Application guideContent
LabCyte EPI-MODEL

1

Tissue structure

3

Protein expression analysis by immunohistochemistry5

Protocol5

Results

8

Protein expression analysis by Western blotting

12

Protocol12

Results

13

Gene expression analysis by qPCR

14

Protocol14

Results

16

OECD Test Guideline 439 and 431

19

LabCyte EPI-MODEL 6D

20

Immunohistochemistry of cultured EPI-MODEL 6D

20

Western blotting analysis of cultured EPI-MODEL 6D

24

Gene expression proﬁle of cultured EPI-MODEL 6D

25

Test of mild irritants using EPI-MODEL 6D

28

Permeability and Percutaneous absorption tests

29

EPI-KIT (preparation of 3D epidermal models)30

Gene expression knockdown using EPI-KIT

31

Protocol31

Results

32

LabCyte CORNEA-MODEL34

Eye irritation test

35

Histological features36

3-D cultured human epidermis
LabCyte EPI-MODEL
LabCyte EPI-MODEL is a 3-D human cultured epidermis produced from normal human epidermal cells(keratinocytes) that are cultured at the air-liquid interface to become stratiﬁed. LabCyte EPI-MODEL wasdeveloped as an alternative to animal experimentation performed in skin irritation tests. Because it iscomposed of human epidermal cells, the structure of LabCyte EPI-MODEL is morphologically similar to thatof the human epidermis. Furthermore, LabCyte EPI-MODEL has a high in vivo-in vitro correlation, and very
low lot-to-lot variability. LabCyte EPI-MODEL can be used to determine the skin irritation potential of a wide
range of chemicals through the analysis of cell viability using the MTT assay. Moreover, since LabCyte
EPI-MODEL is metabolically active, it can also be used to evaluate biological processes, such as the
production of cytokines and growth factors, in response to diﬀerent test substances.

A cultured model that closely reproduces human epidermal features
Model structure

10.5 mm (EPI-MODEL 12)
6.4 mm (EPI-MODEL 24)
corniﬁed layer

Culture insert

Epidermal tissue

PET membrane ﬁlter

Tissue structure
LabCyte EPI-MODEL

LabCyte EPI-MODEL 6D

Normal human epidermis (forearm)

Courtesy of Dr. Y. Kitajima (Gifu Univ.)

EPI-MODEL has a structure similar to that of the epidermis, with a basal layer,
a spinal layer, a granulous layer, and a corniﬁed layer.
EPI-MODEL 6D is a cultured epidermal model with an under-developed corniﬁed layer.

Protein expression pattern analysis
The expression pattern of proteins speciﬁcally expressed at diﬀerent layers of the epidermis
Corniﬁed layer (Filaggrin)

Basal lamina (Laminin)Ｄiﬀerentiation marker (Tranglutaminase)Customer support
Japan Tissue Engineering Co. Ltd is responsible for the development, manufacture and sales of
LabCyte products, and is able to oﬀer broad support to all customers through our highly trained
technical specialists.

Skin Irritation Test and other applications
LabCyte EPI-MODEL is an alternative to animal experimentation in skin irritation and toxicity tests.
It can be used in pharmaceutical, dermatological and basic research. The EPI-MODEL 6D, a 3D
epidermal model with under-developed corniﬁed layers, can be used in the risk assessment ofmild irritants, and the analysis of ceramide production in vitro.

Production of highly reproducible models using gold standard techniques
LabCyte EPI-MODEL is produced by highly trained and specialized staﬀ. We guarantee the delivery ofhigh quality products by implementing strict in house quality control tests.

*EPI-MODEL data (taken in house)

Live cell count (within-lot, lot-to-lot variation)

Barrier function analysis (SLS 18 h exposure)

Data demonstrating the low within-lot andData demonstrating the high reproducibility

lot-to-lot variation in cell viability.

of results between diﬀerent lots.

Tissue structure
EPI-MODEL has a structure similar to that of the epidermis,
comprising of a basal layer, a spinal layer, a granulous layer, and a corniﬁed layer.

Corniﬁed layer
Granular layer
Spinous layer
Basal layer

Normal human epidermis (forearm)

LabCyte EPI-MODEL
Courtesy of Dr. Y. Kitajima (Gifu Univ.)Morphological and structural changes of the diﬀerent epidermal layers during culture.
Day 1

Day 4

Day 7

Day 10Day 14Day 18Day 21

Day 28

Immunohistochemistry
Detection of proteins expressed in the corniﬁed and viable layers of the epidermis.
Involucrin

Cytokeratin-1/10

Transglutaminase

Claudin-1

Ultrastructure
Ultrastructure of epidermal layers observed by transmission electron microscopy
Cell membrane
Basal lamina-like structure

Desmosome

Basal
Layer

Spinous
Layer
Membraneﬁlter
Magniﬁed area:
presence of immature hemidesmosomes

Granular
Layer

Corniﬁed
Layer

Keratohyalin granules

Intercellular lamelar lipid structures

Ceramide analysis
EPI-MODEL produces high levels of ceramide

Courtesy of Dr. Y. Tokudome (Josai Univ.)
Skin Pharmacol Physiol 2011;24:218-223

Protein expression analysis by immunohistochemistry
ProtocolParaﬃn embedding① Transfer the culture insert containing the EPI-MODEL to a tube with 4% Paraformadelhyde (PFA).
② Leave it overnight at 4℃ to ﬁx the epidermal tissue.
③ Cut the PET membrane oﬀ from the culture insert using a scalpel.
④ Transfer enough 4% agar solution to ﬁll half of a well of a 6-well plate.
⑤ Transfer the epidermal tissue to the well before the agar completely solidiﬁes.
⑥ Top the well with 4% agar solution and wait until it completely solidiﬁes.
⑦ Trim the excess of agar with a scalpel.
⑧ Transfer the tissue embedded in agar to a biopsy bag.
Biopsy bag
Epidermis⑨ Close the biopsy bag surrounding the tissue using a stapler.

⑩ Sandwich the biopsy bag in between a folded Tissue-Tek foam biopsy sheet.
⑪ Put the tissue in paraﬃn embedding cassettes.
⑫ Rinse the tissue in tap water.
⑬ Remove the excess of water from the cassette and proceed with the paraﬃn embedding protocol.

