General Step Procedure

Preparation of Caco-2 cells for
cell seeding
• Remove the T75 flask from the incubator
• Place the T75 flasks in the BSC
• Remove the media from T75 flask by suction (Caution: Since the cells are attached to the base of the
flask, pipet out the media by tilting the flask and careful not to touch the tip of the pasteur pipet with the
base of the flask)
• Add 10mL PBS to the cells slowly.
• Remove PBS.
• Add 2 mL trypsin
• Incubate for 5-10 minutes to dislodge the cells.
• Add 10mL media (DMEM + FBS + PenStrep)
• Remove media including cells and transfer to centrifuge tube.
• Dispose T75 flask.
• Centrifuge for 3 minutes at 1750 rpm.
• Get new T75 flask.
• Remove supernatant carefully.
• Place 10mL media to the new T75 flask
• Place 10mL media to the suspended cells in the centrifuge tube.
• Pipet up and down to dislodge cells.
• Get 10 µl and count cells using hemocytometer.
• Add Trypan blue
• Count the cells that have not been stained by Trypan blue in each quadrant
• Get the sum of the cells divided by 4
 Example: Total # of cells from the 4 sets = 1080
1. Divide 1080 by 4
1080/4 = 270
2. Multiply by 10,000 (104) è The final value is the number of viable cells/mL in the original cell
suspension (protocol in using hemocytometers)
270 x 104 cells/mL
3. Each well should contain 80,000 cells (0.08 x 106 cells) è optimized cell density
4. Since there are 24 wells in one plate, [(0.08 x 106 cells) x (24)]
1.92 million cells
5. Take an extra # of wells (make it 26)
[(0.08 x 106 cells) x (26)] = 2.08 million cells
6. To get the volume of cell suspension needed to have 2.08 million cells
2.7 million cells: 1 mL = 2.08 millions cells: x mL
0.770 mL or 770 µL
7. Total volume of mixture needed = 13 mL
26 wells x 0.5 mL = 13 mL (pipet 0.5 mL into each insert)
8. Get the volume of media (DMEM)
13 mL – 0.770 mL = 12.23 mL
9. Mix 0.770 mL of cell suspension and 12.23 mL of media in a centrifuge tube.
10. Vortex mixture.
11. Pipet 0.5 mL of the mixture into each insert.
Permeability Assay Example:

Prepare the drug solution of the High Permeability Internal Standard: Metoprolol tartrate
a. Calculation: MW: 684.81 g/mole
Dose: 100 mg; Volume of Water: 250 mL; Concentration: 0.4 mg/mL or 584 µM

To get 584 µM: 0.4 mg/mL x 1 g/1,000 mg x 1 mole/684.81 g x 1,000 mL/1 L x 1,000 millimole/1 mole =
0.584 mM

0.584 mM x 1,000 µM/1 mM = 584 µM

Cont… Area of Filter Support: 1.12 cm2

AP–BL plate
• Remove blank buffer (DMEM) from BL side and replace it with 1.5 mL HBSS
• Remove blank buffer from AP side (upper) and replace with 300 µL of drug solution
• Incubate at 37 °C
• Remove 100ul samples from BL side after 15 min
• Place the sample in empty microcentrifuge tubes for the 3 drugs and scintillation vial for mannitol
• Replace 100 µL HBSS into the BL side
• Incubate, wait for the next sampling point (after 30, 45, 60, 90, and 120 min)
• Mix manually.
• Take sample from the donor side (transwell insert or apical side) at 120 min.
• Store the samples at 4 °C until use
• Prewarm to 37 °C before use
• Analyze test compounds by HPLC. Analyze Mannitol by scintillation counting.

BL–AP plate
• Remove blank buffer (HBSS) from AP side and replace it with 300 µL HBSS
• Remove blank buffer from BL side and replace with 1.5mL of drug solution
• Incubate at 37 °C
• Remove 100ul samples from AP side after 15 min
• Place the sample in empty microcentrifuge tubes for the 3 drugs and scintillation vial for mannitol
• Replace 100 µL HBSS into the AP side
• Incubate, wait for the next sampling point (after 30, 45, 60, 90, and 120 min)
• Mix manually.
• Take sample from the donor side (basolateral side) at 120 min.
• Store the samples at 4 °C until use
• Pre-warm to 37 °C before use
• Analyze test compounds by HPLC. Analyze Mannitol by scintillation counting
• Generate pre- and post- Absorbance-Concentration Curves of the Standard solutions
• Record the areas of the curves generated by the 9 standard solutions
• Plot Peak Area vs Concentration (ug/mL) of the standard solutions
• Convert the concentration expressed in ug/mL to uM
• Get the average areas for each standard solution
• Plot Average Peak Area vs Concentration (uM)
 • Using the equation [Average Peak Area vs Concentration (uM)], calculate the fitted concentration by
finding for “x”
• Calculate the difference between expected/theoretical concentration and fitted concentration
Apparent Permeability • For each well and time point, record the areas generated by the test compound
Coefficient • Using the equation, calculate the concentration (uM) of the test compound in each well and time point
• Get the average concentration of the 3 wells for each time point
• Convert the concentration (uM) to umoles by multiplying the concentration (uM) by 0.0015 (L) è 1.5
mL is the volume of solution in the receiver (basolateral) compartment for the A à B transport
• Convert the concentration (uM) to umoles by multiplying the concentration (uM) by 0.0003 L è 300 µL
is the volume of solution in the receiver (apical) compartment for the B à A transport
• Get the average concentration (umoles) of the 3 wells for each time point
• Convert the time (min) to seconds
• Get the cumulative concentration (umoles) for well
• Get the average concentration (umoles) of the 3 wells for each time point
• Compute for the permeability coefficient of the test compound in each well è P = ((dm/dt)/(Area of well
x Concentration of API))
• A test drug substance is highly permeable if its Papp is greater or equal to the Papp of the high
permeability internal standard
Efflux Ratio • Calculate the efflux ratio by dividing the average permeability coefficient of the BL to AP by the AP to
BL direction. An efflux ratio greater than 2 means the API is an efflux transporter substrate.
Mass Balance • Determine the concentration in µM (after 120 min) and initial volume of the test compound (L) in the
donor side.
• Multiply the concentration (after 120 min) and initial volume of the test compound (L) in the donor side
èA
• Determine the concentration in µM (120 min) and initial volume of the test compound (L) in the receiver
side as well.
• Multiply the concentration (120 min) and initial volume of the test compound (L) in the receiver side as
well è B
• Determine the concentration in µM and initial volume of the test compound (L) in the donor side at time
0
• Multiply the concentration and initial volume of the test compound (L) in the donor side at time 0 è C
• Copy/rewrite the average concentrations (uM) of the test compound in each time point as well as the
volume (L) of sampled test compound
• Multiply the average concentrations of the test compound in each time point by the volume of sampled
test compound and get their sum è D
• Calculate % recovery for each direction by summing up (A, B, & D) divided by C x 100]