SkinAxis LLC

2D anti-melanoma activity of drugs

Using SK-Mel28 or A375 melanoma cell (express mutant B-Raf (V600E) and wild type N-Ras) to determine proliferation and differentiation in response to treatment with different compounds (Count cell numbers at 24, 48,72hr) as a positive control use TNFα or IFNβ. Using RT-qPCR to determine mRNA expression of adhesion molecules ICAM-1, LFA-3, and VLA-2.

Testing Anti-Acne Activity of the Compounds

• Lipid production by primary human sebocytes (lipid production using Oil Red staining assay) • Androgen metabolism by measuring 5-alpha reductase activity (ELISA assay, using finasteride as positive control);

• Anti-inflammatory activity by qRT-PCR and ELISA for IL-6 and IL-8)

• Primary human epidermal keratinocytes (qRT-PCR for filaggrin, IL-6)

• Anti-microbial peptide release (ELISA for β-defensin, Elafin release)

• Primary human dermal fibroblasts

• Reconstructed human epidermis and full-thickness skin

Testing Anti-Psoriasis activity of the Compounds

• CD4+ T cells, cytokine release (anti-CD3/anti-CD28 stimulation using Anti-CD3 10 µg/ml + Anti-CD28 3µg/ml) IL-17 Elisa, as a control Cyclosporin A

• PBMC, anti-CD3/anti-CD28-stimulated T cells, IL-17 cytokine release (anti-CD3/anti-CD28 stimulation) ELISA as a control Cyclosporin A.

• Reconstructed epidermis stimulated with IL-17 3ng/ml, Oncostatin M-OSM 3ng/ml, and TNFa 3ng/ml, as a control Jak 1 inhibitor. Detection of expression S100A7, beta-defensin 2/4, RNAse 7 by IHC

• Normal human keratinocytes stimulated with IL-17 3ng/ml, Oncostatin M-OSM 3ng/ml, and TNFα 3ng/ml, control compound Jak1 inhibitor. Detection of CXCL-1 and IL-8 by ELISA

• Induction in keratinocytes (or reconstructed epidermis) of a psoriasis-like profile by cytokines by RT-qPCR or microarray

• NF-kB translocation-activation assay (immunofluorescence, ELISA or QuantiSig assay)

• Production of chemokines, antimicrobial peptides (AMPs) and expression of epidermal differentiation markers

• Stat3 activation by immunofluorescence assay as a control Jak 1 inhibitor

Testing wound healing and skin regeneration

• PBMC, LPS-stimulated monocytes, cytokine release (LPS stimulation) Endpoint Cytokine release (IFN-g, IL-1β, IL-6, IL-8, TNFα, MIP-1α, IL-12. Method FACs multiplex or ELISA. Stimulation with 1ug/ml of LPS as an inhibitor using Dexamethasone

• CD14 monocytes LPS (1ug/ml) stimulated control Dexamethasone, endpoint IL-1b, IL-6, TNFα, ELISA or FACs

• NHEK migration/proliferation, as a positive control EGF (GFP-Fluorescently labeled NHK, after trauma, treated with EGF or control compound)

• NHK differentiation markers RT-qPCR with low Ca2+

• Co-culture of human endothelial cells HMVEC (Red) + normal human dermal fibroblasts NHDF (GFP-labeled), differentiation/pseudotube formation (basal, pro-angiogenic agents) as a control VEGF, fluorescence imaging.

Assays for Aging

• Dermal fibroblasts stimulated with TGF-β as a positive control or compounds and release of Hyaluronic acid is measured by ELISA

• Dermal fibroblasts, stimulated with TGF-β as a positive control or compounds and Pro-collage I and MMP-1 synthesis and/or release is measured by ELISA or EIA

• Dermal fibroblasts, stimulated with H2O2 followed with a positive control TGF-b or Dexamethasone or compounds treatment and Pro-collage I and MMP-1 synthesis and/or release is measured by ELISA or EIA

• Full-thickness reconstructed skin, SkinAxis, stimulated with UVA+UVB every 24 hours for 4 days, IL-8 release, MMP-3, PGE2, Pro-collagen I synthesis/release.

• NHEK (Normal Human Epidermal Keratinocytes) stimulated with H2O2 (250uM) followed with treatment with either positive control alpha-tocopherol or test compounds to determine Reactive oxygen species content

• NHDF (Normal Human Dermal Fibroblast) UVA or UVB treatment control Dexamethasone. RT-qPCR for genes of interest

Testing Anti-Oxidant activities of the Compounds

• Measurement of DPPH radical scavenging activity • Transcription factor Nrf2 is activated in response to oxidative stress and coordinates the expression of antioxidant gene products. QuantiSig testing system developed by SkinAxis is another quantitative measurement of anti-oxidant

DNA damage assays

• SkinAxis evaluates morphological changes after UVA or UVB exposure by H&E staining of skin equivalents and DNA damage by immunohistochemical staining.