Paraﬃn embedding program
Perform this step according to the instructions of the equipment being used in your laboratory.
① 70% ethanol

60 min② 100% ethanol

90 min × 5 cycles③ Xylene60 min × 4 cycles④ Paraﬃn (63℃)

45 min × 4 cycles

Preparation of tissue sections① Prepare paraﬃn blocks according to the instructions of the equipment being used in your laboratory.
② Store the paraﬃn blocks at 4℃.
③ Cut 3 mm-thick tissue sections.
④ Mount sections on glass slides.
⑤ Dry the slides at 40 - 60℃ overnight.

Immunohistochemistry
Deparaﬃnization① Incubate slides in xylene three times for 2 min each.
② Incubate slides in 100% ethanol three times for 1 min each.
③ Wash slides in pure water for 1 min.
④ Leave slides in pure water.

Antigen retrieval (choose according to the antibody being used)❶ Heat-induced antigen retrieval① Put slides in a container with the buﬀer of your choice:・For pH 6: Target Retrieval Solution, Citrate pH 6（Dako）
・For pH 9: Target Retrieval Solution, Tris-EDTA pH 9（Dako）② Bring slides to 95℃ and incubate them according to the antibody being used.
③ After incubation, leave the slides in the buﬀer to cool down at room temperature.
④ Transfer the slides to PBS.
⑤ Circle the tissue sections with a PAP pen.

❷ Proteinase K-induced antigen retrieval① Put slides in a container with PBS.
② Circle the tissue sections with a PAP pen.
③ Transfer the slides to a humidiﬁed chamber.
④ Add Proteinase-K solution (Dako) to the tissue sections.
⑤ Incubate at room temperature.
⑥ Wash slides in DPBS-T three times for 2 min each.

Endogenous peroxidase blocking① Incubate slides in 0.3% hydrogen peroxide for 30 min at room temperature.
② Wash slides in DPBS-T three times for 2 min each.

Blocking① Transfer the slides to a humidiﬁed chamber.
② Add 100 µl of Blocking One (Nacalai Tesque) to each slide.
③ Block slides for at least 30 min at room temperature.

Primary antibody① Dilute the primary antibody with Dako REAL antibody diluent (Dako).
② Remove the blocking solution and add 100 µl of the diluted antibody solution to each slide.
③ Incubate for 1 h at room temperature.

Secondary antibody
Secondary antibody solution:
・Anti-rabbit secondary antibody (HRP-conjugated)
ImmPRESS Reagent, Anti-Rabbit Ig（VECTOR）
・Anti-mouse secondary antibody (HRP-conjugated)
ImmPRESS Reagent, Anti-Mouse Ig（VECTOR）① Wash slides in DPBS-T three times for 2 min each.
② Add 100 µl of the appropriate secondary antibody solution.
③ Incubate for 30 min at room temperature.
④ Wash slides in DPBS-T three times for 2 min each.
⑤ Wash slides in DPBS for 2 min.
Signal staining using Histogreen (AbCys)⑥ Add 2 drops of Histogreen-Chromogen (No. 1) to 1 ml of Histogreen-Buﬀer (No. 2) and mix well.
⑦ Prepare HistoGreen by adding two drops of H2O2 (No. 3) to the solution prepared above. Mix well.
⑧ Add 100 µl of HistoGreen to each slide.
⑨ Incubate for 1 - 5 min at room temperature.
⑩ Wash slides in DPBS three times for 2 min each.
⑪ Shortly wash in pure water.

Dehydration and mounting① Incubate slides in 100% ethanol three times for 30 sec each.
② Incubate slides in ethanol : xylene (1:1) mixture for 30 sec.
③ Incubate slides in xylene three times for 30 sec each.
④ Mount sections with coverslips using VectaMount™ Permanent Mounting Medium (Vector)

Human skinTransglutaminase

EPI-MODEL24
Day 0

Day 3

Day 7

Expression detected from spinous to corniﬁed layer.

Laminin-332

Expression detected in the basal and spinous layers, as culture progressed.

Collagen IV

Expression detected in the basal layer.

Collagen VII

Expression detected in the basal layer.

Ki-67

Ki67 is a widely used proliferation marker.
Expression detected in the cells of the basal layer, as it is observed in human skin.

Human skin
p63

EPI-MODEL24
Day 0

Day 3

Day 7

p63 is often referred to as a marker for epidermal stem cells.
Expression detected in the basal and spinous layers, as it is described in human skin.

PCNA

PCNA is a widely used proliferation marker.
Expression detected in the cells of the basal layer, as it is described in human skin.

Claudin-1

Claudin-1 is a tight junction marker.
Expression detected from basal to granular layer, similar to human skin.

Claudin-4

Claudin-4 is a tight junction marker.
Expression detected from basal to granular layer, similar to human skin.

E-cadherin

E-cadherin is an adherens junction marker.
Expression detected from basal to granular layer, similar to human skin.

Human skinDesmoglein 1

EPI-MODEL24
Day 0

Day 3

Day 7

Desmoglein is a desmosome marker.
Expression detected between the basal and granular layer, similar to human skin.

Filaggrin

Expression detected from the granular to corniﬁed layer, similar to human skin.

Loricrin

Expression detected in the granular layer, similar to human skin.

Involucrin

Involucrin is an epidermal diﬀerrentiation marker.
Unlike in human skin, expression was detected in all layers of the epidermal tissue.

Keratin 1

Keratin 1 is an epidermal diﬀerentation marker.
Expression detected from spinous to corniﬁed layer, similar to human skin.

Human skin
Keratin 5

EPI-MODEL24
Day 0

Day 3

Keratin 5 is a basal layer marker.
Expression detected in the basal layer, similar to human skin.

Keratin 10

Keratin 10 is an epidermal diﬀerentation marker.
Expression detected from spinous to corniﬁed layer, similar to human skin.

Keratin 14

Keratin 14 is a basal and spinous layer marker.
Expression detected in the basal and spinous layers, similar to human skin.

