Vitrigel-EIT Method Protocol Version 1.81e

Standard Protocol for the Vitrigel-EIT method
Version 1.81e January 18, 2019
Hiroyuki Yamaguchi1, 2 and Toshiaki Takezawa1
1Institute of Agrobiological Sciences, National Agriculture and Food Research Organization
2Kanto Chemical Co., Inc.

Address correspondence to: Dr. Toshiaki Takezawa, Division of Biotechnology,
Institute of Agrobiological Sciences, National Agriculture and Food Research Organization,
1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
E-mail: t.takezawa@affrc.go.jp1Vitrigel-EIT Method Protocol Version 1.81e

1.

Principle of the method

The Vitrigel-EIT method is a highly sensitive means of testing for eye irritation potency by analyzing aseries of transepithelial electrical resistance (TEER) measurements taken over time after exposure to atest chemical. A typical protocol for this test method is as follows.

2.

Applicability domain

The following substances are not suitable for testing with this method and are excluded from theapplicability domain.
1.

Chemicals that have a pH level of 5 or less in solution (see section 5.5.3, step 3).

2.

Solids that have both a logP value of at least 2.5 and a density of either less than 0.95 g/cm3 or
more than 1.10 g/cm3.

3.

Materials to be procured by the user


HCE-T cells (Cell Bank, Riken BioResource Center, RCB2280)



Clean bench or biological safety cabinet

CO2 incubator (37°C, 5% CO2 in air)Phase-contrast microscopeWater bathCentrifuge (150 × g or greater)Safety pipette filler

Micropipettes (100 μL, 1000 μL)

Tweezers

70% EthanolBalanceComputer (Windows OS with USB port)

Liquid nitrogen freezer (for cell storage)

Universal pH test paper (ADVANTEC, 07011030)

2

Vitrigel-EIT Method Protocol Version 1.81e

4.

Vitrigel-EIT kit contents (for 72 tests)*Other equivalent reagents or materials are acceptable.
Name
Maker
TM
ad-MED Vitrigel comprise 12 collagen Kanto
vitrigel membrane (CVM) chambers set in
a 12-well plate

Culture medium (prepared by Kanto)
Composition
D-MEM/F-12
5% fetal bovine serum,
5 μg/ml human insulin,
10 ng/ml human epidermal growth factor,
2.5 % dimethyl sulfoxide,
100 U/ml penicillin, and100 μg/ml streptomycin

Item code
08360-96

Quantity
6 plates

Store atr.t.1

1 bottle

2–8°C

LT2
Sigma LTLT
Sigma LT11330-032
F2442
12585-014
PHG0311
D2650
15140-148

0.05% trypsin-0.02% EDTA-2 Na solution

LT

25300-054

10 bottles

PBS

Sigma

D8537

1 bottle−20°C or
lower
2–8°C

15250-061

1 vial1 vial

r.t.
2–8°C

B6294

1 vialr.t.

14033-00

1 vial

r.t.

D2650

1 vial
1 set
3 packages
1 package1 package

r.t.
r.t.
r.t.
r.t.
r.t.

1 package
1 set

r.t.
r.t.

0.4% trypan blue solution
LT
Negative control reagent (saline)(prepared by Kanto)
Positive control reagent (benzalkonium Sigma
chloride)
Reference control reagent
Kanto(99.5% ethanol)
Dimethyl sulfoxide
Sigma
TEER recorder (prototype)
Kanto
Tissue culture flask (T-75)
BD Falcon
5ml Pipette
BD Falcon
15ml Plastic tube
BD Falcon
1.5ml Plastic tube
As oneHemocytometer
TGK
Chambers without membrane and 12-well Kanto
plate for TEER recorder check
NB1: room temperature, NB2: Life Technologies

33530243575293520961-7521-01
OC-C-S02

Vitrigel-EIT Method Protocol Version 1.81e

5.

Procedure

5.1

Thawing and initial culturing of HCE-T cells

1.

Warm culture medium in a water bath at 37°C.

2.

Pour 14 ml of the culture medium into a T-75 tissue culture flask and spread it over thebottom surface.

3.

