Caco-2 cell permeability assay is the golden standard for in vitro prediction of intestinal drug permeability and absorption.

1. Caco-2 Cell
Permeability Assay
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2. Contents
Caco-2 Monolayer
Caco-2 Cell Culture
Measuring TEER
Assay Procedure
Papp Calculation

3. 3Part 1 1.1 Characteristics of Caco-2 Cells
1 Origin:
Human colorectal adenocarcinoma
2 Growth in culture:
Monolayer epithelial cells
3 Differentiation:
21 days after confluence in standard
culture medium
4
Morphology:
Polarised cells, with tight junctions, apical,
brush border

4. 4Part 1 1.2 Advantages of Caco-2 Monolayer
01
Spontaneously differentiate
to express morphological
and functional
characteristics of mature
small-intestinal enterocytes
02
Four times higher in
trans-epithelial
resistance compared to
HT 29-cell monolayer 03
Expressing various drug
metabolizing enzymes
such as aminopeptidase,
esterase, and sulfatase.

5. 5Part 1 1.3 Limitation of Caco-2 Monolayer
Limitation
of Caco-2
P-gp
LC/MS
Time
Cost
Enzyme
Mucus
Tissue

6. 6Part 1 2.1 Caco-2 Cell Culture Materials
1
2
Caco-2 Culture Media
Transport Media
DMEM (with L-glutamine, without sodium
pyruvate)+FBS (10%)+Non essential amino acids
(1%)+D-glucose (4.5g/L)+Penicillin
(10000U/mL)+Streptomycin (10mg/mL)
DMEM (with L-glutamine, phenol red, without sodium
pyruvate)+HEPES (4.76g/L)+NaCl (1.987g/L)+D-
glucose (4.5g/L)

7. 7Part 1 2.2 Cultivation of Caco-2 Cell Monolayers
Trypsinize Caco-2 cells and spin down the cellsTrypsinize
Seed
Incubate
Differentiate
Place the desired number of filters and seed by dispensing
0.5 mL of the re-suspended cell solution on each filter.
Fill the basolateral chamber with transport media and
incubate the plate with the filter supports.
Media should be changed on days 4, 8, 12, 16 and 18 and
cells will be fully polarized by day 21.
1
2
3
4

8. 8Part 1 3.1 Measuring TEER
01
Washing the cell
monolayer with
37℃ tempered D-
PBS (with Ca2+,
Mg2+)
Transport media
was added into
the apical and the
basal chamber.
Allow for
equilibrium for 60
min in the cell
culture incubator.
After
measurement,
Caco-2
monolayer with
TEER values
exceeding 250 Ω
x cm2 were used
for transport
experiments.
Background
TEER may be
recorded in wells
without cell
monolayers, and
can be
subtracted from
the raw TEER
values with cells.
02 03 04

9. 9Part 1 4.1 Caco-2 Permeability Assay Procedure
A B C D
Wash
Wash the
monolayer with
sterile HBSS, pH7.4,
and transfer the
filter plate to a 12-
well transport
analysis plate.
Add analytes
Add the test
compounds to the
filter well. Drug
concentrations
typically ranging
from 10 μm to 200
μm.
Incubate
Join the filter and
receiver plates once
all drugs and buffer
have been added.
Incubate at 37℃
shaking at 60 rpm
on a rotary shaker.
For analysis
At the end of the
incubation, remove
a fixed volume
(typically 50-100 μL)
directly from the
apical and
basolateral wells to
a clean plate.

10. 10Part 1 5.1 Papp Calculation
Papp=
𝒅𝑸
𝒅𝒕
𝟏
𝑨·𝑪 𝟎
𝒅𝑸
𝒅𝒕
C0
A
Transport rate over time
Area of Caco-2 covered filter
Initial concentration in the
donor chamber

11. THANK YOU
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