Day 7

Protein expression analysis by Western blotting
Protein extraction① Rinse the epidermal tissue with PBS and cut the membrane oﬀ from the culture insert with a scalpel.
② Transfer the tissue to a 1.5 ml microtube.
③ Leave the microtube on ice. If necessary, store the tube at -80℃, and thaw the tissue on ice prior use.
④ Transfer the tissue to a 10 cm dish, and cut it in small pieces with a scalpel.
⑤ Transfer the tissue fragments to a new 1.5 ml microtube.
⑥ Add 200 - 300 µl of RIPA buﬀer to each microtube.
⑦ Homogenize the tissue using a BioMasher®II (Nippi).
⑧ Sonicate for 30 min (37 W).
⑨ Centrifuge the microtube for 10 min (15,000 x g, 4℃).
⑩ Transfer the supernatant to a new 1.5 ml microtube.
⑪ Centrifuge the microtube for 10 min (15,000 x g, 4℃).
⑫ Transfer the supernatant to a new 1.5 ml microtube.
⑬ Estimate the protein concentration by BCA or Bradford assay.

Immunoblotting① Mix 10 µg of protein sample to 2x Laemmli buﬀer (SIGMA-ALDRICH) and mix well.
② Heat the sample to 100℃ for 2 min.
③ Brieﬂy centrifuge the tube.
④ Load onto a SDS-PAGE gel (1 mm gel, Tris-Glycine, 15 mA).
⑤ Rinse the gel in Tris-Glycine buﬀer for 10 min.
⑥ Prepare a PVDF membrane (Amersham Hybond P PVDF 0.45) by incubating it in methanolfor 1 min, and washing it in distilled water for 5 min.
⑦ After transfer, block the membrane in 5% skim milk in TBS-T (1 h at room temperature).
⑧ Dilute the primary antibody in 5% skim milk in TBS-T.
⑨ Transfer the membrane to the primary antibody solution and incubate overnight at 4℃.
⑩ Wash the membrane in 5% skim milk in TBS-T three times for 15 min each.
⑪ Wash the membrane in TBS-T for 5 min.
⑫ Dilute the secondary antibody in TBS-T.
⑬ Transfer the membrane to the secondary antibody solution and incubate for 2 h at room temperature.
⑭ Wash the membrane in TBS-T three times for 15 min each.
⑮ Treat the membrane with ECL Western Blotting Detection Reagents (GE Healthcare).
⑯ Reveal the signal using a LAS4000 or equivalent.

Day 7

Results indicate theproduction of collagen IV

Involucrin

Day 7

Day 3

Day 0

EPI-MODEL24
monolayer

Day 7

Day 3

Day 3

monolayer

Day 7

Day 3

EPI-MODEL24

EPI-MODEL24

Day 0

Day 7

Day 3

Day 0

Day 0

Filaggrin

EPI-MODEL24
monolayer

Day 7

Day 3

Day 0

monolayer

EPI-MODEL24

Native-PAGE

monolayer

E-Cadherinmonolayer

Day 7

Day 3

Results indicate thepresence of tight junctions

Collagen XVIIEPI-MODEL24

EPI-MODEL24
monolayer

Day 7

Day 3

Day 0

monolayer

EPI-MODEL24

Collagen IV

Day 0

Claudin-1

Day 0

CBB staining

Results indicate theproduction of collagen XVIIResults indicate thepresence of adherens junction

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

Keratin 5

Keratin 10

Keratin 14

Loricrin

Results demonstrate the maintenanceof the basal layer during culture.

Results demonstrate the diﬀerentiation process
induced by the 3D culture of epidermal cells.

PCNA

Transglutaminase

Results demonstrate the decreaseof proliferating cells during culture.

Day 7

Day 3

Day 0

EPI-MODEL24
monolayer

Day 7

Day 3

Day 0

monolayer

EPI-MODEL24

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate the maintenanceof the basal layer during culture.

Day 7

Day 3

Day 0

EPI-MODEL24
monolayer

Day 7

Day 3

Day 0

EPI-MODEL24
monolayer

Day 7

Day 3

Day 0

EPI-MODEL24
monolayer

Day 7

Day 3

Day 0

monolayer

EPI-MODEL24

Results demonstrate the diﬀerentiation process
induced by the 3D culture of epidermal cells.

Gene expression analysis by qPCRRNA extraction① Rinse the epidermal tissue with PBS.
② Cut the membrane oﬀ from the culture insert with a scalpel, and transfer the tissue to a 1.5 ml microtube.
③ Leave the microtube on ice. If necessary, store the tube at -80℃, and thaw the tissue on ice prior use.
④ Transfer the tissue to a 10 cm dish, and cut it in small pieces with a scalpel.
⑤ Transfer the tissue fragments to a new 1.5 ml microtube.
⑥ Extract the RNA using PureLink® RNA Mini Kit (Thermo Fisher).
⑦ Add 200 µl of lysis buﬀer to each tube.
⑧ Homogenize the tissue using a BioMasher®II (Nippi).
⑨ Using a seringe, pass the tissue fragments through a 21G needle at least 10 times.
⑩ Centrifuge the microtube at 2 min (12,000 x g, room temperature).
⑪Transfer the supernatant to a fresh microtube, add the same volume of 70% ethanol
and mix well by vortexing.
⑫ Transfer 700 µl of the solution above to a spin column.
⑬ Centrifuge the microtube for 15 sec (12,000 x g, room temperature) and discard the ﬂow through.
⑭ Repeat steps 12 and 13 if necessary.
⑮ Add 350 µl of Wash buﬀer I.
⑯ Discard the ﬂow through and transfer the column to a fresh microtube.
⑰ Add 80 µl of PureLink DNA mixture.
⑱ Incubate for 15 min at room temperature.
⑲ Add 350 µl of Wash buﬀer I.
⑳ Centrifuge the microtube for 15 sec (12,000 x g, room temperature) and discard the ﬂow through.
㉑ Add 500 µl of Wash buﬀer II.
㉒ Centrifuge the microtube for 15 sec (12,000 x g, room temperature) and discard the ﬂow through.
㉓ Add 500 µl of Wash buﬀer II.
㉔ Centrifuge the microtube for 15 sec (12,000 x g, room temperature) and discard the ﬂow through.
㉕ Centrifuge the microtube for 1 min (12,000 x g, room temperature) and discard the ﬂow through.
㉖ Transfer the column to a fresh microtube.
㉗ Add 50 µl of Rnase-free water to the center of the ﬁlter of the spin column.
㉘ Incubate for 1 min at room temperature.
㉙ Centrifuge the microtube for 2 min (12,000 x g, room temperature).
The ﬂow through contains the extraced RNA.
㉚ Use the Quant-iT™ RNA Assay Kit (Thermo Fisher) to estimate the RNA amount.

