Frequently Asked Questions

THE USE OF OUR MODELS

1. WHICH SIZE (SMALL OR LARGE) SHOULD I ORDER?

It depends on the test protocol: large cultures allow easiest application of small quantities of creams (2 mg/cm² for example).

2. IF I ORDER THE RECONSTITUTED HUMAN EPIDERMIS (RHE) AT DAY 10 OR 12, CAN I USE THE SAME MEDIUM THAN THE ONE USED FOR THE CULTURES AT DAY 17?

Yes, the medium is the same, but growth medium have to be used for more than 48 hours of culture.

3. WHAT I HAVE TO DO WHEN I RECEIVE THE TISSUES?

Tissue storage
The EPISKIN human tissue models should be removed from the agarose-nutrient solution in the shipping multiwell plate immediately after arrival in the laboratory, according to the guidelines below.

Upon arrival, remove the multiwell plate from the aluminium bag and strip off the white tape. Open the plate under a sterile airflow and remove the sterile filter paper. Carefully take out each insert (or HTS plate) containing the tissue, rapidly remove any remaining agarose that adheres to the outer sides of the insert by gentle blotting on the sterile filter paper, and immediately place in a plate in which each well has previously been filled with an appropriate volume of EPISKIN Maintenance or Growth Medium (at room temperature)(+ make sure that the medium is never above the polycarbonate filter).

Act quickly as the tissue cultures dry out rapidly when not in contact with medium. Make sure that no air bubbles are formed underneath the insert!
Place culture in the incubator at 37°C, 5% CO2, and saturated humidity.

4. HOW CAN I APPLY THE TEST PRODUCTS?

It depends on the test product: powder, liquid, cream, gel...

Nature of test product	Volume or quantity (RHE/S)	Application method
Hydrosoluble (liquid)	Minimal volume: 5 µl	Micropipette + nylon mesh* + spread with an inoculating loop
Hydrosoluble (liquid)	100 µl	Micropipette + spread with an inoculating loop
Oils	Minimal volume: 1 µl	Positive displacement pipette** + spread with an inoculating loop
Creams, gels	1 µl to 4 µl	Positive displacement pipette + spread with an inoculating loop
Creams, gels	5 µl	Positive displacement pipette + spread with an paint brush n°2***
Solids (powder)	According protocol	Applied with syringe and spread with an inoculating loop

*Mesh (Nylon 150 µm, ref: 03150/44)
Mesh is prepared with Biopsy punch d = 8 mm
STIEFEL laboratory

** Microman, Gilson.
***Raphael, n°2 petit gris n° 835.

5. I WANT TO PERFORM A PERMEABILITY ASSAY: CAN I REPLACE THE MAINTENANCE MEDIUM BY ANY OTHER SOLUTION UNDERNEATH THE CULTURES?

No, the maintenance medium has to be used. If another solution is used, a preliminar cytotoxity test has to be performed.

6. HOW CAN I DETACH RECONSTRUCTED HUMAN EPIDERMIS FROM THE POLYCARBONATE FILTER?

Dispase Protocol is used to detach the cultures from the filter:

Put the insert (which has little feet) with the epidermis on 1 to 2 ml of Dispase II solution at room temperature.
After 3-5 minutes, the epidermis will come off the polycarbonate and can be lifted with forceps. You then need to rinse the epidermis by spreading it on a small amount of medium on which it will float.
Product information on Dispase grade II: Boehringer Mannheim, cat. number 295 825. (solution at 2.4 U/ml).

7. CAN WE OBTAINED THE EPISKIN MODEL IN OTHER STADE OF CULTURE THAN 13 DAYS?

The EpiSkin^TM model can be provided at a youngest stage. In this case no controls can be provided to the users. Only controls at day 13 are performed and demonstrate a good differentiation of the EpiSkin^TM model in normal conditions.

8. IS THE SkinEthic^TM RHE MODEL VALIDATED FOR CORROSIVITY?

STATEMENT ON THE APPLICATION OF THE SKINETHIC™ HUMAN SKIN MODEL FOR SKIN CORROSIVITY TESTING

At its 25th Meeting, held on 16-17 November 2006 at the European Centre for the Validation of Alternative Methods (ECVAM), Ispra, Italy, the non-Commission members of the ECVAM Scientific Advisory Committee (ESAC)1 unanimously endorsed the following statement: On the basis of a peer review of the results of an inter-laboratory study3 with the SkinEthic™ reconstituted human epidermal (RHE) model, the Committee endorses the conclusion that the SkinEthic™ human skin model can be used for distinguishing between corrosive and non-corrosive chemicals within the context of the OECD test guideline, TG 431.