Transfer a cryotube of human corneal epithelium (HCE) T cells from the liquid nitrogen
freezer to the water bath and agitate gently until completely thawed.

4.

Open the cryotube, make sure any sunken HCE-T cells are suspended in the medium, then
add the total cell suspension to the culture medium in the T-75 flask, and mix until uniform.
Then, transfer the flask to the CO2 incubator (37°C, 5% CO2 in air) to start culturing the
HCE-T cells.

5.

After culturing the cells for at least 2 but no more than 6 hours, observe the morphology of
the cells under a phase-contrast microscope. If
most of the cells are well attached, as shown in Fig.
1 (a few non-attached cells can be ignored),
replace the culture medium with a fresh one andcontinue the culturing. If not well attached,dispose of the failed cell culture and repeat steps 1

Fig. 1 Phase-contrast microphotograph of
HCE-T cells cultured for two hours.

to 4 using a different cryotube of HCE-T cells.
6.

After culturing the cells for a few days, observe the morphology of the cells under a phasecontrast microscope. If the cells have proliferated
into a monolayer of between 80 and 100%confluency, as shown in Fig. 2, proceed to section
5.2 to passage the cells as described. If not, disposeof the failed culture and repeat steps 1 to 5 using a
different cryotube of HCE-T cells.

5.2

Passaging HCE-T cells

Fig. 2 Phase-contrast microphotographof HCE-T cells in a min. 80%
confluent monolayer appropriate
for passaging.

1.

Prepare four new T-75 flasks with 14 ml of fresh culture medium in each flask.

2.

Warm the culture medium bottles, PBS, and trypsin solution in a water bath at 37°C.

3.

Remove the culture medium from the flask of HCE-T cells that have proliferated into a min.
80% confluent monolayer. Pour 10 ml of PBS into the flask and spread it over the bottom
surface to rinse the cells.

4.

Remove the PBS from the flask. Pour 5 ml of trypsin solution into the flask, spread it overthe bottom surface, and the remove 4.3 ml of the solution. Incubate the flask in a CO₂
incubator for five minutes. Surplus trypsin solution can be stored in a freezer and reused.
4Vitrigel-EIT Method Protocol Version 1.81e
5.

Tap the bottom and side walls of the flask several times to detach the cells from the bottom
surface, as shown in Fig. 3.

6.

Pour 4 ml of fresh culture medium into the flask andsuspend the cells by pipetting as quickly as possible.

7.

Add 1.0 to 1.2 ml of the cell suspension to the culture
medium in each of the four new flasks described in step
1 and mix until uniform. Next, place the flasks in a CO₂
incubator to start passaging HCE-T cells in the four new
flasks. Thereafter, change the culture medium every
other day.

8.

Fig. 3 Phase-contrast microphotograph
of
HCE-T
cells
after
trypsinization

Allow the subculture to proliferate in as many or as few flasks as needed (ordinarily, two toeight).

5.3

Cryopreservation of HCE-T cells

Prepare eight cryotubes of HCE-T cells from the four T-75 flasks prepared in section 5.2 bytransferring half of the cells in each flask to a cryotube, as described below.
1.

Prepare a culture medium with 20% DMSO by adding 0.8 ml of DMSO to 3.2 ml of culture
medium and mixing until uniform.

2.

Follow steps 2 to 6 of the procedure described in section 5.2 to create four flasks, in which
HCE-T cells have proliferated into a monolayer of between 80 and 100% confluency.

3.

Collect the cell suspension in a 50 ml centrifuge tube and spin at 150 × g for three minutesat room temperature.

4.

Remove the supernatant and tap the tube several times to loosen the multicellular pellet.

5.

Suspend the cells in 4 ml of a fresh culture medium.

6.

Add 4 ml of the culture medium with 20% DMSO to the cell suspension and gently mix bypipetting. Quickly dispense 1 ml of the cell suspension to each cryotube.

7.

Place the cryotubes in a programmable freezer (or equivalent) and cool at a rateof -1°C/minute to a temperature of -80°C. Preserve the cryotubes in a liquid nitrogen freezer.

5.4
1.

Preparation of Vitrigel-HCE models
Follow the procedure described in section 5.1. to thaw HCE-T cells preserved in the
cryotubes.