Reverse transcription and qPCR
① Transfer 100 ng of RNA to a PCR tube.
② Perform a reverse transcription reaction using the SuperScript® VILO™ cDNA Synthesis Kit (Thermo Fisher).

Temperature

Time

25℃

10 min42℃

60 min85℃

5 min③ Dilute and aliquot the cDNA if necessary.
④ Perform qPCR using 5 - 10 ng of cDNA and Power SYBR® Green PCR Master Mix (Thermo Fisher).

Temperature

Time

Number of cycles

95℃

10 min1 cycle94℃

15 sec60℃

1 min

40 cycles

Primers were designed using the following ProbeLibrary.
https://qpcr.probeﬁnder.com/input.jsp?organism=h̲sap

FLG

Monolayer

Normal
Skin

Day 0

LOR

Day 3

Day 7

Monolayer

Normal
Skin

Day 0

KRT1

Day 3

Day 7

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

EPI-MODEL24

EPI-MODEL24

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

KRT10

IVL

TGM1

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

EPI-MODEL24

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

KRT5

KRT14

ΔNp63

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

EPI-MODEL24

Results demonstrate the maintenanceof the basal layer during culture.

Results demonstrate the maintenanceof the basal layer during culture.

Results demonstrate the maintenance of
epidermal stem cells during culture.

PCNA

MKI67

TJP1

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

Results demonstrate the decrease of
proliferating cells during culture.

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

Results demonstrate the decrease of
proliferating cells during culture.

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

Results demonstrate the formation oftight junctions during culture.

TJP2

Monolayer

Normal
Skin

Day 0

CLDN1

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

CLDN4

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

EPI-MODEL24

Results demonstrate the formationof tight junctions during culture.

Results demonstrate the formationof tight junctions during culture.

Results demonstrate the formationof tight junctions during culture.

OCLN

CDH1

DSG1

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

EPI-MODEL24

Results demonstrate the formationof tight junctions during culture.

Results demonstrate the formationof adherens junctions during culture.

Results demonstrate the formationof desmosomes during culture.

DSG2

DSG3

DSG4

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

EPI-MODEL24

Results demonstrate the formationof desmosomes during culture.

Results demonstrate the formationof desmosomes during culture.

Results demonstrate the formationof desmosomes during culture.

CDSN

ITGA6

ITGB4

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

Results demonstrate the formationof desmosomes during culture.

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

Results demonstrate the maintenanceof the basal layer during culture.

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

Results demonstrate the maintenanceof the basal layer during culture.

COL4A1

Monolayer

NormalSkin

Day 0

COL4A2

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

COL7A1

Day 3

Day 7

Monolayer

Normal
Skin

Day 0

Day 3

EPI-MODEL24

Day 7

EPI-MODEL24

Detection of COL4A1
expression during culture.

Detection of COL4A2
expression during culture.

Detection of COL7A1
expression during culture.

COL17A1

LAMA3

LAMB3

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

Monolayer

NormalSkin

Day 0

EPI-MODEL24

Day 3

Day 7

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

EPI-MODEL24

Detection of COL17A1
expression during culture.

Detection of the Laminin-332
component LAMA3.

Detection of the Laminin-332
component LAMB3.

LAMC2

LAMA1

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

Monolayer

Normal
Skin

Day 0

LAMB1

Day 3

Day 7

Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

EPI-MODEL24

EPI-MODEL24

Detection of the Laminin-332
component LAMC2.

Detection of the Laminin-111
component LAMA1.

Detection of the Laminin-111
component LAMB1.

LAMC1Monolayer

Normal
Skin

Day 0

Day 3

Day 7

EPI-MODEL24

Detection of the Laminin-111
component LAMC1.

1

Accepted
Model

Skin Irritation and corrosionTest

Accepted
Model

LabCyte EPI-MODEL 24 was accepted by the OECD to be used in the irritancy and corrosivity assessmentof chemical substances under the test guideline 439 and 431.
The OECD Guidelines are internationally agreed testing methods usedby government, industry and independent laboratories to identify andcharacterize potential hazards of chemicals.
OCED Test Guideline 439 and 431 describe in vitro procedures that may be
used for the hazard identiﬁcation of chemicals (substances and mixtures)using reconstructed human epidermis that closely mimics the biochemicaland physiological properties of the outermost layer of the human skin.
Skin irritation test set(Product No.: 401151)

Concordance with in vivo classiﬁcation- Skin irritation testin vivo classiﬁcationin vitro
prediction

irritant

Non-irritantTotal

irritant

16

11

27

Non-irritant

1

26

27

Total

17

37

54

Sensitivity(%)

94.1

Speciﬁcity(%)

70.3

Accuracy(%)

77.8- Skin corrosion testin vivo classiﬁcationin vitro
prediction

1A

1B/C

Non-corrosive

Total

1A

31

27

3

61

1B/C

5

63

21

89

Non-corrosive

0

0

87

87

Total

36

90

111

237

Correct Classiﬁcations:

1A correctly classiﬁed
1B-and-/1C correctly classiﬁed
NC correctly classiﬁed
Overall Accuracy

86.1%
70.0%
78.4%
76.4%

Immunohistochemistry of cultured EPI-MODEL 6D
Human skinTransglutaminase

EPI-MODEL24 6D
Day 0

Day 3

Expression detected from spinous to corniﬁed layer.

Laminin-332

Expression detected in the basal and spinous layers
as culture progressed.

Collagen IV

Expression detected in the basal layer.

Collagen VII

Expression detected in the basal layer.

Ki-67

Ki67 is a widely used proliferation marker.
Expression detected in the cells of the basal layer,
as it is observed in human skin.