• Highly sensitive quantitative measurement of DNA damage by ELISA to determine cyclobutane pyrimidine dimers (CPD)

• QuantiSig assay for DNA damage

Pigmentation assay

• Tyrosinase inhibition assay using Tyrosinase inhibitor Assay kit (Cojic acid as a positive control Colorimetric detection)

• Melanasome transfer assay. Keratinocyte melanocyte co-culture. FACS melanosome transfer (Pmel17 (melanosomes) in CD49f-positive keratinocytes, co-labeling; Dexamethasone stimulates melanin transfer)

• Melanin content detection assay (pigmented normal human epidermal melanocytes; positive controls L-tyrosine or IBMX) Colorimetric detection

Barrier Function

SkinAxis evaluates the formation of functional barriers as a result of differentiation using protein or RNA expression of specific differentiation markers in 2D or 3D skin models by PCR, immunoblot, or immunohistochemical staining.

Epidermal Markers

SkinAxis evaluates the expression of specific differentiation markers in 2D or 3D models, inflammatory cytokine release, cell viability and metabolism using the MTT test, ATP content by luminescence assay, and detoxification enzymes by ELISA.

Dermal Markers

SkinAxis evaluates collagen production in human dermal fibroblasts, MMP activity using colorimetric assays, and MMP expression using PCR or ELISA.

Skin Irritation Assay

Skin irritation assay is used to determine reversible damage to the skin by application of a test compound to the reconstructed human epidermis. Compound exposure that induces skin irritation evaluated by the release of inflammatory mediators such as the cytokine Interleukin 1 alpha (IL-1a) or MTT assay.

QuantiSig Skin Technology

The measurement of different biological responses of the skin is of great importance in the activity and toxicity testing of molecules employed in skin product development. Various methods have been developed to assess the effects of compounds on the various skin features. These methods rely mainly on the measurement of changes in the expression of RNA or protein markers within treated skin cellular components (e.g. keratinocytes, dermal fibroblasts, melanocytes). Different markers are assessed as indicative endpoints of specific skin responses using mostly immunohistological and immunohistological approaches. These methodologies are offer often limited sensitivity and are highly variable and difficult to objectively quantify.

To circumvent these limitations, SkinAxis has developed the QuantiSig technology that allows the analysis of the activation of different pathways representative of specific biological responses of the skin in a reproducible, highly sensitive, and measurable fashion. QuantiSig is a cell engineering technology that can be applied both in 2D cell cultures as well as in the reconstitution of 3-dimensional skin models without interfering with the normal physiological responses of the cells. QuantiSig allows the different cell types composing the skin tissue to stably carry a reporter gene whose expression is controlled by genetic elements activated during the stimulation of various pathways. The activation of the pathway following the application/exposure to chemical and biological compounds can then be monitored enzymatically in the engineered cells. Using different reporter genes, different cells and/or pathways can be evaluated at the same time.
The QuantiSig technology can be used to monitor the following cellular pathways in 2D cultures and reconstituted 3D tissues:

DNA damage response QuantiSig-DDR provides a very sensitive measurement of the DNA damage response pathway. The test can be applied to determine the effects of UV irradiation, sun protection applications on the DNA damage response pathway in the skin as well as anti-cancer drug testing affecting the p53 pathway.

Antioxidative response QuantiSig-AOR allows testing the anti-oxidative response pathway involved in the modulation of the Nuclear factor erythroid-related factor 2 (Nrf2) and the expression of cytoprotective genes. Constitutive activation of Nrf2 has been found in many cancers, resulting in resistance against chemotherapy and radiotherapy in cancer cells. Thus, screening for Nrf2 inducers and inhibitors is important to develop therapies for stress-induced diseases including cancer, and for the identification of anti-oxidant components for skincare products.

PPARs (PPARα PPARβ/δ, and PPARγ) pathway QuantiSig-PPAR identifies the activation of Peroxisome Proliferator-Activated Receptors (PPARs), members of the nuclear receptor family of ligand-activated transcription factors. The system detects all three-member subfamily PPARα, PPARβ/δ, and PPARγ control the expression of genes involved in lipid and carbohydrate metabolism, vascular biology, tissue repair, cell proliferation and differentiation. Testing of PPAR agonists is suitable for drug discovery applications for various conditions (e.g. diabetes, coronary artery disease, obesity, and cancer) and the identification of agents supporting cell regeneration.

XRE- xenobiotic responsive element (XRE) QuantiSig-XRE allows testing the activity of the aryl hydrocarbon receptor (AhR). AhR is a cytosolic ligand-activated transcription factor that directly interacts with drug metabolites and environmental toxins collectively known as xenobiotics. This receptor is widely distributed in vertebrates and is expressed in skin cells, in which exogenous and endogenous ligands are abundant, where it is involved in the metabolism of xenobiotics as well as in the regulation of skin pigmentation and skin inflammation.