2.

Initiate culturing and allow the HCE-T cells to proliferate into a monolayer of between 50
and 100% confluency. About 80 models can be prepared from a single T-75 flask in which
the cells have proliferated into a confluent monolayer.

5Vitrigel-EIT Method Protocol Version 1.81e
Caution:
Do not use HCE-T cells in an over-confluent state, because cell-to-cell aggregatesare difficult to separate.

3.

Follow steps 2 to 6 of the procedure described in section 5.2.

4.

Observe the morphology of the cells under a phase-contrast microscope. Proceed to step 5
only if the culture comprises well-separated, individual cells. If cellular aggregatescomprising six cells or more are observed over more than 20% of the surface area, disposeof the failed cell culture and return to step 1 of this section.

5.

Transfer the cell suspension to a 50-ml tube. Resuspend the cells uniformly and then
immediately dispense 100 μL of the cell suspension into a 1.5-ml tube.

6.

Add 100 μL of trypan blue solution to the cell suspension and mix well by pipetting. Countthe numbers of living and dead cells by using a hemocytometer and calculate the survival
rate and density of the cell suspension. If the cell survival rate is lower than 95%, disposeof the failed cell culture and return to step 1 of this section.
Caution:
Do not push a collagen vitrigel membranewith the pipette tip. Use the guide on theside wall of the chamber to fix the pipette
tip temporarily.

7.

For each collagen vitrigel membrane chamber pre-set in a well of a 12-well plate, first pour1.5 ml of culture medium into the well on the outside the chamber and then pour 0.5 ml ofculture medium directly into the chamber itself. Let stand at room temperature for at least
10 minutes but no more than 2 hours.

8.

Remove the culture medium from the inside of each collagen vitrigel membrane chamber
and pour 0.5 ml of the cell suspension at a density of 1.2 × 105 cells/ml. Transfer the plate
to a CO₂ incubator and culture for two days.

9.

Change the culture medium in the cell outside the chamber and remove the culture medium
6Vitrigel-EIT Method Protocol Version 1.81efrom inside the chamber to start the air–liquid interface culture, then culture for four daysin a CO₂ incubator.
10.

Change the culture medium in the cell outside the chamber on the fifth day after seeding.

11.

On the sixth day after seeding, pour 0.5 ml of culture medium directly into the inside of thechamber. Adjust the temperature of the model to 28±2°C. Set the electrode to the TEER
recorder on the chamber. Measure an initial TEER value for each HCE model before
exposure to a test chemical and record the measured values on the data sheet.

12.

HCE models with an initial TEER value of 140–220 Ω·cm2 are acceptable for use in testing.
HCE models that pass the above acceptance criterion should be used in a chemical exposure
test within the same day.
Caution:Although no technique for measuring temperature is specified, we recommend
pouring culture medium into an empty well of the 12-well plate before adjusting thetemperature for all models, then measuring the temperature of the medium inside thewells. If there is no empty well available, use an empty well on a new 12-well plate.

theshould stand verticallythe7Vitrigel-EIT Method Protocol Version 1.81e

5.5

Performing tests using the Vitrigel-EIT method

5.5.1

Checking the TEER recorder

1.

Pour 3 ml of a 0.45% NaCl aqueous solution at 25±5°C in one well of a 12-well plate andinsert a chamber without membrane in the well.

2.

Pour 3 ml of a 0.9% NaCl aqueous solution at 25±5°C in one well of 12-well plate and insert
a chamber without membrane in the well.

3.

Measure the TEER value in both wells. If the TEER recorder is functioning normally, themeasured values will satisfy the following conditions:(TEER value of 0.45% NaCl aqueous solution) − (TEER value of 0.9% NaCl aqueous
solution) ≥ 60 Ω·cm2

5.5.2

Testing positive, negative, and reference control reagents

1.

Equilibrate the culture medium to an ambient temperature of between 22 and 30°C.

2.