Human skin
p63

EPI-MODEL24 6D
Day 0

Day 3

p63 is often referred to as a marker for epidermal stem cells.
Expression detected in the basal and spinous layers,
as it is described in human skin.

PCNA

PCNA is a widely used proliferation marker.
Expression detected in the cells of the basal layer,
as it is described in human skin.

Claudin-1

Claudins are tight junction markers.
Expression detected from basal to granular layer,
similar to human skin.

Claudin-4

Expression detected from basal to granular layer,
similar to human skin.

E-cadherin

E-cadherin is an adherens junction marker.
Expression detected from basal to granular layer,
similar to human skin.

Human skinDesmoglein 1

EPI-MODEL24 6D
Day 0

Day 3

Desmoglein is a desmosome marker.
Expression detected between the basal and granular layer,
similar to human skin.

Filaggrin

Expression detected from the granular layer.

Loricrin

Expression detected in the granular layer, similar to human skin.

Involucrin

Involucrin is an epidermal diﬀerrentiation marker.
Unlike in human skin, expression was detected in all layers of
the epidermal tissue.

Keratin 1

Keratin 1 is an epidermal diﬀerentation marker.
Expression detected from spinous to corniﬁed layer,
similar to human skin.

Human skin
Keratin 5

EPI-MODEL24 6D
Day 0

Day 3

Keratin 5 is a basal layer marker.
Expression detected in the basal layer, similar to human skin.

Keratin 10

Keratin 10 is an epidermal diﬀerentation marker.
Expression detected from spinous to corniﬁed layer,
similar to human skin.

Keratin 14

Keratin 14 is a basal and spinous layer marker.
Expression detected in the basal and spinous layers,similar to human skin.

Western blotting analysis of cultured EPI-MODEL 6D

Day 3

monolayer

Day 3

Day 0

monolayer

Day 3

EPI-MODEL 6D

Native-PAGE

Results indicate theproduction of collagen IV

E-Cadherin

Filaggrin

Involucrin
Day 3

Day 0

EPI-MODEL 6D
monolayer

Day 3

Day 0

EPI-MODEL 6D
monolayer

Day 3

EPI-MODEL 6D
monolayer

Day 3

Day 0

EPI-MODEL 6D

Results indicate thepresence of tight junctions

EPI-MODEL 6D
monolayer

Day 0monolayer

Day 3

Day 0

monolayer

Collagen XVIICollagen IV

EPI-MODEL 6D

Day 0

Claudin-1

EPI-MODEL 6D

Day 0

CBB staining

Results indicate theproduction of collagen XVIIResults indicate thepresence of adherens junction

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

Keratin 5

Keratin 10

Keratin 14

Loricrin

Results demonstrate the maintenanceof the basal layer during culture.

Results demonstrate the diﬀerentiation process
induced by the 3D culture of epidermal cells.

PCNA

Transglutaminase

Results demonstrate the decreaseof proliferating cells during culture.

Day 3

Day 0

EPI-MODEL 6D
monolayer

Day 3

Day 0

monolayer

EPI-MODEL 6D

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate the maintenanceof the basal layer during culture.

Day 3

Day 0

EPI-MODEL 6D
monolayer

Day 3

Day 0

EPI-MODEL 6D
monolayer

Day 3

Day 0

EPI-MODEL 6D
monolayer

Day 3

Day 0

monolayer

EPI-MODEL 6D

Results demonstrate the diﬀerentiation process
induced by the 3D culture of epidermal cells.

Gene expression proﬁle of EPI-MODEL6D during culture
FLG

Monolayer

Normal
Skin

LOR

Day 0

Day 3

Monolayer

Normal
Skin

KRT1

Day 0

EPI-MODEL6D

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3
EPI-MODEL6D

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

KRT10

IVL

TGM1

Monolayer

Normal
Skin

Day 0

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3
EPI-MODEL6D

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

Results demonstrate thematuration of the corniﬁed layer.

KRT5

KRT14

ΔNp63

Monolayer

Normal
Skin

Day 0

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3
EPI-MODEL6D

Results demonstrate the maintenanceof the basal layer during culture.

Results demonstrate the maintenanceof the basal layer during culture.

Results demonstrate the maintenance of
epidermal stem cells during culture.

PCNA

MKI67

TJP1

Monolayer

Normal
Skin

Day 0

Day 3
EPI-MODEL6D

Results demonstrate the increase of
proliferating cells during culture.

Monolayer

Normal
Skin

Day 0

Day 3
EPI-MODEL6D

Results demonstrate the increase of
proliferating cells during culture.

Monolayer

Normal
Skin

Day 0

Day 3
EPI-MODEL6D

Results demonstrate the formation oftight junctions during culture.

TJP2

Monolayer

Normal
Skin

Day 0

CLDN1

Day 3

Monolayer

Normal
Skin

CLDN4

Day 0

EPI-MODEL6D

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3
EPI-MODEL6D

Results demonstrate the formationof tight junctions during culture.

Results demonstrate the formationof tight junctions during culture.

Results demonstrate the formationof tight junctions during culture.

OCLN

CDH1

DSG1

Monolayer

Normal
Skin

Day 0

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3
EPI-MODEL6D

Results demonstrate the formationof tight junctions during culture.

Results demonstrate the formationof adherens junctions during culture.

Results demonstrate the formationof desmosomes during culture.

DSG2

DSG3

DSG4

Monolayer

Normal
Skin

Day 0

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3
EPI-MODEL6D

Results demonstrate the formationof desmosomes during culture.

Results demonstrate the formationof desmosomes during culture.

Results demonstrate the formationof desmosomes during culture.

CDSN

ITGA6

ITGB4

Monolayer

Normal
Skin

Day 0

Day 3
EPI-MODEL6D

Results demonstrate the formationof desmosomes during culture.

Monolayer

Normal
Skin

Day 0

Day 3
EPI-MODEL6D

Results demonstrate the maintenanceof the basal layer during culture.

Monolayer

Normal
Skin

Day 0

Day 3
EPI-MODEL6D

Results demonstrate the maintenanceof the basal layer during culture.

COL4A1

Monolayer

NormalSkin

Day 0

COL4A2Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

COL7A1Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3
EPI-MODEL6D

Detection of collagen 4A1
expression during culture.

