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Caco 2 Cell Permeability Assay
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- 2. Contents Caco-2 Monolayer Caco-2 CellCulture Measuring TEER Assay Procedure Papp Calculation
- 3. 3Part 1 1.1Characteristics 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 1.2Advantages 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 1.3Limitation of Caco-2 Monolayer Limitation of Caco-2 P-gp LC/MS Time Cost Enzyme Mucus Tissue
- 6. 6Part 1 2.1Caco-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 2.2Cultivation 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 3.1Measuring 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 4.1Caco-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 5.1Papp 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
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