Interest of ex vivo human skin for the

evaluation of activity and safety

assessment of cosmetic products

Philippe Gasser
Centre de Recherches Biologiques et dExprimentations Cutanes
Lonjumeau, France
Le laboratoire BIO-EC est agr par le ministre de la Recherche
au titre du Crdit Impt Recherche.

Comet congres, Cergy-Pontoise, 8th-9th June 2015 COMET 2015

 Evaluation of cosmetic product activity

1993: first EU provisions (directives) on a marketing BAN of

cosmetics tested on animals. Deadline to BAN: 1998.

Alternative methods

In vitro Ex vivo In vivo

2D 3D 3D 3D
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 Skin 3D models

There exists:

- Reconstructed epidermis on a synthetic support.

- Collagen lattice that form an equivalent dermis

with or without reconstructed epidermis.

- Reconstructed epidermis on a de-epidermized

dead dermis.

- Reconstructed epidermis on collagen cellularized

sponge

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 Differences vs human skin

These models are different compared to in vivo human skin :

The stratum corneum barrier function is altered.

The dermal-epidermal junction is more or less present.
All cellular and tissular structures of the human skin are not present.
The expression of keratinocyte differentiation markers is modified.

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Human living skin explant model

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 Living skin explants

Separated
epidermis

Epidermis
Dermis

Epidermis
Dermis
Adipose tissue

Adipose tissue

Isolated hair follicle

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 Human living skin explants

Ex vivo model

Human Skin Topical application of formulations

Solubilization of actives
in the culture medium

Explants for histology and/or

for biochemichal assays

Culture medium for

biochemichal assays

Living skin explants

(epidermis + dermis)

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 Model validation

This model has been validated in studies on which 10 parameters

has been examined:
Capacits et limites du modle ex vivo BIO-EC
Capacits et limites du modle ex vivo BIO-EC
Param tres

Morphologie
Param tres

Transglutaminase membranaire
Morphologie

Flilaggrine
Transglutaminase membranaire

Kratine Basale CK14

Flilaggrine

Ki67
Kratine Basale CK14

Connexine 43
Ki67

Aquaporine43
Connexine 3

Glycosaminoglycanes
Aquaporine 3

Collagne VII
Glycosaminoglycanes

Collagne VII
Collagne IV

PECAM
Collagne IV

PECAM 0 5 10 15 20 25 30
Jours de survie
0 5 10 15 20 25 30
Jours de survie

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 Model advantages

Possibility to choose old or young plasties and

different phototypes.

Cellular and tissular structures identical to

human skin in vivo.

Application of different kind of formulations, UV

medical devices, light stress, laser pulse,

injectable product, peeling, strippings,
mecanical and chemical alterations.

Possibility of biochemical assays.

Important histological material.

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 Quantification

To support these revendications, BIO-EC Laboratory have impressive tools:

Histology with panoptic, specific stainings or immunostainings, transmission

and scanning electron microscopy.

Biochimical assays of SOD, catalase, TNF, PGE2, cytokins, growth

factors, glycerol, MDA.

Raman spectroscopy for hydration activity, lipidic class characterisation,

protein conformation analysis, skin penetration.

Cosmetogenomic for gene expression profile modifications after product

application.

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 Image analysis

In complement of the stainings and immunostainings, the quantification of

protein expression is possible thanks to the image analysis method
providing to the customer photos and quantified results.

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Transmission electron microscopy

Hydrolipidic bi-layers

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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation

Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation
Acidic GAGs

Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation
CK14
Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation
Collagen
CK14 I

Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation
CK14
p53
Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation
CK14
Melanin
Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation
CK14
Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation
CK14
Nrf2
Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation

AQP3
Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation

Filaggrin
Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation

Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation

Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation

CML
Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 Many claims

Solar protection

Anti-ageing Pigmentation/depigmentation

Defensin -2
Anti-bacterial Wound healing

Anti-glycation Anti-stress

Hair growth Hydration

Slimming Cutaneous barrier protection

And more
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 RAMAN spectroscopy

Skin hydration

Lipid and protein conformation

Skin penetration

-50
0 0 x10-3

-40
120
5 5

-30
110

Water content
10 10

-20 SC 15 15 100
Depth into epidermis, Y (m)

Depth into skin, Y (m)

-10
20 20
90
0
25 25
80
10
30 30

20 70
35 35

30 60
40 40

40
e 50
45 1 m 45
4 m
50
-40 -20 0 20 40 0 5 10 15 20 25 30
Across epidermis, X (m)
Across skin, X (m)

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 Cosmetogenomic

Treatment of skin explants or scalp explants

Product objectivation

Fonc*onnal
Genomic study

Extraction and
validation of ARNs

Quantification and
normalization of data

Microarrays

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 Conclusion

Raw material, cosmetic formulations and food complements.

Choice of age and phototype of the donor.
Many kind of stress.
Quick observation of the searching activities (repairing or preventive).
Results close to those observed in vivo.
Model allowing a large histological evaluation and investigation.

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 Remerciements
Merci toute lquipe Bio-EC et Genex:

Caroline Durand
Giuseppe Percoco
Tatiana Judith
Cynthia Delpy
Pauline Meunier
Stphanie Scalvino
Emelline Blondet
Doriane Reby
Annie Verdier
Angela Patapian

Laurent Peno-Mazzarino
Magalie Daniel
Thomas Bader Philippe Benech

Philippe Transon Elian Lati Philippe Gasser

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