Manual EST1000 Skin Corrosion Validation Kit - CellSystems ...

Manual
EST1000
Skin Corrosion
Validation Kit
CellSystems Biotechnologie Vertrieb GmbH
Langeler Ring 5 . D-53842 Troisdorf
Phone +49 (0) 2241.25515 - 0 . Fax +49 (0) 2241.25515 - 30
Internet www://cellsystems.de . Email info@cellsystems.de

Table of Content
1. Introduction 1
2. Kit Contents 2
3. Additionally Required Materials 3
4. Instructions 4
5. Protocol 5
5.1 Application of test sample 5
5.2 Addition of MTT 5
5.3 Extraction of the formazan crystals 6
6. Result Protocol 7
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1. Introduction
Due to legal restrictions for animal testing, three dimensional in vitro models are
increasingly used to measure effects of skin-active substances.
For routine corrosion testing CellSystems ® provides the epidermis model Epidermal Skin
Test EST1000. The cellular structure of EST1000 resembles that of natural epidermis
showing a base membrane, proliferating keratinocytes and stratum corneum with an
intact barrier function. It shows an excellent in vitro/in vivo comparison and allows a
high reproducibility of results.
CellSystems ® Epidermal Skin Test EST1000 is validated for the classification of
compounds concerning skin corrosion according to the OECD test guideline 431 for the
testing of chemicals: “In Vitro Skin Corrosion: Human Skin Model Test”.
The European Centre for the Validation of Alternative Methods (ECVAM) has published
the ESAC statement on the scientific validity of EST1000 method for skin corrosion
testing (June, 12 th , 2009). It can be used for reliably predicting the corrosive potential of
chemical substances (http://ecvam.jrc.it/ look for publications / ESAC statements).
One major parameter to evaluate the skin corrosive potential of a given substance is
the viability of the treated skin equivalents after exposure to the test
substances. A compound is classified as corrosive if the cell viability is reduced
below 50 % after 3 min of exposure. If the viability after 3 min exposure exceeds 50 %
a compound is corrosive if the viability is reduced below 15 % after 1 hour. For
statistical relevance 3 skin models should be used for each time point and compound.
As a control PBS is used in triplicates per time point. According to the definition of skin
corrosion, skin models should be incubated with the compound for 3 min and 1 h. It is
crucial to evenly apply the compound on the surface of EST1000. After the indicated
time skin models are rinsed thoroughly.
Finally, cell viability is measured by performing a standard MTT assay. Metabolically
active cells reduce the yellow tetrazolium salt MTT (3-[4,5–dimethylthiazol-2-yl]–2,5–
diphenyltetrazolium bromide) to insoluble purple formazan crystals. The addition of
isopropanol dissolves the crystals and the change in colour can be quantified by
standard absorbance measurement. The relation between the absorbance values of the
treated skin models and of the control models defines the rate of cell survival.
The validation kit is designed to confirm interlaboratory reproducibility and to gain
reliable results in your laboratory. It consists of 2x 18 epidermis models of the same
production lot. The corrosive potential of two blinded test substances is determined in
parallel at your facility and at CellSystems ® ISO certified laboratories in Germany. After
successful performing this validation assay you will be certified. This certificate will prove
your expertise in the use of EST1000, in the context of the OECD test guideline 431.
CellSystems ® laboratory facility has implemented the Quality-Management-System and
is certified according to DIN ISO 9001:2008, register number: 266714 QM08.
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2. Kit Contents
One half of the material will be delivered to your laboratory; one half will be delivered
to the CellSystems ®´ R&D department for parallel testing.
You receive:
• 18 human skin equivalents in 24 well plates,
embedded in transport medium
• 50 ml Maintenance Medium
• 20 mg MTT-Reagent
• 20 ml MTT-Assay Medium (enough medium to prepare a 1 mg/ml MTT
solution as specified on the label)
• Test Item A
• Test Item B
• Negative Control (PBS)
• 3x 6-well plates
• 3x 24-well plates
• 1x 96-well plate
Note
The test items purchased are toxic and corrosive. Please, be careful while handling
these compounds and establish suitable security precautions for your own safety.
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3. Additionally Required Materials
• Class II biological safety cabinet
• Incubator (37 °C, 5 % CO 2 , 95 % humidity)
• Water bath (37 °C)
• Vertical shaker
• 96-well plate reader spectrophotometer
• Micro-Pipettor (sterile)
• Pipette tips (sterile)
• Pair of tweezers (sterile)
• Squeeze bottle
• 250 ml beaker
• 1 x PBS (Phosphate buffered saline), sterile
• Isopropanol
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4. Instructions
Preparation on receipt
Immediately upon receipt check the kit for completeness and possible transport
damages. Read the instructions completely and carefully before handling the skin
equivalents as described below:
• Please, inform CellSystems ® when the package has arrived and you are going to
begin with the testing to ensure the same conditions.