The Vitrigel-EIT method uses a saline solution as negative control, a benzalkonium-chloride
solution as positive control, and an ethanol solution as reference control. Prepare each in a
2.5% (w/v) reagent solution by adding 0.1–0.2 g of sodium chloride, benzalkonium chloride,and ethanol to an appropriate volume of culture medium in a 15-ml tube and mixing until
uniform. As long as the proper concentration is maintained, the total quantity of the preparedreagent is unimportant.
To minimize volatilization, 15-ml tubes should be sealed tightly when not in use.

3.

Adjust the temperature of the reagent solution to 28±2°C.

4.

Adjust the temperature of a model to 28±2°C.
Caution:Measure temperature by immersing a thermometer in the reagent solution or culture
medium.

5.

Remove the culture medium from inside the chamber and set the electrode to the TEER
recorder on the chamber.
Caution:
Add the control reagent within 5 minutes of removing the culture medium from inside
the chamber.

6.

Bearing in mind the importance of performing the test within the required timeframe,
quickly pour 500 μL of the control reagent into the inside of a chamber (within 2-secondstime) and click the Start icon on the measurement software within 2 to 5 seconds of exposure
to the control reagent. TEER values are automatically recorded every 10 seconds for 3
minutes thereafter. If the differential between the initial TEER value and the TEER value at
0 seconds is more than ±40 Ω·cm2, reject the results and retest using a different HCE model.
8Vitrigel-EIT Method Protocol Version 1.81e
7.

Input the measured TEER values to the data sheet (software), as described in section 5.5.4.

8.

Once the measured TEER values are input, the data is automatically analyzed to determine
whether or not the success criteria have been satisfied as show below. If the data is marked
Pass on the data sheet, proceed to the next test. If the data is marked NG on the data sheet,redo the test starting with preparation of a Vitrigel-HCE model, as described in section 5.4.
Success criteria
Negative control: The plateau level is 5% or less of the TEER value at 0 seconds.
Positive control: The plateau level is 40% or more of the TEER value at 0 seconds.
Reference control: The plateau level is 10% or more of the TEER value at 0 seconds.

9.

Rinse the electrode with pure water, and wipe it with an absorbent paper towel after every
test.

5.5.3

Testing test chemicals

1.

Equilibrate the culture medium to an ambient temperature of between 22 and 30°C.

2.

Prepare a 2.5% (w/v) test chemical solution by adding 0.1–0.2 g of each test chemical to an
appropriate volume of culture medium in a 15-ml tube. As long as the proper concentration
is maintained, the total quantity of the prepared reagent is unimportant. Mix manually until
the test chemical dissolves or for a maximum of one minute. If the test chemical does notdissolve readily, try using the following techniques in the following order to dissolve it: a)mix mechanically for a maximum of one minute using a vortex mixer, b) sonication for a
maximum of 20 minutes, or c) heating to a maximum temperature of 70°C. After trying eachtechnique, adjust the temperature of each test chemical solution to 28±2°C and checksolubility. Move to the next step of the procedure once the test chemical solution is welldissolvedorhomogeneously

dispersed.

Caution:
To minimize volatilization, the 15-ml tubes should be tightly sealed when not in use.
The parameters (time, temperature, intensity, etc.) for dissolving test chemicals may
be adjusted to accommodate the physiochemical properties of each test chemical.
3.

Measure the pH level of the test chemical solution using universal pH test paper.

4.

Adjust the temperature of the test chemical solution to 28±2°C.

5.

Adjust the temperature of a model to 28±2°C.
Caution:Measure temperature by immersing a thermometer in the test chemical or culture
medium.

6.

Remove the culture medium from inside the chamber and set the electrode to the TEER
recorder on the chamber.

7.

Bearing in mind the importance of performing the test within the required timeframe,
quickly pour 500 μL of the test chemical solution into the inside of a chamber (within
9Vitrigel-EIT Method Protocol Version 1.81e
2-seconds time) and click the Start icon on the measurement software within 2 to 5 secondsof exposure to the test chemical solution. TEER values are automatically recorded every 10
seconds for 3 minutes thereafter. If the differential between the initial TEER value and the
TEER value at 0 second is more than 40 Ω·cm2, reject the results and retest using a different
HCE model.
Caution:For test chemicals that are insoluble, a homogeneous suspension of the test chemical
may be prepared immediately before testing.
8.

