Dec 10, 2024

Public workspaceAssessing Intestinal and CNS Permeability with MDR1-MDCKII Cells

DOI
dx.doi.org/10.17504/protocols.io.n2bvjne6ngk5/v1
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DOI: dx.doi.org/10.17504/protocols.io.n2bvjne6ngk5/v1
Protocol Citation: Anzhela Rodnichenko, Yurii Kheilik 2024. Assessing Intestinal and CNS Permeability with MDR1-MDCKII Cells . protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjne6ngk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: August 12, 2024
Last Modified: December 10, 2024
Protocol Integer ID: 106087
Keywords: Caco-2 cells, MDR1-MDCKII cultures, TEER measurements, LC-MS/MS analysis, HPLC, Efflux ratio, ADMET, permeability
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Abstract
The protocol allows measurement of the rate of flux of a compound to identify intestinal or CNS permeability. The assay measures the rate of flux across Canine MDR1 Knockout, Human MDR1 Knockin MDCKII cells (MDR1-MDCKII) cell monolayers to identify intestinal or CNS permeability.

By measuring how quickly a compound passes through MDR1-MDCKII cell monolayers, researchers can estimate its intestinal permeability and predict its potential to reach the brain.

Interpreting Results:
  • High Permeability: A compound that readily crosses the MDR1-MDCKII monolayer is likely to be well-absorbed from the intestine and may have a high potential to reach the brain.
  • Low Permeability: A compound that does not readily cross the monolayer may have limited intestinal absorption or be actively pumped out of the brain by the MDR1 protein.
Guidelines
Introduction:

MDR1-MDCKII cells are often used instead of Caco-2 cells and tend to require a relatively short culture period (3–5 days) while displaying interpassage homogeneity and a high level of human P-gp expression. MDR1-MDCKII cells stably transfer and express the MDR1 gene (ABCB1) in MDCK cells, which encodes and expresses the efflux protein P-gp that causes multiple drug resistance. This protocol describes the permeability in the bidirectional permeability assay.

CALCULATION OF PERMEABILITY COEFFICIENT:

The apparent permeability coefficient (Papp, unit: cm s-1) is determined from the amount of compound transported per time; Papp is usually calculated according to the following equation:

Papp is expressed in 10-6cm/sec.



VA – volume of transport buffer in acceptor well,
Area – surface area of the insert (0.7 sq.cm)
Time – time of the assay,
[drug]acc – peak area of the test compound in acceptor well,
[drug]initial,d – initial peak area of the test compound in a donor well.


CALCULATION OF EFFLUX RATION:

Usually, perform transport experiments in both directions across the cell monolayers. The ratio between the two permeability coefficients obtained can then be used as a first indication of the involvement of an active transport process. If there is no clear difference between the permeability coefficients in the two directions, the transport might be passive. The Efflux ratio reveals the difference in Papp as a result of active transport.

Efflux ratio is calculated according to the following equation:

Papp(BA)/Papp(AB)
Note
The intestinal barrier is composed of multiple parallel transport processes including passive diffusion (transcellular and paracellular) and active diffusion (carrier-mediated absorption and carrier-mediated efflux). If the efflux ratio is less than 2, this indicates that the compound is passively transported. If the efflux ratio is greater than 2, this indicates the occurred active efflux.


CALCULATION OF MASS BALANCE:

The % recovery (mass balance) can be useful in interpreting the MDR1-MDCKII data. If the recovery is very low, this may indicate poor solubility, binding of the compound to the test plate materials, metabolism by the MDR1-MDCKII cells, or accumulation of the compound in the cell monolayer. The % recovery was calculated using the following equation:



Vacc – volume of compound solution in acceptor well (cm3),
Vd – volume of compound solution in donor well (cm3),
Cacc – peak area of test compound in acceptor well,
Cinitial,d – initial peak area of test compound in a donor well.

Note
The recovery should be kept within limited ranges in order to assure high-quality and accurate Papp values. Limits for recovery are between 80 and 120%.


