Ex Vivo objectivation

Our laboratory has developed several ex vivo models based on keeping human skin alive.

  • Whole skin
  • Epidermis + Dermis
  • Separate skin tissue
  • Adipose tissues
  • Isolated hairs
  • Scalp explants

These ex vivo models enable the activity and tolerance to be evaluated, both of raw materials and finished products

Advantages of ex vivo cutaneous explants 

  • Models close to the actual conditions of use of the products to be tested.
  • The skin conserves a normal morphology, structure and metabolism.

  • Several application modes are possible: topically or solubilised in the survival medium, single or chronic application. Possibility of making injections, laser treatments, etc.

  • Possibility of reproducing external interactions: exposure to UV or pollutants, chemical attacks, wound, sensitisation, hair removal, etc.

  • Carrying out histological analyses

Our laboratory has a battery of stainings and immunomarkers that enable different cellular and tissue markers to be visualised. The marked parameters may be quantified by image analysis software.

  • Carrying out biological analyses

On whole skin, separate epidermises, homogenates, extracts or in the culture medium. All types of extractions, separations, chromatograms and assays may be envisaged.

  • Carrying out genomic studies (in partnership with the Genex Laboratory)

Large-scale screening and analysis of genomic data. Identification of the effects of the products on human skin thanks to the study of the genic expression (DNA chip, miRNA chip, qPCR) correlated with the protein expression of the genes of interest.

Interest of ex vivo
Studies

Ex vivo objectivation enables the action mechanisms of a product or active ingredient on the skin to be understood.

Furthermore, thanks to the images obtained from histological processes, our studies provide you with excellent materials for your marketing claims.

BIO-EC laboratory also develops specific, customised studies to prove your claims.

Our model

BIO-EC Laboratory has developed an ex vivo mode based on keeping explants of human skin alive for 10 to 12 days. This model enables pertinent replies to your cosmetic and dermatological objectivation requirements to be obtained.

This model enables us:

  • To work in conditions very close to the actual physiology of the skin, with, inter alia, all the cellular population and their communications remaining active.

  • To choose the phototype, the age and the gender of the donor.

  • To put different experimental protocols in place in order to best refine your objectivations

  • To rapidly observe the effects of your product on the skin ahead of in vivo studies (on volunteers).

  • To provide you with answers on understanding the action mechanism of your product that you can illustrate by all the photographs provided in the final study report.

    Ex vivo appears to be a first-line technique, both in the diversity of the personalisable parameters for your study and in the quality of the results that you will obtain for the objectivation of your active ingredient or finished product.

The model developed by BIO-EC Laboratory can be adapted on the basis of the products to be tested. Indeed, it is possible to carry out studies on whole skin, on isolated cutaneous compartments and on hair.

Our expertise gives us the possibility of exposing our models to different treatments and, after applying your products, different biological markers can be studied by immunomarking or assays.

All the histological studies can be accompanied by an OMICs study thanks to our collaboration with the Genex Laboratory.

Anti-aging

General morphology

  • Thickness of the epidermis

  • Epidermal and dermal stimulation

Immunomarking

Restructuring and/or regenerate activity

  • Epidermal markers

– Mitotic division (Ki67)

– Cytokeratins (CK14, CK10, CK1)

– Keratinocyte differentiation (involucrin, loricrin, caspase 14)

  • Dermal-epidermal junction markers

– Integrin B4

– Laminin 5

  • Dermal markers

– Extracelluar matrix (GAGs)

– Synthesis of hyaluronic acid (HAS1 and HAS2)

– Heparanase 1

– Hyaluronic acid

– Versican

– Elastin

– Fibrillin

– Emilin

– Vimentin

– Collagens (types I, III, IV, V, VII, VIII et XII)

Other markers: progerin, NDST1 and NDST2, UGT1A, etc.

 Anti-collagenase activity

Anti-elastase activity

Anti-glycation activity

  • Advanced Glycation End products or AGEs (pentosidine, Carboxymethyl-Lysine CML)
  • Fibrillin-1

Filling effect topography

(In paternship with the Cosmetomics platform)

With the help of this device BIO-EC Laboratory can carry out topographic studies on the surface of explants with an accuracy evaluated at a tenth of a nanometre. This enables various parameters to be analysed such as the depth and width of wrinkles and micro-lines and the roughness of the skin in order to evaluate a filling effect.

