Monolayer Integrity and Drug Transport Guidelines

Millicell-24 Cell Culture Insert Plate

Millicell-24 cell culture insert plate is a 24-well general purpose device designed to support cell growth, attachment, differentiation, or other desired applications. The procedure described below details how to measure the formation of a differentiated cell monolayer and the rate of drug transport across the cell barrier. All procedures are designed to be carried out in a single device and, if desired, can be performed using automation for cell seeding, cell feeding, washing, and other experimental procedures.

Materials and Reagents

Millicell-24 cell culture insert plate (PCF or PET membrane ) — Millipore cat. nos. PSHT 010 R5, PSRP 010 R5
Millicell ERS System—Millipore cat. no. MERS 000 01
24-well receiver plate—Millipore cat. no. PSMW 010 R5
24-well feeder tray—Millipore cat. no. PSSW 010 R5
Lucifer Yellow, 100 µg/mL concentration
Hanks balanced salt solution (HBSS)
Radioactive drug transport:
- Wallac/Perkin Elmer 96-well flexible plate
- Microbeta® Trilux® Counter or equivalent
- Scintillation cocktail

Note: Although the following methods have been optimized for monolayer integrity and cell based drug transport on epithelial cell lines such as Caco-2 or MDCK, they can be applied to any applicable cell system.

Methods

Measurements of Monolayer Integrity

A. Trans-Epithelial Electrical Resistance (TEER)

At the end of the desired growth period, remove the plates from the incubator and allow them to equilibrate to room temperature (approximately 0.5 hours).
Measure the electrical resistance across the monolayer using the Millicell ERS system. Position the probe such that the shorter prong is immersed in the media inside the filter well and the longer prong is placed through the basolateral access hole into the media in the growth plate.

Record the electrical resistance for each well. Take care not to touch the filter during TEER measurements, as it can damage the cell monolayer.

Note: If applicable, background TEER may be recorded in wells without cell monolayers, and can be subtracted from the raw TEER values with cells.

B. Lucifer Yellow (LY) Rejection

Using the same methodology as when feeding, rinse the monolayer three times with 300 µL HBSS in the apical wells and 28 mL in the feeder tray.
Add 300 µL of LY solution to each well in the filter plate.
Add 600 µL HBSS to each well of a 24-well receiver tray.
Assemble the filter plate and 24-well receiver plate and incubate for 1–2 hours at 37°C.
Remove the filter plate from the receiver plate and place the receiver plate into a fluorescent plate reader. Determine the LY fluorescence using an excitation wavelength of 485 nm and an emission wavelength of 535 nm.
Calculate the percent of LY rejection across the cell monolayer by measuring fluorescence in the receiver plate as compared to an ‘equilibrium’ standard.

Note: The standard plate should consist of 4 wells with 600 µL HBSS (blank) and 4 wells with 200 µL LY (100 µg/mL) + 400 µL HBSS (equilibrium samples).

Cell Drug Transport Template (Four Compounds, Three Replicates)

Filter Plate (Apical) Template

300 µL Drug 1	300 µL Drug 1	300 µL Drug 1	300 µL Drug 2	300 µL Drug 2	300 µL Drug 2
300 µL HBSS	300 µL HBSS	300 µL HBSS	300 µL HBSS	300 µL HBSS	300 µL HBSS
300 µL Drug 3	300 µL Drug 3	300 µL Drug 3	300 µL Drug 4	300 µL Drug 4	300 µL Drug 4
300 µL HBSS	300 µL HBSS	300 µL HBSS	300 µL HBSS	300 µL HBSS	300 µL HBSS

Receiver Plate (Basolateral) Template

600 µL Drug 1	600 µL Drug 1	600 µL Drug 1	600 µL Drug 2	600 µL Drug 2	600 µL Drug 2
600 µL HBSS	600 µL HBSS	600 µL HBSS	600 µL HBSS	600 µL HBSS	600 µL HBSS
600 µL Drug 3	600 µL Drug 3	600 µL Drug 3	600 µL Drug 4	600 µL Drug 4	600 µL Drug 4
600 µL HBSS	600 µL HBSS	600 µL HBSS	600 µL HBSS	600 µL HBSS	600 µL HBSS

Calculate the LY rejection using the following equation:

Find the LY passage using this equation:

Example: If the measured values for each of these solutions were:

Then the percent LY passage would equal:

The calculated LY rejection would therefore be:

Note: The cell seeding protocol is commonly optimized by choosing the density that results in the highest average electrical resistance with the least variability (e.g., lowest CV) combined with the lowest LY passage. After the seeding density has been optimized, monolayer integrity can be tested using TEER reading, LY rejection, or transport of a paracellular drug compound such as atenolol or mannitol.

