Frequently Asked Questions

THE USE OF OUR MODELS

UPDATED NOVEMBER, 2024

1. WHICH SIZE (S, M, L) 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 has 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 bag and strip off the white tape. Open the plate under a sterile airflow. 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 a sterile paper, and immediately place in a plate in which each well has previously been filled with an appropriate volume of Skinethic 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

*Circular nylon mesh Ø= 7.5 mm (Sefar Fyltis, # Sefar Nitex 03-150/38) or equivalent

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: (ex ref D4693, Sigma. (solution at 2.4 U/ml).

7. 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.

Please follow this direct link to the validated protocol: SkinEthic^TM Human Skin Model for Corrosivity Testing

8. 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.

Please follow this direct link to the validated protocol: SkinEthic^TM validated protocol for Skin Irritation

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

Always leave them at room temperature.

10. 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 1 mg/mL in maintenance medium.

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

Carefully cut the polycarbonate filter out of its plastic ring with a sharp scalpel.

Fill a 1.5 ml vial with buffered formaldehyde 4% (W/V) (ex: MMFrance ref F/40877-29 ) and put the sample in it.

The sample must not dry (at any step of the procedure). Be aware that formaldéhyde is a dangerous product : read its MSDS before use.

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

For whitening tests : we recommend to use phototype VI.

For pigmentation induced tests, we recommend to use phototype IV.

Be aware that media specific for RHPE contain pigmentation factors. Ask our Sales team for further information.