You can either call +49 (0)2241 25515- 0 or send the enclosed fax form
to +49 (0)2241 25515-30.
• The culture dishes with the cooled skin equivalents are in the inner transport box.
• Set up the culture dishes (3x 6-well) and pipette 1000 μl cold maintenance
medium (4 °C to 8 °C) into each well.
• Remove the ParafilmTM from the transport plate with the skin equivalents and open
the culture dish under sterile conditions.
• Lift the inserts with a sterile pair of tweezers and transfer them into the prepared
6-well plates filled with maintenance medium. Make sure not to transfer any agarose.
• Avoid bubbles between the insert and the bottom of the culture dish by setting
obliquely the insert into the culture dish.
• Incubate the culture dishes at least 2 hours at 37 °C, 5 % CO 2 , 95 % humidity before
performing first experiments. It is also possible to incubate overnight. In this case it is
recommended to change medium before applying the test substances.
• After this adaption-phase, your test substances can be applied onto the stratum
corneum.
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5. Protocol
5.1 Application of test sample (under sterile conditions)
• Apply 50 μl each of test item A, test item B and PBS topically to the centre of the
skin equivalents by using a sterile micropipette. It is crucial to evenly apply the
compound on the surface of EST1000.
For each incubation period three skin models are required.
• Cultivate the skin models in the incubator (37 °C, 5 % CO 2 , 95 % humidity) for 3 min
and 1 h, respectively.
5.2 Addition of MTT
• Prepare a 1.0 mg/ml solution of MTT in MTT-Assay Medium and add 300 μl of the
MTT medium to 18 wells of a new 24-well plate.
• Prepare a second 24-well plate as a “holding plate” with 300 μl Maintenance
Medium in 18 wells.
• Pick up each insert with tweezers. To remove the test substance rinse the tissue
gently with 1x PBS (20 times) using a squeeze bottle. Remove excess PBS by
gently shaking the insert and blotting the bottom with a piece of paper towel.
• Place the insert into the prepared holding plate.
• When every treated tissue has been rinsed transfer the inserts quickly into one
well each of the prepared 24-well plate with the MTT-Assay Reagent.
• Incubate the 24-well plate for 3 hrs (37 °C, 5 % CO 2 , 95 % humidity).
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5.3 Extraction of the formazan crystals
• Pick up the insert with tweezers.
• Remove excess MTT-Assay Reagent by blotting the bottom with a piece of paper
towel.
• Transfer the inserts to a new 24-well plate.
• Add 2 ml of isopropanol directly to each insert. The insert in the well should be
submerged completely.
• Shake the plate for 1 hour at RT on a vertical shaker or store at 2 – 8 °C overnight.
• Puncture the membrane of the inserts with a pipette tip and remove the inserts to
combine the isopropanol extraction of each well.
• Shake carefully on a vertical shaker for about 10 min.
• Dispense duplicates of 200 μl of each sample dilution to an appropriate well of a
96-well flat bottom plate.
• Using a 96-well plate reader spectrophotometer read the absorbance at
540 – 570 nm using acidified isopropanol as a blank.
• If necessary dilute the sample extract 1:3 to 1:5 in isopropanol and repeat the
photometric measurement.
• Viability is calculated as follows:
viability (%) = (absorbance test substance /absorbance neg. control) x 100
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6. Result Protocol
Please, send a copy of this protocol to CellSystems ®
Fax: +49 (0)2241 25515-30
Date:
Model: EST1000
Assay: Skin Corrosion Study
Experimenter:
Method: EST1000 Skin Corrosion Validation
Device Verification:
CO 2
5% ±0.5
Incubator Water bath Micro-Pipettes
Temp
37°C± 1
Water reservoir
check
Temp
37°C± 1
1 ml H 2 O
weight
1.: 1.:
50 μl H 2 O
weight
2.: 2.:
3.: 3.:
Mean
SD
CV %
You may also provide a certificate of pipette calibration!
Quality Evaluation:
Remarks
Macroscopic
Microscopic
other
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Schedule:
Date
Preincubation
60 min exposure to
test compound incl.
appl. + washing
3 min exposure to
test compound incl.
Appl. + washing
MTT-Assay medium
incubation
Formazan extraction
Start Stop Start Stop Start Stop Start Stop Start Stop
Results:
Mean
OD 550nm
Compound A
3 min
Compound A
1 h
Compound B
3 min
Compound B
1 h
PBS
3 min
PBS
1 h
Insert 1
Insert 2
Insert 3
Mean
Viability
Compound A
3 min
Compound A
1 h
Compound B
3 min
Compound B
1 h
PBS
3 min
PBS
1 h
Insert 1
Insert 2
Insert 3
Summarizing remarks:
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