Materials
Reagents and equipment:

  1. ReagentCelCulture® Incubator, CO2ESCO lifesciencesCatalog #CCL-170B-8
  2. Centrifuge 5804R (Eppendorf, USA)
  3. Etched Hemacytometer, dark line counting chamber (Hausser Scientific, USA; Cat# 3500)
  4. Innova 4080 Incubator Shaker (New Brunswick Scientific, USA)
  5. Millicell-ERS system ohm meter (Millipore, Cat# MERS 000 01)
  6. Gradient HPLC system Prominence UFLC XR (Shimadzu, Japan)
  7. VWR Membrane Nitrogen Generators N2-04-L1466, nitrogen purity 99%+ (VWR, USA)
  8. ReagentChannel Manual Pipette pipette.comCatalog #FA16-50R
  9. Multichannel Electronic Pipettes 2-125 µL, 5-250 µL, 15-1250 µL, Matrix (Thermo Scientific, USA)
  10. PIPETMAN pipettes 2-20 µl, 50-200 µl, 200-1000 µl (Gilson, USA)
  11. Water purification system Millipore Milli-Q Gradient A10 (Millipore, France)
  12. Opentrons OT-2 (Opentrons Labworks, USA)
  13. Triple quadrupole mass-detector API 3000 with TurboIonSpray Ion Source (AB Sciex, Canada) or Hybrid triple quadrupole/linear ion trap mass-detector API 4000 QTRAP with Turbo V ion source
  14. ReagentDMEM/HIGH with L-glutamineHyCloneCatalog #SH30003.04
  15. ReagentFetal Bovine SerumMerck MilliporeSigma (Sigma-Aldrich)Catalog #F7524
  16. ReagentSodium bicarbonateMerck MilliporeSigma (Sigma-Aldrich)Catalog #S5761
  17. ReagentPenicillin-StreptomycinMerck MilliporeSigma (Sigma-Aldrich)Catalog #P4333
  18. ReagentTrypsin-EDTA solutionMerck MilliporeSigma (Sigma-Aldrich)Catalog #T4174
  19. ReagentHEPESSanta CruzCatalog #SC-29097
  20. ReagentDPBS, powder, no calcium, no magnesiumThermo FisherCatalog #21600044
  21. ReagentHanks′ Balanced SaltsMerck MilliporeSigma (Sigma-Aldrich)Catalog #H4891
  22. ReagentMillicell® 24 well PlateMerck MilliporeSigma (Sigma-Aldrich)Catalog #PSRP010R5
  23. Reagent24 well Collection TrayMerck MilliporeSigma (Sigma-Aldrich)Catalog #PSMW010R5 )
  24. ReagentUltraCruz® Centrifuge Tube, Conical, 50 ml, bulkSanta CruzCatalog #sc-200251
  25. Serological Pipettes 5 ml, 10 ml, 25 ml (Greiner Bio-One)
  26. Disposable pipettor tips (Thermo Scientific, Fisherbrand, Eppendorf USA)
  27. ReagentMatrix™ Blank and Alphanumeric Storage TubesThermo Fisher ScientificCatalog #4140
  28. ReagentKetoprofenMerck MilliporeSigma (Sigma-Aldrich)Catalog #PHR1375
  29. ReagentAtenololMerck MilliporeSigma (Sigma-Aldrich)Catalog #74827
  30. ReagentQuinidineMerck MilliporeSigma (Sigma-Aldrich)Catalog #Q3625
  31. ReagentDimethyl sulfoxide (DMSO)Merck MilliporeSigma (Sigma-Aldrich)Catalog #34869
  32. ReagentAcetonitrileMerck MilliporeSigma (Sigma-Aldrich)Catalog #34851
  33. ReagentFormic Acid (FA)Merck MilliporeSigma (Sigma-Aldrich)Catalog #94318
  34. ReagentMethanol suitable for HPLC ≥99.9%Merck MilliporeSigma (Sigma-Aldrich)Catalog #34860
  35. ReagentHeptafluorobutyric acidMerck MilliporeSigma (Sigma-Aldrich)Catalog #52411
  36. ReagentInfinityLab Poroshell 120, EC-C18, 2.1 x 50 mmAgilent TechnologiesCatalog #699770-902 or Phenomenex Luna® C18 HPLC column, 2.1x50 mm, 5 µm (Cat# 5291-126) or ReagentZORBAX RR HILIC Plus Column, 2.1 x 50 mm, 3.5 µmAgilent TechnologiesCatalog #959743-901 or ReagentPursuit XRs 100Å C18, 2.0 x 50 mm, 5 µmAgilent TechnologiesCatalog #A6000050X020


Reagent Preparation:

  • Complete medium: DMEM high glucose (4500 mg/l) with L-glutamine 4 mM (584 mg/l) supplemented by 10% heat-innactivated FBS, 1% of non-essential amino acids, 10 mM HEPES buffer and 1% Penicillin/Streptomycin solution.

  • Trypsin/EDTA solution prepare from 10x stock solution by addition of DPBS.

  • Transport buffer preparation:
Hanks’ BSS (1x) without Ca & Mg, without Phenol Red, with NaHCO3 (final concentration 0.35 g liter) supplemented by MgSO4 (final concentration 0.81 mM), CaCl2 (final concentration 1.26 mM), HEPES (final concentration 25 mM).