Note: We do not recommend performing LY rejection in tandem with drug transport as it may interfere with radiometric or LC/MS analysis. It is recommended to run LY post drug transport to assess the integrity of the monolayer.

C. Cell-based Drug Transport

After the desired cell growth period, remove the Millicell-24 plate from the incubator and determine the electrical resistance for each well (as described above). Wash the monolayer, exchanging the volume three times using sterile HBSS, pH 7.4. After washing, remove the buffer from the filter plate and feeder tray.
Transfer the filter plate to a 24-well transport analysis plate.
To determine the rate of drug transport in the apical to basolateral direction, add 300 µL of the test compounds to the filter well. Drug concentrations typically ranging from 10 µm to 200 µm may be used (achieve desired concentration using HBSS, pH 7.4 or an alternative buffer of desired pH).
Fill the wells of the 24-well receiver plate with 600 µL buffer.
To determine the rate of drug transport in the basolateral to apical direction, add 600 µL of the test compounds to the 24-well receiver plate.
Fill the filter wells (apical compartment) with 300 µL of buffer.
Combine the filter and receiver plates once all drugs and buffer have been added. Begin timing the experiment.
Incubate at 37°C shaking at 60 rpm on a rotary shaker. Typical incubation times are 1 to 2 hours. We recommend a 2-hour incubation for optimal results.
For LC/MS analysis: At the end of the incubation, remove a fixed volume (typically 50–100 µL) directly from the apical and basolateral wells (using the basolateral access holes) or by disassembling the plates. Transfer the volume to a clean plate.

For radiolabeled drug evaluation: Remove 25 µL or applicable volume from each compartment and transfer to a plate containing 100 µL of scintillation fluid. Mix, then determine the radioactivity per sample using a Multiwell Plate Scintillation Reader such as the Trilux from Perkin Elmer. Add 25 µL of your initial drug to 100 µL of scintillation fluid to obtain your standard counts.

Calculating Drug Transport Rates

The apparent permeability, in units of centimeter per second, can be calculated for Millicell-24 plate drug transport assays using the following equation.

Where:

VA=the volume in the acceptor well

Area=the surface area of the membrane (0.7 cm2 for Millicell-24 plates)

Time=the total transport time in seconds.

For radiolabeled drug transport experiments the CPM units obtained from the Trilux Multiwell Plate Scintillation Counter are used directly for the drug acceptor and initial concentrations such that the formula becomes:

Note: Caco-2 or MDCK monolayer differentiation is evaluated by the transport of compounds that are effluxed, such as digoxin and vinblastine. The (B to A)/(A to B) ratios are good measurements of expression and localization of P-glycoprotein (P-gp) to the apical plasma membrane. Optimization of seeding densities may also be assessed by monolayer differentiation.

Note: The growth, integrity and differentiation of the cell monolayers need to be carefully monitored when optimizing the assay for use in a drug transport analysis. Many factors may contribute to the assay variability. Note that the cell passage number and culture medium can influence how the cells perform on the Millicell-24 cell culture plate. These factors may cause a shift in the behavior of both tight junction formation and polarized expression of membrane proteins, as the cell passage number increases. How this will ultimately affect the measurement of drug transport rates needs to be carefully considered in the experimental design.

References

Artursson, P., Karlsson, J. (1991) Correlation between oral drug absorption in humans and apparent drug permeability coefficients in human intestinal epithelial (Caco-2) cells. Biochem. Biophys. Res. Comm. 175:880–85.
Artursson, P. (1990) Epithelial transport of drugs in cell culture. I: A model for studying the passive diffusion of drugs over intestinal absorptive (Caco-2) cells. J. Pharm. Sci. 79:476–82.
Artursson, P., Palm, K., and Luthman, K. (2001) Caco-2 monolayers in experimental and theoretical predictions of drug transport. Adv. Drug Deliv. Rev. 46:27–43.
Bailey, C.A., Bryla, P., Malick, A.W. (1996) The use of intestinal epithelial cell culture model, Caco-2, in pharmaceutical development. Adv. Drug Deliv. Rev. 22:85–103.
Arena, A.A., et.al. (2005) Development of a higher throughput, permeability model system using MDR1-transfected MDCK cells in 24- and 96-well formats. 2005 American Association of Pharmaceutical Sciences Annual Meeeting, Nashville, TN.

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