General morphology

Cytokeratin 14

Elastin

Hydration / barrier

General morphology

  • Thickness of the stratum corneum
  • Exfoliation

Immunomarking

  • Filaggrin (NMF precursor)
  • Membrane Transglutaminase – TGM (formation and cohesion of the stratum corneum)
  • Tight junctions

– ZO-1

– Occludin-1

– Claudin-1

  • Kallikreins
  • Hyaluronic acid
  • Small proline-rich proteins – SPRRs (corneal envelope assembly)
  • Ceramides
  • Neutral lipids
  • Terminal differentiation of keratinocytes

– Loricrin

– Involucrin

– Caspase 14

  • Serine proteases inhibitor – LEKTI (desquamation regulation)
  • Aquaporins (protein regulating the hydric flow between the dermis and the stratum corneum)

Analysis by Raman spectroscopy

BIO-EC Laboratory may, using this device, evaluate the kinetics of the tissue penetration and distribution of your product, the skin modifications caused by your product across various parameters, the hydration rate in the stratum corneum and the epidermis, the distribution of the NMF components, of the cutaneous lipids, the carotenoids, etc.

Transmission electron microscopy

Thanks to our partnership with BIC (Bordeaux Imaging Center), it is possible to complement our histological studies with TEM observations, such as the evaluation of the quality of lipid bilayers.

Filmogenic effect topography

(In paternship with the Cosmetomics platform)

With the help of this devic BIO-EC Laboratory can carry out topographic studies on the surface of explants with an accuracy evaluated at a tenth of a nanometre. This enables various parameters to be analysed such as the depth and width of wrinkles and micro-lines and the roughness of the skin in order to evaluate a filmogenic effect.

Filaggrin
Ceramides
Aquaporin 3
Raman

Pigmentation

Staining

Visualisation of melanin by Fontana-Masson.

Immunomarking

melanogenesis

  • Tyrosinase-Related Protein-1 and 2 (melanin synthesis)
  • Tyrosinase (melanin synthesis)
  • Melan-A (synthesis of melanosomes)
  • MelanoCortin-1 Receptor (migration of melanosomes)
  • Premelanosome protein – PMEL17 (formation of melanosomes)

Screening

in vitro

Screening test to evidence melanin produced by melanocytes: DOPA oxidase test

Test carried out on separate epidermis.

Melanin
PMEL17
DOPA control
DOPA + active ingredient

Photoprotection / Solar protection

irradiation sources

UVs (UltraViolets)

IR (infrared)

Visible light

SOLARBOX®

Process exclusive to BIO-EC Laboratory which enables us to expose the skin solely to visible light. It is also possible to expose the skin to each luminous spectrum independently:

– Full visible domain: 420-650 nm

– Band in the blue: 420-580 nm

– Band in the yellow-green: 90-650 nm

– Band in the red: 575-645 nm

General morphology

after irradiation

Observation of the appearance of Sun Burn Cells (SBCs).

Immunomarking

  • DNA protection after UVB irradiation

    • p53
    • Caspase-3
    • Thymine dimers
    • H2AX
  • Evaluation of the oxidative stress after UVA irradiations

    • Oxidative-Stress response-1 – OXSR-1
    • 8-hydroxy-2-deoxyguanosine – 8-OHdG
    • Hème oxygénase – HO-1 (mitochondrial enzyme)
  • Markers evaluated after infrared ray irradiation

    • Tropoelastin (elastin precursor)
    • Metalloproteinases – MMPs (remodelling of the extra-cellular matrix)
    • Fibrillin (principal constituent of the extra-cellular matrix microfibrils)
  • Markers evaluated after irradiation by visible light (blue light)

    • MMP-1 (protease involved in the remodelling of the ECM)
    • Involucrin (protein involved in the stratum corneum organisation and connections)
    • Sirtuin 1 – SIRT1 (protein involved in the cellular renewal process)
    • NF-E2-related factor – Nrf2 (anti-oxidant)

Assays

  • MDA (malonedialdehyde acid), lipid peroxidation marker.

  • Cytokines

MMP1

Tropoelastin

H2AX

Thymin dimers

Lipolytic activity / Lypogenesis

Assays

In the culture medium:

  • Fatty acids
  • Glycerol
  • Triglycerides

Image analysis

  • Masson’s Trichrome Staining
  • Measurement of the average size of the adipocytes

Immunomarking

Of Thermogenin – UCP-1 (specific mitochondrial protein of brown adipocytes).

This marking enables demonstrating the conversion of white adipocytes into smaller, more easily eliminated brown adipocytes.

Adipocytes
UCP1

Sensitisation / Tolerance

Sensitisation

General morphology

After topical application of the product.

Immunomarking

Langerhans cells: CD1a and langerin.

Tolerance

 

General morphology

Determination of the irritant character in compliance with GLP (OECD method 439)

CD1a control
CD1a + sensitiser
CD1a + sensitiser + active ingredient

Control tolerance

SLS 10%

Strong irritant

Cicatrisation / Repair

Three cicatrisation models

  • Localised burn caused by a strong dose of UV in the centre of the explant
  • Burn by application of chemical products
  • Mechanical injury to the explant

General morphology

Evaluation of the re-epithelialisation speed (structure of the growth bud).