  • 1M stocks solution:

  1. MgSO4 - Dissolve 120.37 g of MgSO4 (anhydrous) or 246 g of MgSO4x 7H2O to 1 liter of distilled H2O.
  2. CaCl2 – Dissolve 110.98 g of CaCl2 or 147,01 g of CaCl2x 2H2O to 1 liter of distilled H2O.
  3. HEPES - Dissolve 238.30 g of HEPES in 1 liter of distilled H2O.
  4. For preparation of 5 L Transport buffer add: 4.05 ml 1 M MgSO4, 6.3 ml 1M CaCl2, and 125 ml 1M HEPES. Adjust the pH to 7.4 and then filter sterile the solution using a 0.22 filter.


Safety warnings
Always wear appropriate PPE for this protocol
Refer to Material Safety Data Sheets for additional safety and handling information.
CULTIVATION OF THE MDR1-MDCKII CELLS BEFORE SEEDING ON THE FILTER
CULTIVATION OF THE MDR1-MDCKII CELLS BEFORE SEEDING ON THE FILTER
15m
15m
Trypsinize MDR1-MDCKII cells from maximally (90%) confluent MDR1-MDCKII cultures: remove the medium, rinse with DPBS (Amount10 mL per 75 cm2 flask) 2-3 times, decant DPBS and add the trypsin/EDTA solution (Amount3 mL per 75 cm2 flask); the remaining trypsin must wet the entire cell layer.
Incubate the flask at for Duration00:04:00 –Duration00:10:00 (use the shortest time possible) and check the detachment of the cells from the plastic surface by mildly knocking the sidewall of the flask with your palm.

  • As soon as the cells are detached, immediately stop trypsinization by resuspending the cells in a complete medium.

14m
Incubation
Culture MDR1-MDCKII cells in complete medium in 75 cm2 flasks to 80-90% confluence according to the humidified atmosphere at Temperature37 °C and 5% CO2.

Spin down the cells (Centrifigation1000 rpm, 00:05:00 ) and remove the supernatant.

5m
Centrifigation
Take an aliquot and count the cells.



Resuspend in the complete medium and seeded at a density 6-7x103 cells/cm2.

Note
After thawing a vial of MDR1-MDCKII cells from the stock, cultivate the cells for two passages before seeding cells on the filter support to stabilize the cell phenotype.

CULTIVATION OF THE MDR1-MDCKII CELLS ON FILTER SUPPORTS
CULTIVATION OF THE MDR1-MDCKII CELLS ON FILTER SUPPORTS
6h 15m
6h 15m

Note
Used MDR1-MDCKII cells of passage number 10-20.

Trypsinize MDR1-MDCKII cells from maximally (90%) confluent MDR1-MDCKII cultures: remove the medium, rinse with DPBS (Amount10 mL per 75 cm2 flask) 2-3 times, decant DPBS and add the trypsin/EDTA solution (Amount3 mL per 75 cm2 flask); the remaining trypsin must wet the entire cell layer..

Incubate the flask at Temperature37 °C for Duration00:04:00 –Duration00:10:00 (use the shortest time possible) and check the detachment of the cells from the plastic surface by mildly knocking the sidewall of the flask with your palm.

  • As soon as the cells are detached, immediately stop trypsinization by resuspending the cells in a complete medium.

10m
Incubation
Transfer the cells to a test tube and allow the large cell aggregates (if any, use 70-μm cell strainer) to sediment. Transfer the supernatant to a new test tube, take an aliquot, and count the cells.

Note
The percentage of dead cells must not exceed 5%.

Spin down the cells (Centrifigation1000 rpm, 00:05:00 ) and remove the supernatant.

5m
Centrifigation
Resuspend the cells in complete medium (at a concentration of 400x103 cells/ml).

Seed by dispensing Amount0.4 mL of the resuspended cell solution on each filter and Amount25 mL of prewarmed complete medium add to the feeder tray.

Note
Filters with a pore diameter of 3 µm are not recommended, as they allow the cells to crawl through the pores to the opposite side of the filter, resulting in a double monolayer.

Incubate the plate with the filter supports at Temperature37 °C and 5% CO2 in a humid atmosphere for Duration06:00:00 (if seeding is done in the morning) or DurationOvernight (16 h; if seeding is done at the end of the day).

6h
Incubation
Remove and replace the medium.

Note
This step is done to remove non-adherent cells and to reduce the risk of multilayer formation.

Maintain cells, every second day: aspirate the medium from the basolateral side and then carefully and slowly from the apical side (of all filters in a plate). Replace the aspirate medium with fresh, first in the apical compartments and then in the basolateral compartments.

