scholarly journals Fundamental in vitro 3D human skin equivalent tool development for assessing biological safety and biocompatibility – towards alternative for animal experiments

4open ◽  
2021 ◽  
Vol 4 ◽  
pp. 1
Author(s):  
Ayesha Idrees ◽  
Inge Schmitz ◽  
Alice Zoso ◽  
Dierk Gruhn ◽  
Sandra Pacharra ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Human Skin ◽  
Barrier Function ◽  
Culture Conditions ◽  
Dermal Fibroblasts ◽  
Skin Equivalent ◽  
Epidermal Keratinocytes ◽  
Normal Human ◽  
Human Skin Equivalent

Nowadays, human skin constructs (HSCs) are required for biomaterials, pharmaceuticals and cosmetics in vitro testing and for the development of complex skin wound therapeutics. In vitro three-dimensional (3D) dermal-epidermal based interfollicular, full-thickness, human skin equivalent (HSE) was here developed, recapitulating skin morphogenesis, epidermal differentiation, ultra-structure, tissue architecture, and barrier function properties of human skin. Different 3D cell culture conditions were tested to optimize HSE maturation, using various commercially available serum/animal component-free and/or fully defined media, and air-liquid interface (ALI) culture. Optimized culture conditions allowed the production of HSE by culturing normal human dermal fibroblasts (NHDFs) for 5–7 days in CELLnTEC-Prime Fibroblast (CnT-PR-F) medium and then culturing normal human epidermal keratinocytes (NHEKs) for 3 days in CELLnTEC-Prime Epithelial culture (CnT-PR) medium on them. Co-culture was then submerged overnight in CELLnTEC-Prime-3D barrier (CnT-PR-3D) medium to stimulate cell-cell contact formation and finally placed at ALI for 15–20 days using CnT-PR-3D medium. Histological analysis revealed uniform distribution of NHDFs in the dermal layer and their typical elongated morphology with filopodia. Epidermal compartment showed a multi-layered structure, consisting of stratum basale, spinosum, granulosum, and corneum. NHDFs and keratinocytes of basal layer were positive for the proliferation marker Kiel 67 (Ki-67) demonstrating their active state of proliferation. The presence of typical epidermal tissue proteins (keratins, laminins, filaggrin, loricin, involucrin, and β-tubulin) at their correct anatomical position was verified by immunohistochemistry (IHC). Moreover, transmission electron microscopy (TEM) analyses revealed basement membrane with lamina lucida, lamina densa, hemidesmosomes and anchoring fibers. The epidermal layers showed abundant intracellular keratin filaments, desmosomes, and tight junction between keratinocytes. Scanning electron microscopy (SEM) analyses showed the interwoven network of collagen fibers with embedded NHDFs and adjacent stratified epidermis up to the stratum corneum similar to native human skin. HSE physiological static contact angle confirmed the barrier function. The developed HSE represents a fundamental in vitro tool to assess biocompatibility of biomaterials, pharmacotoxicity, safety and effectiveness of cosmetics, as well as to investigate skin biology, skin disease pathogenesis, wound healing, and skin infection.

Desmosomal distribution in the epidermis of a non-contracting human skin equivalent

1992 ◽  
Vol 50 (1) ◽  
pp. 896-897
Author(s):  
L.X. Oakford ◽  
S.D. Dimitrijevich ◽  
R. Gracy
Keyword(s):  
Human Skin ◽  
Basal Layer ◽  
Skin Equivalent ◽  
Tissue Component ◽  
Intact Skin ◽  
Similar Sequence ◽  
Sequence Of Events ◽  
Suprabasal Keratinocytes ◽  
Human Skin Equivalent

In intact skin the epidermal layer is a dynamic tissue component which is maintained by a basal layer of mitotically active cells. The protective upper epidermis, the stratum corneum, is generated by differentiation of the suprabasal keratinocytes which eventually desquamate as anuclear comeocytes. A similar sequence of events is observed in vitro in the non-contracting human skin equivalent (HSE) which was developed in this lab (1). As a part of the definition process for this model of living skin we are examining its ultrastructural features. Since desmosomes are important in maintaining cell-cell interactions in stratified epithelia their distribution in HSE was examined.

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.

Use of Human Skin Equivalent Apligraf for in Vitro Assessment of Cumulative Skin Irritation Potential of Topical Products

2000 ◽  
Vol 164 (1) ◽  
pp. 38-45 ◽  
Author(s):  
Jesús Medina ◽  
Anne de Brugerolle de Fraissinette ◽  
Salah-Dine Chibout ◽  
Maryelle Kolopp ◽  
Rahel Kammermann ◽  
...  
Keyword(s):  
Human Skin ◽  
Skin Irritation ◽  
Skin Equivalent ◽  
In Vitro Assessment ◽  
Human Skin Equivalent

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.

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