A Human Epidermal Model that can be Used in an Automated Multiple Endpoint Assay

1992 ◽  
Vol 20 (2) ◽  
pp. 313-323
Author(s):  
Jeffery R. Cook ◽  
Joseph Gabriels ◽  
Laura M. Patrone ◽  
Laura S. Rhoads ◽  
Robert G. Van Buskirk
Keyword(s):  
Membrane Integrity ◽  
Collagen Gel ◽  
Liquid Interface ◽  
Dermal Fibroblasts ◽  
Epidermal Keratinocytes ◽  
Apical Region ◽  
Basal Region ◽  
Human Epidermal Keratinocytes ◽  
Endpoint Assay ◽  
Air Liquid Interface

Normal human epidermal keratinocytes were differentiated on Millipore Millicell CM microporous membranes overlaid with an acellular cross-linked collagen gel. The extracellular calcium concentration was elevated, and stratification of the monolayer to a 20-cell thick multilayer ensued within 5–7 days. Transmission electron microscopy of this stratified, human epidermal model (HEM) revealed typical ultrastructural markers of differentiation, including numerous desmosomes, keratinohyalin granules, keratin filaments, lamellar-type bodies, and squamous-shaped cells in the apical region, but not the basal region, of this HEM. Preparations lifted to the air–liquid interface had electron-dense apical cells suggestive of cornification. An electron-dense lamina appeared on the basal surface in both submerged and air–liquid interface cultures, suggesting that this structure can be produced without the help of underlying dermal cells and is independent of the air–liquid interface conditions. The uniform thickness of the epidermis, the absence of dermal fibroblasts, the lack of contraction of the epidermis, and the thinness of the underlying collagen gel, made this HEM a suitable candidate for use in an automated, fluorescence multiple endpoint assay. Fluorescent probes monitoring lysosomal integrity, epidermal permeability and plasma membrane integrity were monitored using the CytoFluor 2300 fluorescent plate reader. Our data suggest the feasibility of using multilayered human tissues for automated multiple endpoint analysis.

Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism

1999 ◽  
Vol 112 (12) ◽  
pp. 1843-1853 ◽  
Author(s):  
N. Maas-Szabowski ◽  
A. Shimotoyodome ◽  
N.E. Fusenig
Keyword(s):  
Neutralizing Antibodies ◽  
Growth Regulation ◽  
Keratinocyte Growth Factor ◽  
Dermal Fibroblasts ◽  
Tissue Homeostasis ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Keratinocyte Proliferation ◽  
Normal Human ◽  
Kinetics Of

Epithelial-mesenchymal interactions play an important role in regulating tissue homeostasis and repair. For skin, the regulatory mechanisms of epidermal-dermal interactions were studied in cocultures of normal human epidermal keratinocytes (NEK) and dermal fibroblasts (HDF) rendered postmitotic by alpha-irradiation (HDFi). The expression kinetics of different cytokines and their receptors with presumed signalling function in skin were determined at the RNA and protein level in mono- and cocultured NEK and HDFi. In cocultured HDFi, mRNA and protein synthesis of keratinocyte growth factor (KGF) (FGF-7) was strongly enhanced, whereas in cocultured keratinocytes interleukin (IL)-1alpha and -1beta mRNA expression increased compared to monocultures. Thus we postulated that IL-1, which had no effect on keratinocyte proliferation, induced in fibroblasts the expression of factors stimulating keratinocyte proliferation, such as KGF. The functional significance of this reciprocal modulation was substantiated by blocking experiments. Both IL-1alpha and -1beta-neutralizing antibodies and IL-1 receptor antagonist significantly reduced keratinocyte proliferation supposedly through abrogation of KGF production, because IL-1 antibodies blocked the induced KGF production. These data indicate a regulation of keratinocyte growth by a double paracrine mechanism through release of IL-1 which induces KGF in cocultured fibroblasts. Thus IL-1, in addition to its proinflammatory function in skin, may play an essential role in regulating tissue homeostasis.

scholarly journals Persea americanaMill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

2013 ◽  
Vol 2013 ◽  
pp. 1-12 ◽  
Author(s):  
Maria del R. Ramos-Jerz ◽  
Socorro Villanueva ◽  
Gerold Jerz ◽  
Peter Winterhalter ◽  
Alexandra M. Deters
Keyword(s):  
Chlorogenic Acid ◽  
Dermal Fibroblasts ◽  
Persea Americana ◽  
Epidermal Keratinocytes ◽  
Hacat Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Residual Solvent ◽  
Keratinocyte Proliferation ◽  
Normal Human

