EpiPackTM culture system (Clonetics Corporation, Boulder, CO). For scanning electron microscopy (SEM) and transmission electron microscopy (TEM), cells were seeded on sterile 35 mm Falcon plastic dishes; for elemental microanalysis, cells were plated on polished pyrolytic carbon discs (E. Fullam, Latham, NY) placed in the culture dishes.

scholarly journals Antioxidant and Pro-Oxidant Capacities as Mechanisms of Photoprotection of Olive Polyphenols on UVA-Damaged Human Keratinocytes

Molecules ◽  
2021 ◽  
Vol 26 (8) ◽  
pp. 2153
Author(s):  
Raffaella Marina Lecci ◽  
Isabella D’Antuono ◽  
Angela Cardinali ◽  
Antonella Garbetta ◽  
Vito Linsalata ◽  
...  
Keyword(s):  
Antioxidant Activities ◽  
Biological Activities ◽  
Human Keratinocytes ◽  
Ldl Oxidation ◽  
Trolox Equivalent Antioxidant Capacity ◽  
Epidermal Keratinocytes ◽  
Olive Cultivar ◽  
Human Epidermal Keratinocytes ◽  
Apoptotic Effect

A wide variety of polyphenols are reported to have considerable antioxidant and skin photoprotective effects, although the mechanisms of action are not fully known. Environmentally friendly and inexpensive sources of natural bioactive compounds, such as olive mill wastewater (OMWW), the by-product of olive-oil processing, can be considered an economic source of bioactive polyphenols, with a range of biological activities, useful as chemotherapeutic or cosmeceutical agents. Green strategies, such as the process based on membrane technologies, allow to recover active polyphenols from this complex matrix. This study aims to evaluate the antioxidant, pro-oxidant, and photoprotective effects, including the underlying action mechanism(s), of the ultra-filtered (UF) OMWW fractions, in order to substantiate their use as natural cosmeceutical ingredient. Six chemically characterized UF-OMWW fractions, from Italian and Greek olive cultivar processing, were investigated for their antioxidant activities, measured by Trolox Equivalent Antioxidant Capacity (TEAC), LDL oxidation inhibition, and ROS-quenching ability in UVA-irradiated HEKa (Human Epidermal Keratinocytes adult) cultures. The photoprotective properties of UF-OMWW were assayed as a pro-oxidant-mediated pro-apoptotic effect on the UVA-damaged HEKa cells, which can be potentially involved in the carcinogenesis process. All the UF-OMWW fractions exerted an effective antioxidant activity in vitro and in cells when administered together with UV-radiation on HEKa. A pro-oxidative and pro-apoptotic effect on the UVA-damaged HEKa cells were observed, suggesting some protective actions of polyphenol fraction on keratinocyte cell cultures.

scholarly journals Regulatory Mechanism for Exfoliative Toxin Production inStaphylococcus aureus

2011 ◽  
Vol 79 (4) ◽  
pp. 1660-1670 ◽  
Author(s):  
Fuminori Kato ◽  
Noriko Kadomoto ◽  
Yuko Iwamoto ◽  
Katsuaki Bunai ◽  
Hitoshi Komatsuzawa ◽  
...  
Keyword(s):  
Mouse Model ◽  
Regulatory Mechanism ◽  
Toxin Production ◽  
Neonatal Mouse ◽  
Epidermal Keratinocytes ◽  
Global Regulators ◽  
Exfoliative Toxin ◽  
Blistering Disease

ABSTRACTThe exfoliative toxin (ET) is a major virulence factor ofStaphylococcus aureusthat causes bullous impetigo and its disseminated form, staphylococcal scalded-skin syndrome (SSSS). ET selectively digests one of the intracellular adhesion molecules, desmoglein 1, of epidermal keratinocytes and causes blisters due to intraepidermal cell-cell dissociation. MostS. aureusstrains that cause blistering disease produce either ETA or ETB. They are serologically distinct molecules, where ETA is encoded on a phage genome and ETB is enocded on a large plasmid. ETA-producingS. aureusstrains are frequently isolated from impetigo patients, and ETB-producingS. aureusstrains are isolated from SSSS. ET-induced blister formation can be reproduced with the neonatal mouse. To determine the regulatory mechanism of ET production, we investigated the role of the two-component systems and global regulators foretaoretbexpressionin vitroandin vivowith the mouse model. Western blot and transcription analyses using a series of mutants demonstrate ETA production was downregulated bysigB,sarS, andsarA, while ETB production was downregulated bysigBandsarAbut not bysarS. Production of both toxins is upregulated bysaeRS,arlRS, andagrCA. Furthermore, by thein vivoneonatal mouse model,sigBandsarSbut notsarAnegatively regulate the exfoliation activity of the ETA-producing strain, whilesarAnegatively regulates the ETB-producing strain. In both strains,saeRS,arlRS, andagrCApositively regulate the exfoliation activityin vivo. The data illustrate similar but distinct regulatory mechanisms for ETA and ETB productionin S. aureus in vitroas well asin vivo.

scholarly journals Adipose-derived stromal/stem cells improve epidermal homeostasis

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Mariko Moriyama ◽  
Shunya Sahara ◽  
Kaori Zaiki ◽  
Ayumi Ueno ◽  
Koichi Nakaoji ◽  
...  
Keyword(s):  
Stem Cells ◽  
Wound Healing ◽  
Culture System ◽  
Wound Repair ◽  
Cell Types ◽  
Skin Wound ◽  
Epidermal Keratinocytes ◽  
Stromal Stem Cells ◽  
Collagen Vitrigel