Detection of collagen 4A2
expression during culture.

Detection of collagen 7A1
expression during culture.

COL17A1

LAMA3

LAMB3

Monolayer

Normal
Skin

Day 0

Day 3

Monolayer

NormalSkin

Day 0

EPI-MODEL6D

Day 3

Monolayer

Normal
Skin

Day 0

Day 3

EPI-MODEL6D

EPI-MODEL6D

Detection of collagen 17A1
expression during culture.

Detection of the Laminin-332
component LAMA3.

Detection of the Laminin-332
component LAMB3.

LAMC2

LAMA1

LAMB1

Monolayer

Normal
Skin

Day 0

Day 3

Monolayer

Normal
Skin

Day 0

Day 3

Monolayer

Normal
Skin

Day 0

Day 3

EPI-MODEL6D

EPI-MODEL6D

EPI-MODEL6D

Detection of the Laminin-332
component LAMC2.

Detection of the Laminin-111
component LAMA1.

Detection of the Laminin-111
component LAMB1.

LAMC1Monolayer

Normal
Skin

Day 0

Day 3
EPI-MODEL6D

Detection of the Laminin-111
component LAMC1.

Test of mild irritants
Histology

Corniﬁed
layer

LabCyte EPI-MODEL 6D

LabCyte EPI-MODEL

Barrier function assessment
Ceramide analysis

TEWL (g/m2/h)

TEWL

Transepidermal
water loss meter
Chamber

EPI-MODELEPI-MODEL 6D

Ceramide2 EPI-MODEL

EPI-MODEL 6D
Data courtesy of Nihon Kolmar Co., Ltd.

(J Soc Cosmet Chem Jpn. 2013, Vol 47, No. 1)

Test of detergents
Test substances: Sodium lauryl sulfate (SLS), Polyoxyethylene(10) octylphenyl ether (Triton X-100)
Protocol: 15 min exposure, 42 h post-exposure incubation

SLS
IC50 EPI-MODEL: 0.32%
EPI-MODEL 6D: 0.19%

Triton X-100
IC50 EPI-MODEL: 0.61%
EPI-MODEL 6D: 0.19%

Sodium lauryl sulfate concentration (%)

Polyoxyethylene(10) octylphenyl ether concentration (%)

Sodium lauryl sulfate concentration (%)

Polyoxyethylene(10) octylphenyl ether concentration (%)

Data courtesy of Nihon Kolmar Co., Ltd.
(J Soc Cosmet Chem Jpn. 2013, Vol 47, No. 1)

Test of mild irritants
Evaluation of cosmetic ingredients
Test substances
Test substanceSkin irritation index
of human patch test

Lotion 1
Lotion 2
Lotion 3
Essence 1
Essence 2
Essence 3
Cream 1 (w/o)
Cream 2 (o/w)
Cream 3 (w/o)

10
0
1.3
3.8
1.3
0
0
0
2.5

LotionClass Ingredients

Butylene glycol
9
PEG-8
3
PEG-60 hydrogenated castor oil
0.5※(Methylparaben)(0.2)(Phenoxyethanol)(Pentylene glycol)(Ethylhexylglycerin)
Citric acid
0.01
Sodium citrate
0.09
Water
remaining vol.

A

Cream (w/o)

Cream (o/w)
Conc. (%)

Class Ingredients

A

B

100
※ Only when phenoxyethanol is also included

Conc. (%)

Polyglyceryl-2-isostearate
2
Dextrin palmitate
1
Petrolatum
2
Mineral oil
40
Beeswax
2(Ethylhexylglycerin)
Glycerin
10
Monosodium glutamate
1※(Methylparaben)(0.2)(Phenoxyethanol)(Pentylene glycol)
Waterremaning vol.
100

Class Ingredients

A

B
C
D

Conc. (%)

2.1
Polyglyceryl-10 myristate
Hydrogenated lecithin
0.6
1.6
Behenyl alcohol
0.5
Stearic acid
6.6
Cetyl ethylhexanoate
2
Mineral oil
0.2
Dimethicone
5Glycerin(0.2)※(Methylparaben)(Phenoxyethanol)(Pentylene glycol)(Ethylhexylglycerin)
Waterremaining vol.
0.1
Carbomer
10
Water
0.04
Sodium hydroxide
4.96
Water
100

Modiﬁcation of exposure time

Evaluation of antiseptic ingredients

Test substances: cosmetic ingredients evaluated by the human patch test.
Protocol: 24 h exposure

Test substances: cosmetic ingredients containing antiseptics.
Human patch test index unkown.
Protocol: 24 h exposure
24 h exposure

24 h exposure

Test
substances

EPI-MODELEPI-MODEL 6D

EPI-MODELEPI-MODEL 6D

Cell viability (%)

Cell viability (%)

Cell viability (%)

Cell viability (%)

Antiseptic

Concentration andingredient

formulation

Mean SD Result Mean SD Result Mean SD Result Mean SD Result(0.2% Methylparaben)

15 min exposure
42 h post incubation

EPI-MODELEPI-MODEL 6D

Cell viability (%)

Cell viability (%)

Mean SD Result Mean SD Result

0.5% Lotion

113.6±27.8

NI74.7±35.6

NI

0.1% Lotion

122.5±3.7NI119.1±16.3

NI

0.5% Cream (o/w)

78.6±7.7NI

19.6±5.6

I

0.1% Cream (o/w)

115.0±10.0

NI52.0±18.3

NI

0.5% Cream (w/o)

74.3±24.7NI64.8±23.4

NI

0.1% Cream (w/o)

107.7±13.3

NI107.5±11.8

NINI

85.2±15.1NI

12.5±2.0I

5.8±0.5

I

Lotion 2

101.9±9.7

NI89.8±11.3

NI

107.4±3.6NI34.2±24.7

I

Lotion 398.5±4.6NI88.5±15.7

NI

125.5±13.4NI78.2±11.6

NI

Essence 1

103.2±6.9

NI105.5±7.1

NI

40.0±34.0I

5.5±3.8

I

Essence 2

105.0±8.7NI93.9±27.5NI

36.3±16.3I

17.4±8.5

I

Essence 3

103.3±1.8NI96.0±8.3

NI

107.6±12.0

NI104.6±9.1

NI

5% Lotion

111.4±16.1

NI

33.9±6.9

I

Cream 1 (w/o)