Methanolic avocado (Persea americanaMill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Theirin vitroinfluence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fractionM.2composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore,M.2increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fractionM.6increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fractionM.7contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

PREPARATION AND EVALUATION OF GAG-INCORPORATED SKIN SUBSTITUTE: AN IN VITRO STUDY

2006 ◽  
Vol 18 (03) ◽  
pp. 153-157 ◽  
Author(s):  
TZU-WEI WANG ◽  
HSI-CHIN WU ◽  
YI-CHAU HUANG ◽  
JUI-SHENG SUN ◽  
FENG-HUEI LIN
Keyword(s):  
Lower Layer ◽  
In Vitro Study ◽  
Dermal Fibroblasts ◽  
Skin Equivalent ◽  
Skin Substitute ◽  
Epidermal Keratinocytes ◽  
Different Temperatures ◽  
Air Liquid Interface ◽  
Epidermis Structure

A bi-layered gelatin-C6S-HA membrane with different pore sizes was prepared by freeze-drying at different temperatures - 20°C and -196°C, respectively Glycosaminoglycans (GAGs) were incorporated within the gelatin matrices to mimic the dermal composition and to create an appropriate environment for cell growth. The gelatin-C6S-HA membrane was cross-linked by 1-ethyl-3(3-dimethylaminopropryl) carbodiimide (EDC) to resist rapidly biodegradation by matrix enzymes. In this study, the lower layer of the sponge was inoculated with dermal fibroblasts for dermis development and as the feeder layer for epidermal keratinocytes. The upper layer was seeded with keratinocytes for epidermalization. After cultured for a period of time in air-liquid interface, the upper layer was developed into an epidermis structure with stratified epidermal layers. The lower part was developed into dermis-like structure synthesized by dermal fibroblasts surrounding with its own secreted extracellular matrix. In brief, the bi-layered skin equivalent with biological dermal analog and epidermal analog would be a suitable tool for autologous skin equivalent tissue engineering.

scholarly journals In Vitro and Clinical Evaluation of Cannabigerol (CBG) Produced via Yeast Biosynthesis: A Cannabinoid with a Broad Range of Anti-Inflammatory and Skin Health-Boosting Properties

Molecules ◽  
2022 ◽  
Vol 27 (2) ◽  
pp. 491
Author(s):  
Eduardo Perez ◽  
Jose R. Fernandez ◽  
Corey Fitzgerald ◽  
Karl Rouzard ◽  
Masanori Tamura ◽  
...  
Keyword(s):  
Human Skin ◽  
Cannabis Sativa ◽  
Transepidermal Water Loss ◽  
Dermal Fibroblasts ◽  
Ultraviolet B ◽  
Epidermal Keratinocytes ◽  
Yeast Fermentation ◽  
Lauryl Sulfate ◽  
Human Epidermal Keratinocytes

Cannabigerol (CBG) is a minor non-psychoactive cannabinoid present in Cannabis sativa L. (C. sativa) at low levels (<1% per dry weight) that serves as the direct precursor to both cannabidiol (CBD) and tetrahydrocannabinol (THC). Consequently, efforts to extract and purify CBG from C. sativa is both challenging and expensive. However, utilizing a novel yeast fermentation technology platform, minor cannabinoids such as CBG can be produced in a more sustainable, cost-effective, and timely process as compared to plant-based production. While CBD has been studied extensively, demonstrating several beneficial skin properties, there are a paucity of studies characterizing the activity of CBG in human skin. Therefore, our aim was to characterize and compare the in vitro activity profile of non-psychoactive CBG and CBD in skin and be the first group to test CBG clinically on human skin. Gene microarray analysis conducted using 3D human skin equivalents demonstrates that CBG regulates more genes than CBD, including several key skin targets. Human dermal fibroblasts (HDFs) and normal human epidermal keratinocytes (NHEKs) were exposed in culture to pro-inflammatory inducers to trigger cytokine production and oxidative stress. Results demonstrate that CBG and CBD reduce reactive oxygen species levels in HDFs better than vitamin C. Moreover, CBG inhibits pro-inflammatory cytokine (Interleukin-1β, -6, -8, tumor necrosis factor α) release from several inflammatory inducers, such as ultraviolet A (UVA), ultraviolet B (UVB), chemical, C. acnes, and in several instances does so more potently than CBD. A 20-subject vehicle-controlled clinical study was performed with 0.1% CBG serum and placebo applied topically for 2 weeks after sodium lauryl sulfate (SLS)-induced irritation. CBG serum showed statistically significant improvement above placebo for transepidermal water loss (TEWL) and reduction in the appearance of redness. Altogether, CBG’s broad range of in vitro and clinical skin health-promoting activities demonstrates its strong potential as a safe, effective ingredient for topical use and suggests there are areas where it may be more effective than CBD.