Note
  • Do not touch the filter surface with the pipettes! Slow pipetting and avoiding physical contact between the pipette tip and the monolayer are essential for the maintenance of monolayer integrity.
  • Permeability testing performed 4 days post seeding.

DRUG TRANSPORT EXPERIMENT
DRUG TRANSPORT EXPERIMENT
1d 3h 10m
1d 3h 10m
Critical step: Change the culture medium Duration12:00:00 –Duration24:00:00 before the experiment.

Note
Longer periods without feeding before starting the experiment should be avoided because the cells may have consumed the essential nutrients and adapted to a more starved phenotype.

1d
Critical
Equilibration plates from Temperature37 °C to TemperatureRoom temperature at Duration00:20:00 -Duration00:30:00 .

Note
  • TEER measurements are temperature dependent. Ideally, TEER measurements should be conducted in an incubator at Temperature37 °C .
  • If TEER measurements are performed at TemperatureRoom temperature , it would be necessary to first equilibrate temperature before performing TEER measurements to avoid any temperature fluctuation-induced TEER changes.
  • Typically, equilibration from Temperature37 °C to TemperatureRoom temperature requires at least Duration00:20:00 .

30m
Measure the TEER values using a Millicell-ERS system ohm meter (Millipore, Cat# MERS 000-01).

One electrode is placed in the upper compartment and the other in the lower compartment, and the electrodes are separated by the cellular monolayer. For correction of the background resistance by subtracting the resistance value obtained with cell-free filters from the TEER of filters with MDR1-MDCKII monolayer.

  • The measurement procedure includes measuring the blank resistance (Rblank) of the semipermeable membrane only (without cells) and measuring the resistance across the cell layer on the semipermeable membrane (Rtotal).
  • The cell-specific resistance (Rtissue), in units of Ω, can be obtained as:
Rtissue (Ω) = Rtotal − Rblank
where resistance is inversely proportional to the effective area of the semipermeable membrane (Marea), which is reported in units of cm2.
  • TEER values are typically reported (TEERreported) in units of Ω cm2 and calculated as:
TEERreported = Rtissue (Ω) x Marea (cm2)

Note
Typically, TEER values > 100 Ω cm2 indicate adequate monolayer integrity.



Prepare donor solutions (containing the compound of interest) and all the reference compounds.

Note
Ketoprofen, Atenolol, and Quinidine used as reference compounds. Ketoprofen is a high permeability standard, Atenolol - is a low permeability standard, and Quinidine is a moderate-high permeability and undergoes active efflux.

Rinse with transport buffer 3 times the filter supports with the cell monolayers to remove residual medium by transferring the cell monolayers (medium decanted, not aspirated).

Wash
The MDR1-MDCKII cells pre-incubate in a transport buffer for Duration00:30:00 at Temperature37 °C in both apical and basolateral compartments.

  • Incubate the filter inserts under gentle shaking (orbital shaker Shaker100 rpm, 37°C, 00:30:00 ).

Note
Too vigorous shaking will affect the cell monolayer integrity.

1h
Incubation
Remove the washing solutions by decanting.

Note
Decanting instead of aspirating the washing solution reduces the risk of compromising the monolayers. Care must be taken to leave as little residual liquid as possible on the monolayers.

Apical-to-basolateral (A-B) transport experiments:

For to determine the rate of compounds transport in apical (A)-to-basolateral (B) direction, Amount300 µL of the test compound dissolved in transport buffer and add into the filter wells (apical compartment); Amount1000 µL of transport buffer add to transport analysis plate wells (basolateral compartment).

Basolateral-to-apical (B-A) transport experiments:

For to determine transport rates in the basolateral (B)-to-apical (A) direction, Amount1000 µL of the test compound solutions add into the wells of the transport analysis plate (basolateral compartment), the wells in the filter plate fill with Amount300 µL of buffer (apical compartment).

Note
The final amount of test and reference compounds Concentration10 micromolar (µM) .

The plates incubate for Duration01:30:00 at Temperature37 °C under continuous shaking at Shaker100 rpm .

  • Then Amount75 µL aliquots taken from the donor and receiver compartments for LC-MS/MS analysis.
  • All samples mix with 2 volumes of acetonitrile followed by protein sedimentation by centrifuging at.
  • Supernatants are analysed using the High-performance liquid chromatography (HPLC) system coupled with a tandem mass spectrometer.

Note
All solutions of test and reference compounds were prepared manually, and further manipulations with the solutions were performed with automation using Opentrons OT-2.