Thomas Hartung
Head of Unit ECVAM
Institute for Health & Consumer Protection Joint Research Centre European Commission Ispra

Scientific Reference:
Assessment of the human epidermis model SkinEthic^TM RHE for in vitro skin corrosion testing of chemicals according to new OECD TG 431
Helena Kandárová(a), Manfred Liebsch(a), Horst Spielmann(a),Elke Genschow(a), Elisabeth Schmidt(a), Dieter Traue(a), Robert Guest(b),Andrew Whittingham(b), Neil Warren(b), Armin O. Gamer(c), Marina Remmele(c),Tanja Kaufmann(c), Elke Wittmer(c), Bart De Wever(d), Martin Rosdy(d)
a ZEBET, Bundesinstitut für Risikobewertung, BfR, Diedersdorfer Weg 1, D-12277 Berlin, Germany
b Safepharm Laboratories, Derby, UK
c Experimental Toxicology and Ecology of BASF AG, Ludwigshafen, Germany d SkinEthic Laboratories, Nice, France
Received 2 June 2005; accepted 21 November 2005.

9. IS THE SkinEthic^TM RHE MODEL VALIDATED FOR SKIN IRRITATION OF CHEMICALS?

At its 29th meeting, the ECVAM Scientific Advisory Committee (ESAC) unanimously endorsed the following statement “It is concluded that the performance of SkinEthic RHE assay in an external catch up validation study (MTT end point), met the criteria outlined to be considered to have sufficient accuracy and reliability for prediction of R38 skin irritating and no-label (non skin irritating) test substances.”(*)

(*)Statement of the scientific validity of in-vitro tests for skin irritation testing, ECVAM, November, 5th, 2008, officially released December, 3rd, 2008.

This statement reinforces the EPISKIN leadership in tissue engineering with two validated methods: EpiSkin^TM in 2007 and SkinEthic^TMRHE in 2008.

The SkinEthic^TM RHE validated protocol, 42 min exposure, 42 hours incubation time, based on a simple cytotoxic end-point (MTT), showed a good specificity (80%) and sensitivity (90%), a low False positive rate (20%) and False negative rate (10%) with an overall accuracy of 85% during the validation process by three lead and participating laboratories.

SkinEthic patented RHE model is a fully differentiated reconstructed epidermis in a chemically defined medium and manufactured according to strict quality controls during mass production process. This ready-to-use model, also validated for skin corrosion, is exclusively commercialized by EPISKIN SA, an ISO 9001 certified company.

SkinEthic^TM RHE and the others patented models proposed worldwide by EPISKIN will reduce the animal use in cosmetics and chemicals testing. They are available to Industries, Contract Research Organizations and Academic Research Institutions that promote in vitro toxicology testing.

10. IS THE EpiSkin^TM MODEL VALIDATED FOR CORROSIVITY?

The EpiSkin^TM model is scientifically validated for corrovivity assessment (TIV 12, 483-524, 1997, Inscription at the Annex V of Directive for Dangerous Substances Publication at the J.O. European Community Commission Directive 2000/33/EC and Directive 86/609/EC Guide line OCDE 431.

11. IS THE EpiSkin^TM MODEL VALIDATED FOR SKIN IRRITATION OF CHEMICALS?

Clichy, 30th April 2007 - ECVAM (European Centre for the Validation of Alternative Methods) has announced the validation of an ‘in vitro’ test as a full replacement method for assessing the skin-irritancy potential of chemicals. This test employs the L’Oréal Episkin model; a human epidermis reconstructed on collagen.

“More than twenty years of research on skin reconstruction have been recognized today” said Jean-François Grollier, Executive Vice-President Research and Development at L’Oréal. “It is a great step towards the elimination of animal use.”

Since the eighties, L’Oréal has been working to develop reconstructed human skin to better understand its biological behaviour, as well as being able, in the future, to assess the tolerance of ingredients and finished products without animal tests.

This work has so far contributed to L’Oréal’s ability to end animal tests on all finished products in 1989. “Today, EpiSkin^TM is routinely used for tolerance evaluation of our products and our commitment to develop alternative methods continues”, added JF Grollier.

The validation of EpiSkin^TM is a particularly important result as the skin-irritancy potential of chemicals is today carried out on animals. Importantly, the test will also enable the replacement of animal tests for skin-irritancy of 10,000 substances, as foreseen under the implementation of REACH.

According to ECVAM, EpiSkin^TM ‘predicts the skin irritancy potential of chemicals with great accuracy and precision and will therefore fully replace tests’ on animals.

12. DO WE FREEZE / STICK IN FRIDGE THE TISSUES ON ARRIVAL?

Always leave them at room temperature.

13. HOW THE MTT SOLUTION IS PREPARED FOR VIABILITY ASSAY?

A stock solution of MTT powder (Sigma Cat No, M5655) at 5 mg/ml is prepared in PBS, filtered and stored at -20°C in 1 ml samples. The day of the experiment, a sample is diluted at 10% in maintenance medium.

14. HOW CAN I FIX THE CULTURES FOR HISTOLOGY ANALYSIS?

Carefully cut the polycarbonate filter or the collagen matrix out of its plastic ring with a sharp scalpel. Fill a 1.5 ml vial with buffered formaldedhyde 4% (W/V) ( ex:Sigma ref HT50-1-1 ; Carlo Erba Ref N°526932) and put the sample in it.

The sample must not dry (at any step of the procedure).

15. WHICH PHOTOTYPE OF THE RECONSTRUCTED HUMAN PIGMENTED EPIDERMIS SHOULD I ORDER FOR MY EXPERIMENT?

For whitenning tests and for pigmentation induced tests, we recommend to use phototype VI and phototype IV.