scholarly journals N-Succinyl-S-Farnesyl-L-Cysteine (SFC): A Novel Isoprenylcysteine Analog with In Vitro Anti-Inflammatory Activity and Clinical Skin Protecting Properties

Cosmetics ◽  
2021 ◽  
Vol 8 (4) ◽  
pp. 110
Author(s):  
José R. Fernández ◽  
Karl Rouzard ◽  
Corey Fitzgerald ◽  
Jason Healy ◽  
Masanori Tamura ◽  
...  
Keyword(s):  
Small Molecule ◽  
Dermal Fibroblasts ◽  
Epidermal Keratinocytes ◽  
Double Blind ◽  
Human Epidermal Keratinocytes ◽  
New Class ◽  
Normal Human ◽  
Anti Inflammatory ◽  
Split Face

Over the past 15 years, small molecule isoprenylcysteine (IPC) analogs have been identified as a potential new class of topical anti-inflammatories. Clinical studies have demonstrated that IPCs are both safe and effective in promoting healthy skin when applied topically. This work aims to demonstrate N-Succinyl-S-farnesyl-L-cysteine (SFC) as a novel IPC molecule that provides a broad spectrum of benefits for skin. Human promyelocytic cell line HL-60, human dermal microvascular endothelial cells (HDMECs), human dermal fibroblasts (HDFs), and normal human epidermal keratinocytes (NHEKs) were exposed in culture to various inducers to trigger reactive oxygen species, cytokines, or collagenase production. A 49-subject randomized double-blind, vehicle-controlled, split face trial was performed with 1% SFC gel, or 5% niacinamide and vehicle applied for 12 weeks to evaluate anti-wrinkle and anti-aging endpoints. We demonstrated that SFC inhibited GPCR and TLR-induced pro-inflammatory cytokine release in NHEKs and HDMECs from several inflammatory inducers such as UVB, chemicals, cathelicidin, and bacteria. SFC successfully reduced GPCR-induced oxidation in differentiated neutrophils. Moreover, photoaging studies showed that SFC reduced UVA-induced collagenase (pro-MMP-1) production in HDFs. Clinical assessment of 1% SFC gel demonstrated improvement above the vehicle for wrinkle reduction, hydration, texture, and overall appearance of skin. N-Succinyl-S-farnesyl-L-cysteine (SFC) is a novel anti-inflammatory small molecule and is the first farnesyl-cysteine IPC shown to clinically improve appearance and signs of aging, while also having the potential to ameliorate inflammatory skin disorders.

scholarly journals A Novel Substance P-Based Hydrogel for Increased Wound Healing Efficiency

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2215 ◽  
Author(s):  
Da Kim ◽  
Ji Jang ◽  
Song Jang ◽  
Jungsun Lee
Keyword(s):  
Wound Healing ◽  
Substance P ◽  
Dermal Fibroblasts ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
In Vivo Experiments ◽  
And Migration ◽  
Proliferation And Migration

The neuropeptide substance P (SP) is known to stimulate wound healing by regulating the production of relevant cytokines as well as cell proliferation and migration. However, the therapeutic application of SP is limited by its low stability under biological conditions and oxidation during purification, formulation, and storage. To address this problem, we developed a novel formulation of SP as an SP gel, and investigated its wound healing activity both in vitro and in vivo. SP in SP gel was stable at various temperatures for up to 4 weeks. In vitro, SP gel exhibited more potential as a candidate wound-healing agent than SP alone, as evidenced by the observed increases in the proliferation and migration of human epidermal keratinocytes and human dermal fibroblasts. In vivo experiments showed that SP gel treatment enhanced the healing of full-thickness wounds in mice as compared to SP alone. These results demonstrate the benefits of SP gel as a promising topical agent for wound treatment.

Sign in / Sign up

Export Citation Format

Share Document