Input the measured TEER values to the data sheet (software), as described in step 5.5.4.

9.

Rinse the electrode with pure water or 70% ethanol, and wipe it with an absorbent paper
towel after every test.

5.5.4
1.

Automatic analysis system utilizing a data sheet
Three individual tests are performed for each test chemical, and the measured TEER values
are input to software data sheet running on PC automatically analyze the data. The mean
TEER values for the three tests are plotted on a timeline to create a profile of TEER values(dP/dT), whichare

analyzedforthree parameters:timelag (t1), intensity(−[P2 − P1]/[t2 − t1]), and plateau level (100 − P2). Time lag (t1) is defined as the maximum
time a profile was maintained at 0 ≥ dP/dT > −0.03%/second. The starting time of plateaulevel (t2) after the profile was maintained at (dP/dT ≤ −0.03%/second) for a particular periodof time was defined as the initial time at which the profile was maintained at
0 ≥ dP (P3 − P2)/dT (t3 − t2) > −0.03%/seconds. The time (t3) is represented in the equation(t3 = t2 + 30 seconds) because the plateau level was evaluated by the profile for 30 seconds.
P1, P2, and P3 are the percentages against the initial TEER value at t1, t2, and t3 after exposure
to the test chemical, as shown in Fig. 4.

Fig. 4 Schematic analysis of a TEER profile after exposure to a test chemical
Here, t1 and t2 represent time lag and starting time of plateau level, respectively. t3 is defined
as t3 = t2 + 30 seconds. P1, P2, and P3 are the percentages against the initial TEER value attimes t1, t2, and t3, respectively.

10Vitrigel-EIT Method Protocol Version 1.81e
2.

Eye irritation potency was predicted using the following criteria.
Criteria

ResultTime lag ≤ 180 or Intensity ≥ 0.05 or Plateau level > 5.0

Irritant (I)Time lag > 180 and Intensity < 0.05 and Plateau level ≤ 5.0

Non-irritant (NI)11

Vitrigel-EIT Method Protocol Version 1.81e

Revision history
Version 1.60e
Date: June 30, 2014 Description of change
4.411, Procedure 4.5.2., step 1, 3 and Procedure 4.5.3., step1, 3 Replaced the word “roomtemperature” to “ambient temperature for the experiment” Added an attention about the ambient
temperature at procedure 4.4., step 11
ReasonControl of the ambient temperature is important for success the experiment.
Procedure 4.5.2., step 1, 4.5.3., step 1
Change the ambient temperature for a TEER test as follows.
between 18 and 30↓
between 22 and 30
ReasonTemperature of a HCE model affects values of time dependent TEER change.
Procedure 4.5.2., step 7
Included the reference control as acceptance criteria, and changed the acceptance criterion as follows.
Plateau level is between 10% and 30%, inclusive.
↓
Plateau level is between 10% and 40%, inclusive. Reason
At the 2nd VMT meeting, all members agreed to should be included the reference control as theacceptance criteria for quality preservative of the model.
Expand the acceptance criteria on ground of the test results by the read laboratory.
Procedure 4.5.3., step 2, 5th sentence
Changed the description of the procedure for propagating a test chemical solution as follows. Old:
If the test chemical has not been dissolved, try to dissolve it by the mechanical mixture for a
maximum 1-minute period using a voltex, by the sonication for a maximum
20-minute period, or by the heating to 70
New:
If the test chemical has not been dissolved, try to dissolve it by selecting an appropriate technique(s)from the following; mechanical mixture for a maximum 1-minute periodusing a vortex mixer, sonication for a maximum 20-minute period, or heating to maximum 70
12Vitrigel-EIT Method Protocol Version 1.81e
ReasonAt the phase 1 study, some members misunderstood the procedure and they thought that all of threetechniques should be done.

Replaced the word “voltex” to “vortex mixer” (corrected a typo).