AbstractWound healing is regulated by complex interactions between the keratinocytes and other cell types including fibroblasts. Recently, adipose-derived mesenchymal stromal/stem cells (ASCs) have been reported to influence wound healing positively via paracrine involvement. However, their roles in keratinocytes are still obscure. Therefore, investigation of the precise effects of ASCs on keratinocytes in an in vitro culture system is required. Our recent data indicate that the epidermal equivalents became thicker on a collagen vitrigel membrane co-cultured with human ASCs (hASCs). Co-culturing the human primary epidermal keratinocytes (HPEK) with hASCs on a collagen vitrigel membrane enhanced their abilities for cell proliferation and adhesion to the membrane but suppressed their differentiation suggesting that hASCs could maintain the undifferentiated status of HPEK. Contrarily, the effects of co-culture using polyethylene terephthalate or polycarbonate membranes for HPEK were completely opposite. These differences may depend on the protein permeability and/or structure of the membrane. Taken together, our data demonstrate that hASCs could be used as a substitute for fibroblasts in skin wound repair, aesthetic medicine, or tissue engineering. It is also important to note that a co-culture system using the collagen vitrigel membrane allows better understanding of the interactions between the keratinocytes and ASCs.

scholarly journals 5-Aminolevulinic Acid-Based Photodynamic Therapy Pretreatment Mitigates Ultraviolet A-Induced Oxidative Photodamage

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Hui Hua ◽  
Jiawei Cheng ◽  
Wenbo Bu ◽  
Juan Liu ◽  
Weiwei Ma ◽  
...  
Keyword(s):  
Aminolevulinic Acid ◽  
Epidermal Keratinocytes ◽  
Control Group ◽  
Hacat Cells ◽  
Ultraviolet A ◽  
Hairless Mice ◽  
Human Epidermal Keratinocytes ◽  
Experimental Group

Aim. To determine whether 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is effective in combating ultraviolet A- (UVA-) induced oxidative photodamage of hairless mice skin in vivo and human epidermal keratinocytes in vitro. Methods. In in vitro experiments, the human keratinocyte cell line (HaCaT cells) was divided into two groups: the experimental group was treated with ALA-PDT and the control group was left untreated. Then, the experimental group and the control group of cells were exposed to 10 J/m2 of UVA radiation. ROS, O2− species, and MMP were determined by fluorescence microscopy; p53, OGG1, and XPC were determined by Western blot analysis; apoptosis was determined by flow cytometry; and 8-oxo-dG was determined by immunofluorescence. Moreover, HaCaT cells were also treated with ALA-PDT. Then, SOD1 and SOD2 were examined by Western blot analysis. In in vivo experiments, the dorsal skin of hairless mice was treated with ALA-PDT or saline-PDT, and then, they were exposed to 20 J/m2 UVA light. The compound 8-oxo-dG was detected by immunofluorescence. Conclusion. In human epidermal keratinocytes and hairless mice skin, UVA-induced oxidative damage can be prevented effectively with ALA-PDT pretreatment.

scholarly journals An in vitro feasibility investigation considering primary human melanocytes for spray- grafting of freshly isolated autologous skin cells for burn treatment and a clinical case report

2019 ◽  
pp. 1-8
Author(s):  
Jörg C. Gerlach ◽  
C. Johnen ◽  
B. Hartmann ◽  
J. Plettig ◽  
K. Bräutigam ◽  
...  
Keyword(s):  
Cell Morphology ◽  
Skin Color ◽  
In Vitro Model ◽  
Primary Cultures ◽  
Cell Suspensions ◽  
Epidermal Keratinocytes ◽  
Cell Counts ◽  
Skin Coloration ◽  
Vitro Model

A skin cell-spray grafting technique that enables the on-site application of freshly isolated autologous single cell suspensions was already applied in many cases on caucasian patients with low skin coloration. Our project hypothesis is that these suspensions contain keratinocytes and vital melanocytes, that are of particular interest for the treatment of patients of darker skin color. To test this, we applied an in vitro model, wherein the feasibility of i) isolating and ii) spraying of freshly isolated autologous melanocyte-keratinocyte cell suspensions was investigated. Primary human epidermal keratinocytes (HEKs) and melanocytes (MCs) were isolated from skin biopsies (n=8). Biochemical parameter, cell counts, cell morphology, growth behavior and immunofluorescence results were compared in two groups using MC cultures and co-cultures of MCs with HEKs. Case information on using the method clinically with one patient is included. The sprayed mixed cell suspensions proliferated in all groups without measurable loss of viability, and MCs exhibited a regular cell morphology in monoculture up to passage 4°. The sprayed MCs and HEKs demonstrated in vitro glucose and lactate metabolism that was comparable to the pipetted controls. In co-culture, well distributed CK14+ HEKs and NKI/beteb+ MCs could be demonstrated, which interacted in the in vitro model. The ratio of HEKs : MCs in our primary cultures were microscopically counted (n=8 each) as mean +/- SD 1,211,000 (+/- 574,343) HEK : 99,625 (+/- 59,025) MC; i.e., a ratio of approx. 12 : 1. Using the isolation method clinically for a patient with dark skin coloration after suffering severe second-degree burns shows a satisfying re-pigmentation of the resulting wound post healing. Freshly isolated spray-on melanocyte/keratinocyte suspensions provide for a considerable amount of viable HEKs and MCs. Using MCs in spray-grafting suspensions could represent a promising approach for treating severe partial-thickness burns and innovative therapy developments that also aim to address cosmetic aspects.

Proliferation of K19+ human epidermal keratinocytes in vitro

2007 ◽  
Vol 416 (1) ◽  
pp. 406-408 ◽  
Author(s):  
E. S. Chermnykh ◽  
E. A. Vorotelyak ◽  
S. B. Tkachenko ◽  
A. V. Vasil’ev ◽  
V. V. Terskikh
Keyword(s):  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes
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