Immunomarking

  • Basal and supra-basal keratins – Stratification
  • Fibronectin – Adhesion
  • Integrin β4 – Adhesion
  • Laminin-5 – Dermo-epidermal junction
  • Perlecan – Dermo-epidermal junction and epidermal differentiation
  • PECAM-1 – Micro-vascularisation
  • αSMA – Papillary dermis below the growth bud

Cicatrisation bud

Fibronectin

Metabolism

Immunomarking

  • G6PDH (protein involved in energy metabolism)
  • Mitochondrial aconitase (Krebs cycle enzyme)
  • Pimonidazole (hypoxia marker)
G6PDH
Aconitase 2
Pimonidazole

Microbiome

Microbiological study of the resident cutaneous flora

 (in parternship with the
Cosmetomics platform)

Impact of the products on the cutaneous microbiome bacteria (commensal or pathogenic), influence of the product on their adhesion (formation of biofilms, proliferation, etc.).

Immunomarking

  • Β defensins 2, 3 (peptides possessing an antimicrobial activity)
  • Cathelicidin LL 37 (peptide possessing an anti-microbial activity)
  • Toll like receptor 2 -TLR-2 (receptor linked to the production of cytokines and cellular activation)

Assays

  • Cytokines (IL8, IL6, TNFα…)
β défensin 2
β défensin 3
TLR2

Pollution

POLLUBOX®

Process exclusive to BIO-EC Laboratory of exposure of explants to atmospheric pollutants:

– Hydrocarbons,

– Heavy metals,

– Diesel particles,

– Ozone,

– Cigarette smoke, etc…

This device enables pollutants to be vaporised into cloud form and to be put into contact only with the surface of the skin to resemble physiological conditions as closely as possible.

Immunomarking

  • Aromatic hydrocarbons receptor – AhR (activation of cytochrome and detoxification enzyme)
  • NF-E2-related factor – Nrf2 (anti-oxidant)
  • Metallothionein – MT-1H 5 (stress resistance and detoxification enzyme)
  • Hème oxygenase – HO-1 (mitochondrial enzyme)

Assays

  • MDA (malonedialdehyde acid), lipid peroxidation marker.
HO-1
MT1H
AhR

Anti-oxydant and anti-inflammatory activity

 
 

Immunomarking

  • NF-E2-related factor – Nrf2 (anti-oxidant)
  • OXSR-1 protein (stress response enzyme)
  • 8-hydroxy-2-deoxyguanosine -8-OHdG (DNA transcription regulation molecule)
  • Mitochondrial aconitase (protection from oxidative stress)
  • Metallothionein – MT-1H (anti-oxidant)
  • TNFα (glycoprotein stress marker)
  • Interleukin 1 alpha – IL1α (inflammatory response cytokine)

Assays

  • MDA (malonedialdehyde acid), lipid peroxidation marker
  • Assays of interleukins (IL-1α, 8, 6), of the TNFα and other proteins involved in the inflammatory response.
OXSR-1
TNF-α
IL1-α

Acne / Séborrhoea

Immunomarking

  • Defensins (peptides possessing an anti-microbial activity)
  • Cathelicidin LL 37 (peptide possessing an anti-microbial activity)
  • Toll like receptor 2 -TLR-2 (receptor linked to the production of cytokines and to cellular activation)

Staining

  • General morphology of the sebaceous glands using Masson’s Trichrome.
  • Staining of the sebum.

Assays

  • Interleukins (IL)

Sebaceous glands  

Sebaceous gland sebum, LipidTox

Hair / Body hair

Two evaluation models

 

  • Explants of scalp or hairy skin kept alive,
  • Isolated hair kept alive.

General morphology

 

Mesurement of the length of isolated hairs

 

Mitotic index Ki67

 

Immunomarking

Growth & Anchorage

  • Hair follicle :
    • CK19
    • CK15
    • IGF-1
    • CD34
    • Tenascin-C
    • Androgen receptor
    • MITF
    • KAP11.1
    • PDGFR-alpha
  • Extra-cellular matrix and fibroblasts :
    • Laminin 5
    • Collagene IV
    • Collagen I et other collagens
    • Fibronectin
    • Vimentin
    • α-SMA
    • GAGs

Pigmentation & Anti-Oxydant

  • Melanin by Fontana Masson…
  • Tyrosinase-Related Protein-1 and 2 (melanin synthesis)
  • Melan-A (synthesis and maturation of melanosomes)
  • MelanoCortin-1 Receptor (migration of the melanosomes), etc.
  • Catalase…

Anti-dandruff

Evaluation after treatment of scalp explants by yeasts involved in the appearance of dandruff.

Immunomarking

  • Staining
  • Epidermal differentiation markers :
    • SPRR,
    • Involucrin…
Bulb

Whole hair

Bulb Ki67
 COL IV Bulb

All the histological studies can be accompagnied by an OMICs study thanks to our collaboration with the Genex Laboratory