95.1±16.6

NI

100.9±0.7

NI108.9±13.0

NI86.3±29.0

NI

2% Lotion

108.2±13.9

NI107.0±3.5

NICream 2 (o/w)

102.7±7.4NI

98.6±7.4NI66.4±33.3

NI

17.3±2.9I

5% cream (o/w)

113.8±14.6

NI49.5±4.0

I

Cream 3 (w/o)

106.5±6.5NI

99.1±0.5

NI

121.4±7.4NI

63.1±15.4

NI

2% cream (o/w)

98.7±10.1

NI

113.9±10.9NI

5% cream (w/o)

104.1±16.4

NI112.5±4.6NI

2% cream (w/o)

97.0±11.7

NI

107.0±4.2NI0.1% Lotion

102.5±3.7NI55.1±4.6

NI

0.02% Lotion

102.5±8.6

NI109.1±6.2

NI

0.1% Cream (o/w)

107.8±4.7

NI107.2±10.3

NI

0.02% Cream (o/w)

101.0±5.3

NI108.6±5.1NI

0.1% Cream (w/o)

92.2±2.8NI94.4±2.3

NI

0.02% Cream (w/o)

94.2±6.2NI

92.6±4.7

NI

Pentylene

Phenoxyethanol

99.8±9.8

glycolLotion 1

Ethylhexyglycerin

Results interpretation
Cell Viability ≦ 50% irritant (I)Cell Viability ＞ 50% non irritant (NI)

Data courtesy of Nihon Kolmar Co., Ltd.
(J Soc Cosmet Chem Jpn. 2013, Vol 47, No. 1)

Permeability test

Percutaneous absorption test

Skin permeability test using LabCyte EPI-MODEL
Skin permeability tests can be performed by sandwiching the
LabCyte EPI-MODEL between silicone elastomer adapters, andthen mounting the adapters between side-by-side diﬀusion cells.

Silicone support

Fresh Buﬀer
LabCyte EPI-MODEL

Donor cell

Percutaneous absorption test cab be performed by simply
attaching the culture insert to a glass vial.
Neck diameter
2.0 cmTest substanceapplication

8.0 cm12-well
culture insert

Sampling
Receptor cell

Sampling

6.5 cmGlass vial

Solution
Stirrer bar

Diameter 3.5 cm

StirrerGlass vial
Dimensions compatible
with EPI-MODEL12

Data courtesy of Dr. T. Hikima (Kyushu Inst. Tech.)
Biol. Pharm. Bull. 35(3) 362̶368 (2012)

Data courtesy of Dr. S. Fukushima (Kobe Gakuin Univ.)Prepare your own epidermal model by simply seeding
and culturing keratinocytes in cell culture inserts

Change culture medium
every 2 or 3 days
Seed keratinocytes

EPI-KIT(Product No.: 401810)

1d

5d

Culture for 14 days

7d

12 d

9d

8d

14 d

Proliferation and diﬀerentiation rates vary according to culture conditions.
The data above should be used as reference only.
Assessing sample histology during culture is highly recommended.

Application example
Apply test substances and perform model analysis at a time of your convenience.
Application of test substance

Keratinocyte seeding

Culture 4 d

Culture 7 d

Culture 14 d

Analysis of tissue morphology
Analysis genes, proteins, lipids

Gene expression knockdown in epidermal models
prepared with LabCyte EPI-KIT■ Introduction
The experiment presented here (fillagrin knockdown) was designed based on
information available in the literature. Please use the protocol below as a reference
for gene expression knockdown in cultured epidermal models.
■Reference
Journal of Investigative Dermatology vol.130 2286-2294 (2010)
Knockdown of filaggrin impairs diffusion barrier function and increases UV sensitivity in a human skin model.
Mildner M et., al.

■ Material
siRNA-1 (filaggrin targetting sequence siRNA-1)
sense：5-GAGGUGGUCUGGGUCUGCUUCCAGA-3
antisense：5-UGGAAGCAGACCCAGACCACCUCUC-3
siRNA-2（filaggrin targetting sequence siRNA-2）
sense：5-ACAGAAAGCACAGUCAUCAUGAUAA-3
antisense：5-AUCAUGAUGACUGUGCUUUCUGUGC-3
siRNA-3（filaggrin targetting sequence siRNA-3）
sense：5-GAGGUUGUCUGGGUCUGCUUCCAGA-3
antisense：5-UGGAAGCAGACCCAGACAACCUCUC-3
ctrl-siRNA（siRNA control sequence）
sense：5-GAGUGGGUCUGGGUCUUCCCGUAGA-3
antisense：5-UACGGGAAGACCCAGACCCACUCUC-3
Opti-MEM®
Life Technologies, Code 31985-070, 500ml
Lipofectamine®2000 Transfection Reagent
Life Technologies, Code11668-027, 0.75ml■ Protocol
The measurements below are for the preparation of one 24-well plate using one siRNA sequence.
Measurements should be adjusted for fewer wells.
1. Preparation of siRNA-Lipofectamine® complex
Prepare the complex in 15 ml tubes, under sterile conditions, using the measurements below.
siRNA mixture
5 ml OPTI-MEM®
50 µl Lipofectamine® 2000
26 µl 100 µM siRNA
Prepare your Mock transfection sample without siRNA at this point, if necessary.
Leave the siRNA-Lipofectamine® complex resting at room temperature for 30 min.

2. Transfection
1. Thaw keratinocytes according to the EPI-KIT instruction manual.
2. Add 7 ml of assay medium to siRNA - Lipofectamine® complexes and mix well.
3. Add thawn keratinocytes to siRNA-containing mixture prepared in step 2.
4. Gently mix the solution prepared in step 3, and add 500 µl to each cell culture insert.

■ Results
1. Epidermal model histology
Media was removed from the cell culture inserts, and keratinocyte culture was done according to
the EPI-KIT instruction manual.