Procedure 4.5.2., step 2 and 4.5.3., step 2
Added an attention “To avoid volatilizing chemicals, the 15ml tube should be tightly lidded after
weighing a test chemical to the tube except for adding a culture medium and sampling the 2.5% test
chemical solution.”.
ReasonClarify the attention that the tube should be tightly lidded during the experiment to avoid volatilizing
chemicals.
Procedure 4.5.2., step 5 and 4.5.3., step 5
Added a sentence “In case the increasing and decreasing of 40 Ω・cm2 and more is occurred
between the initial TEER value and the TEER value at 0 second, reject the experiment and retest
using another HCE model.”ReasonIn case the TEER value at 0 second evidently differs from the initial TEER value, it indicates sometechnical failure in the experiment (e.g., contamination of electrical nose, improper use of electrode).

Version 1.70e
Date: October 15, 2014 Description of change
Procedure 4.4., step 11, Procedure 4.5.2., step 4 and Procedure 4.5.3., step 4
Replaced the word “Let stand for 10 minutes (within 2 hours) at the ambient temperature for theexperiment.” to “Adjust the temperature of the model to 282 Added an attention about the way ofmeasuring temperature at procedure 4.4., step 11
ReasonTemperature control of a model is important for success the experiment.
Procedure 4.5.2., step 1 and Procedure 4.5.3., step 1
Replaced the word “ambient temperature for the experiment” to “between 22 to 30
Remove attentions at 4.5.2., step 1 and Procedure 4.5.3., step 1
ReasonRemove the word “ambient temperature”.

13Vitrigel-EIT Method Protocol Version 1.81e
Procedure 4.5.2., step 3 and Procedure 4.5.3., step 3
Replaced the word “with the ambient temperature for the experiment” to “to 282 Added an attention
about the way of measuring temperature at procedure 4.5.2., step 3 and Procedure 4.5.3., step 3
ReasonTemperature control of a 2.5 w/v% reagent solution is important for success the experiment.
Procedure 4.5.2., step 7
Changed the acceptance criterion of reference control as follows. Plateau level is between 10% and30%, inclusive.
↓
Plateau level is level is 10% or more
ReasonThe upper limit of acceptance criterion will be decided at the end of phase III validation study.

Version 1.80e
March 31, 2015
Description of changes

Section 2. Applicability domain
A new section was added to define an applicability domain based on the test results of 132 chemicals
at the lead laboratory.

Section 3. Materials to be procured by the user
The term universal pH test paper (ADVANTEC, 07011030) was added to section 3, because anadditional step was added to measure the pH level of the test chemical solution using universal pH
test paper.

Section 4. Vitrigel-EIT kit contents
Formerly Section 2, this section was renumbered to accommodate the addition of the new Section
2.

Section 5. Procedure
Formerly Section 5, this section was renumbered to accommodate the addition of the new Section
14

Vitrigel-EIT Method Protocol Version 1.81e
2.

Section 5.5.3, Step 2
Changed the description as follows.
Old:
If the test chemical has not been dissolved, try to dissolve it by selecting an appropriate technique(s)from the following; mechanical mixture for a maximum 1-minute period using a vortex mixer,
sonication for a maximum 20-minute period, or heating to maximum 70°C.
New:
If the test chemical does not dissolve readily, try using the following techniques in the following
order to dissolve it: a) mix mechanically for a maximum of one minute using a vortex mixer, b)
sonication for a maximum of 20 minutes, or c) heating to a maximum temperature of 70°C. Aftertrying each technique, adjust the temperature of each test chemical solution to 28±2°C and checksolubility. Move to the next step of the procedure once the test chemical solution is well dissolved
or homogeneously dispersed.
Also, a condition was added to the Caution box at the end of Step 2, which reads:
The parameters (time, temperature, intensity, etc.) for dissolving test chemicals may be adjusted
to accommodate the physiochemical properties of each test chemical.
This was done due to problems with between-laboratory reproducibility during Phase III, when there
were non-concordant results for some test chemicals. These discrepancies were attributed todifferences in techniques used to dissolve the test chemicals, so we specified procedures fordissolving test chemicals.

Procedure 5.3.3., step 3
Add an extra procedure “Measure pH level of each 2.5% test chemical solution was using Universal
pH test paper.”Reason for revision
Add a procedure in association with setting applicability domains.

Version 1.81e
January 18, 2019
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