The document describes several methods used to study hepatotoxicity in vitro, including hepatocyte cell cultures, Trypan Blue dye exclusion test for determining cell viability, Oil Red O staining to visualize lipid accumulation, MTT assay to assess cell proliferation, RT-PCR to detect gene expression, and Western blotting to analyze protein expression. It then outlines experimental protocols for establishing in vitro models of non-alcoholic fatty liver disease (NAFLD), acetaminophen-induced hepatotoxicity, and hepatotoxicity induced by amiodarone and tamoxifen, involving treating hepatocyte cultures with various concentrations of fatty acids, acetaminophen, amiodarone, or tamoxifen and performing the above assays.

1. Improved Medical Education in Basic
Sciences
for Better Medical Practicing
ImproveMEd
In vitro models of hepatotoxicity

2. Materials and methods used to study
hepatotoxicity in vitro

3. Source: http://cellbank.nibiohn.go.jp/legacy/celldata/jcrb0403.htm
1. Hepatocyte cell cultures
Source: https://www.lgcstandards-atcc.org/products/all/HB-8065.aspx?geo_country=h

4. 2. The Trypan Blue dye exclusion test
Source: https://bitesizebio.com/13687/cell-counting-with-a-hemocytometer-easy-as-1-2-3/
Source:
http://www.ruf.rice.edu/~bioslabs/methods/microscopy/cellcounting.html
Source:https://www.researchgate.net/figure/51450741_
Trypan-blue-staining-Optical-microscopic-images-of-the-
CT-26-cells-treated-with-a
𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠
𝑚𝑙
= 𝑡𝑜𝑡𝑎𝑙 𝑣𝑖𝑎𝑏𝑙𝑒 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 ×
𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠
× 10 000
𝑐𝑒𝑙𝑙𝑠
𝑚𝑙

5. 3. Oil red O staining
Fix cells with cooled
4%
paraformaldehyde.
Remove fixative and
rinse twice with
cooled PBS.
Add Oil red O to
cells by using 0.2
micron syringe filter.
Rinse well with
dH2O until the
water runs clear.
Add hematoxylin
counterstain.
Remove and rinse
well with dH2O until
the water runs clear.
Leave the final
dH2O rinse on the
cells for microscopy.
Lipids will appear
red and the nuclei
will appear blue.
Source:https://atomscientific.com/product/Oil_red_O_Stain_Kit

6. 4. MTT
Plate cells, usually in 96-
well plate (e.g. 2x103 cells
per well for a week long
experiment).
After 24h of incubation
treat cells with agents for
appropriate time.
Remove medium and add
fresh medium with 0.5
mg/ml MTT.
Remove MTT media.
Add equal volume of 0.04 M
HCl in isopropanol or 10%
SDS solution to solubilize
cells.
Read absorbance on
Microplate reader.
Lower absorbance values
signify a reduction in cell
proliferation (comparing to
controls) and higher values
indicate an increase in cell
proliferation.
Source: https://www.slideshare.net/ChanderKNegi/mtt-cell-proliferation-assay

7. 5. RT-PCR
Mix template RNA,
primers and DEPC-H2O
up to 14μl in PCR tube
and incubate.
Add 7.8-8μl of reaction
mix to each sample and
incubate 50 minutes at
42°C.
Add 1μl RNase inhibitor
(10μg) to each sample
and incubate 20 minutes
at 37°C.
Add 10 μl of each primer
to PCR tubes. Afterwards
add 15.5 μl of master
mix.
Add 1-10mg dilution of
RT-PCR cDNA and DEPC
H2O up to 65μl.
Overlay each sample
with 3 drops of mineral
oil and run samples. In
PCR cycle

8. 6. Western blotting
SDS-PAGE electrophoresis
Wet the membrane in
MetOH and the sponge and
filter paper in transfer
buffer.
Create a transfer sandwich.
Move the sandwich to
transfer apparatus and add
transfer buffer to the
apparatus. Then place the
electrodes and transfer for
45-90 minutes.
Block the membrane with
5% skim milk in TBST for 1
hour.
Add primary antibody in 5%
BSA and incubate on shaker.
Wash the membrane 3 times
with TBST and then incubate
with the recommended
dilution of conjugated
secondary antibody in
blocking buffer.
Repeat the washing
procedure.
Detect the membrane by the
signal that HRP-labeled
antibody produced.
Source: https://www.creative-diagnostics.com/Sample-Gel-Preparation.htm

9. Fatty acids- in vitro models of NAFLD
1. Add plain medium.
2. Add medium plus MetOH 99%.
3. Add medium plus 0.33mM PA solution.
4. Add medium plus 0.66mM PA solution.
5. Add medium plus 0.66mM OA solution.
6. Add medium plus 1.32mM OA solution.
Incubate for 24h, and perform abovementioned tests.

10. Acetaminophen induced hepatotoxicity
1. Add plain medium.
2. Add medium plus 100% DMSO.
3. Add medium plus 0.5 mM APAP solution.
4. Add medium plus 5 mM APAP solution.
5. Add medium plus 10 mM APAP solution.
6. Add medium plus 20 mM PA solution.
Incubate for 2, 6 and 24h and perform abovementioned tests.

11. Amiodarone and tamoxifen induced hepatotoxicity
1. Add plain medium.
2. Add medium plus MetOH 99%.
3. Add medium plus 10 μM amiodarone solution.
4. Add medium plus 20 μM amiodarone solution.
5. Add medium plus 5 μM tamoxifen solution.
6. Add medium plus 10 μM tamoxifen solution.
Incubate for 24h and 48h, and perform abovementioned tests.