7d

9d

11 d

Untreated

Mock

siRNA-1siRNA-2siRNA-3Ctrl-siRNA

Cultured models were fixed in 4% Formalin Neutral Buffer Solution, at 7d, 9d, and 11d.
Untreated: Models prepared according to EPI-KIT standard protocols.
Mock: Models prepared with the addition of Lipofectamine®, without siRNA, before cell seeding.
siRNA-1, siRNA-2, siRNA-3, ctrl-RNA: Models prepared with correspondent siRNA-Lipofectamine® complex.
Dotted circles showing cells containing keratohyalin granules.
Cells containing keratohyalin granules not found in models transfected with siRNA-1 and siRNA-3.

2. Immunohistochemistry
Untreated

Mock

siRNA-1siRNA-2siRNA-3Ctrl-siRNA

Immunohistochemistry was performed on epidermal models fixed at 11 d.
Filaggrin staining (red) showed by white arrows.
Primary antibody: Anti-filaggrin antibody (Neuromics MO20041).
Secondary antibody: Alexa FluorR 546 Goat Anti-Mouse IgG (H+L) (Life Technologies A-11030).
Nuclear staining (blue): Hoechst 33258.
Filaggrin was not detected in models transfected with siRNA-1 and siRNA-3.
3. Epidermal barrier function assessment

Live cells (%)

Barrier function test

SLS concentration (%)

Barrier function test was performed using models cultured for 13 d.
25 µl of SLS-PBS solution (SLS concentration: 0%, 0.1%, 0.15%, 0.2%, 0.3%) was added tothe models (n = 3) that were then incubated in a CO2 incubator for 18 h at 37°C.
After 18 h, models were rinsed with DPBS (add 0.5 ml of DPBS to the culture insert and discard - repeat 3
times). Rinsed models were processed for MTT assay. Data were normalized by results from 0% SLS
treatment (0% SLS cell viability = 100%).
Average of 3 readings (n = 3) plotted in the graph above.
Model transfected with siRNA-2 showed higher barrier function compared to untreated model.
Note:
The results of this experiment differ from those of the reference article (siRNA-1, siRNA-2, siRNA-3 show the same results).

LabCyte CORNEA-MODEL is a 3-D human cultured corneal epithelial tissue produced from normal humancorneal epithelial cells. LabCyte CORNEA-MODEL was developed by applying cell culture techniques todiﬀerentiate and stratify corneal epithelial cells to form a tissue structure similar to that of the normal
human cornea. LabCyte CORNEA-MODEL can be used in the hazard identiﬁcation of irritant chemicals
by the eye irritation test. Moreover, CORNEA-MODEL can also be used to evaluate biological processes,
such as the production of mucin and the expression of adhesion molecules, in response to diﬀerent test substances.

Model features
3-D culture of human corneal epithelial cells

Model structure
Φ6.4mmCan be used for eye irritation tests.

Cell culture insert

Enables the analysis of proteins speciﬁcally expressedin the corneal epithelium.
Human corneal epithelial cells

Low lot-to-lot variability

PET membrane ﬁlter

Developed and manufactured at J-TEC using
cell culture techniques fostered in house.

Model appearance

Reproducing the corneal epithelial structure
HE staining
Cell diﬀerentiationand stratiﬁcation.

Transmission electron microscopy
Observation of microvilli andglycocalyx (arrows) on theoutermost layer of cells.

Immunohistochemistry
Detection of proteins expressed at diﬀerent cells layers of the corneal epithelial.
Outermost layer

Mucin-1

Mucin-16

Cell-cell adhesion

Basal layer

Desmoglein-3

Laminin V

Accepted
ModelOECD Test Guideline 492 - Eye Irritation Test

LabCyte CORNEA-MODEL was accepted by the OECD to be used in the eye irritancy assessmentof chemical substances under the test guideline 492.
The OECD test guidelines are internationally accepted testing methods used by government,
industry and independent laboratories to identify and characterize potential hazards of chemicals.
OCED test guideline 492 describes an in vitro procedure that may be used for the hazard
identiﬁcation of eye irritant chemicals using reconstructed human cornea-like epitheliumthat closely mimics the biochemical and physiological properties of the outermost layer of the humancorneum epithelium.

Test procedure① Pre-incubation② Application of test substance,rinsing, and post-exposure incubation(conditions diﬀer according to the physical state of the tested chemical)③ Cell viability measurement (WST-8 assay)④ Determine whether results meet acceptance criteria❶ Cell viability: 0.5 ≦ mean OD (A450/650) measured value for negative control ≦ 1.6

Eye irritation test set(Product No.: 401351)❷ Positive control: mean tissue viability for positive control ≦ 40%❸ SD: SD (negative control and positive control) of tissue viability of three indentical
replicates ≦ 18

Classiﬁcation criteria
Tissue viability ≦ 40%

Category 1 or 2 (irritant)Tissue viability > 40％

Non Category (non-irritant)

Concordance with in vivo classiﬁcationin vivo classiﬁcationin vitro
prediction

irritant

Non-irritantTotal

irritant

76

17

93

Non-irritant

0

46

46

Total

76

63

139

Sensitivity(%)

100

Speciﬁcity(%)

73.0

Accuracy(%)

87.8

LabCyte CORNEA-MODEL can be used to study histological featuresof the corneal epithelium.
LabCyte CORNEA-MODEL reproduces microstructural features of the corneal epithelium.
Desmosome
Glycocalyx

Hemidesmosome

Apoptosis induction by benzalkonium chloride application assessed by TUNEL staining
Benzalkonium chloride concentration
0.005%

0.05%

TUNEL
/
Hoechst

Disruption of cell-cell adhesion promoted by benzalkonium chloride
Benzalkonium chloride
PBS

0.1%

1%

10%

Claudin-1
Tight Junction
component

Desmoglein-3
Tight Junction
component

E-cadherin
Adherens Junction
component

Disruption of cell-cell adhesion by benzalkonium chloride is concentration-dependentMarch 2021.
Product features may change without prior notice.
Through the development of tissue-engineered medical products, J-TEC will contribute to the development of techniquesthat can be used as alternatives to animal testing.

Japan Tissue Engineering Co., Ltd.
6-209-1 Miyakitadori, Gamagori, Aichi 443-0022
Sales and Marketing Department Phone: +81-533-66-2129 FAX: +81-533-66-2018

http://www.jpte.co.jp