1h 30m
Incubation
Centrifigation
CALCULATION OF PERMEABILITY COEFFICIENT
CALCULATION OF PERMEABILITY COEFFICIENT
The apparent permeability coefficient (Papp, unit: cm s-1) is determined from the amount of compound transported per time;
Papp is usually calculated according to the following equation:
Papp is expressed in 10-6cm/sec.





VA – volume of transport buffer in acceptor well,
Area – surface area of the insert (0.7 sq.cm)
Time – time of the assay,
[drug]acc – peak area of the test compound in acceptor well,
[drug]initial,d – initial peak area of the test compound in a donor well.

CALCULATION OF EFFLUX RATIO
CALCULATION OF EFFLUX RATIO
Usually, perform transport experiments in both directions across the cell monolayers. The ratio between the two permeability coefficients obtained can then be used as a first indication of the involvement of an active transport process. If there is no clear difference between the permeability coefficients in the two directions, the transport might be passive. The Efflux ratio reveals the difference in Papp as a result of active transport.
Efflux ratio is calculated according to the following equation:

Papp(BA)/Papp(AB)

Notes: The intestinal barrier is composed of multiple parallel transport processes including passive diffusion (transcellular and paracellular) and active diffusion (carrier-mediated absorption and carrier-mediated efflux). If the efflux ratio is less than 2, this indicates that the compound is passively transported. If the efflux ratio is greater than 2, this indicates the occurred active efflux.
CALCULATION OF MASS BALANCE
CALCULATION OF MASS BALANCE
The % recovery (mass balance) can be useful in interpreting the MDR1-MDCKII data. If the recovery is very low, this may indicate poor solubility, binding of the compound to the test plate materials, metabolism by the MDR1-MDCKII cells, or accumulation of the compound in the cell monolayer.

The % recovery was calculated using the following equation:




Vacc – volume of compound solution in acceptor well (cm3),
Vd – volume of compound solution in donor well (cm3),
Cacc – peak area of test compound in acceptor well,
Cinitial,d – initial peak area of test compound in a donor well.


Notes: The recovery should be kept within limited ranges in order to assure high-quality and accurate Papp values. Limits for recovery are between 80 and 120%.
Protocol references
1. Furubayashi T., Inoue D., Nishiyama N. et al. Comparison of Various Cell Lines and Three-Dimensional Mucociliary Tissue Model Systems to Estimate Drug Permeability Using an In Vitro Transport Study to Predict Nasal Drug Absorption in Rats. Pharmaceutics 2020; 12 (79). doi:10.3390/pharmaceutics12010079.

2. Kuteykin-Teplyakov K., Luna-Tortós C., Ambroziak K. and Löscher W. Differences in the expression of endogenous efflux transporters in MDR1-transfected versus wildtype cell lines affect P-glycoprotein mediated drug transport. British Journal of Pharmacology (2010), 160, 1453–1463.

3. Srinivasan B., Kolli A.R., Esch M.B., Abaci H.E., Shuler M.L., and Hickman J.J. TEER Measurement Techniques for In Vitro Barrier Model Systems. Journal of Laboratory Automation. 2015; 20(2): 107–126. DOI: 10.1177/2211068214561025.

4. Taub M.E., Podila L., Ely D., and Almeida I. Functional assessment of multiple p-glycoprotein (p-gp) probe substrates: influence of cell line and modulator concentration on p-gp activity. The American Society for Pharmacology and Experimental Therapeutics. 2005: 33 (11); 1679–1687.

5. Xiong Xiao-Hong, Huang Li-Hua, Zhong Yun-Ming et al. Absorption mechanism of oxymatrine in cultured Madin–Darby canine kidney cell monolayers. Pharmaceutical Biology. 2016: 54(10); 2168–2175. http://dx.doi.org/10.3109/13880209.2016.1149496.

6. Zhiying W., D.P., Ashaben P., D. K., and Ashim K. M. Influence of overexpression of efflux proteins on the function and gene expression of endogenous peptide transporters in MDR-transfected MDCKII cell lines. Int J Pharm. 2013 January 30; 441(0): 40–49. doi:10.1016/j.ijpharm.2012.12.011.

7.Mitchell E. Taub, Lalitha Podila, Diane Ely, and Iliana Almeida. Functional assessment of multiple p-glycoprotein (p-gp) probe substrates: influence of cell line and modulator concentration on P-gp activity. Drug metabolism and disposition. 2005. Vol. 33, No. 11, P.1679-1687.

8. Harwood M.D., Zettl K., Weinheimer M. et al. Interlaboratory Variability in the Madin−Darby Canine Kidney Cell Proteome. Mol. Pharmaceutics. 2023. Vol.20. P. 3505-3518.