MilliporeSigma's Toni Steiner recently presented a poster at the 2016 AAPS/ITC Transporter Workshop demonstrating how culture conditions can influence drug transporter expression and activity in renal proximal tubule epithelial cells.

1. SA7K, a pseudo immortalized renal proximal epithelial tubule cell line was developed
using Sigma’s Zinc Finger nuclease technology. The SA7K cell line was cultured using
different extracellular matrices. Cell morphology was assessed for formation of tubule
like structures. Gene expression of key uptake and efflux transporters was determined
by qRT-PCR. Uptake of fluorescent substrates for key transporters was visualized using
fluorescence microscopy, while uptake and efflux of nonfluorescent substrates was
assessed by LC/MS following incubation +/- inhibitors.
Materials:
SA7K cell line, RPTEC Complete and Tox supplements were purchased from Sigma
Aldrich (St. Louis). Extracellular matrix products were purchased from BD Bioscience
(San Jose, CA). Substrates (except YM155 - Cayman Chemical, Ann Arbor, MI),
inhibitors, solvents and buffers were purchased from Sigma Aldrich (St. Louis).
Culture Conditions:
SA7K cells were thawed and resuspended in RPTEC Tox Medium and seeded at 125,000
cells per well with or without extracellular matrices. After 2 days in culture, medium
was changed to RPTEC Complete Medium. Medium was changed every 2-3 days until
assay day. For fluorescent imaging, SA7K cells were plated on ibidi µslides using same
culturing conditions. Uptake assay was performed and live cells were visualized using
Nikon fluorescent scope.
Transporter Assays:
Cells were thawed as above and plated in 24 well plates. On day of uptake assay, cells
were rinsed with 37C HBSS x 3. Vehicle +/- inhibitor was added and incubated for 10
minutes at 37C shaking. After incubation, substrate +/- inhibitor was added to wells
and incubated for 10 minutes at 37C with shaking. Cold HBSS was added to stop
reaction. Methanol (0.25mL) was added to each well and the plate placed on a shaker
at room temperature for 20 minutes. After incubation, 0.2mL of the extract was
transferred to a deep well plate and dried under nitrogen. Once dried, 0.125mL of
50% acetonitrile was added. Samples were analyzed by LC-MS/MS. For fluorescent
assays, cells were lysed with RIPA buffer after reaction stopped. Lysate was added to
black well plate and measured at appropriate wavelengths.
OAT Substrates and Inhibitors:
10µM of PAH +/- 10µM or 25µM Probenecid
50µM of 6 carboxyfluorescein +/- 10µM PAH or 10µM Probenecid
50µM Lucifer Yellow
OCT Substrates and Inhibitors:
10µM Amantadine +/- 10µM Doxepin or 10µM Verapamil
10µM MPP+ +/- 10µM Doxepin or 10µM Verapamil
10µM TEA +/- 10µM Doxepin or 10µM Verapamil
10µM YM155 +/- 10µM Doxepin or 10µM Verapamil
METHODS
Culture of Renal Proximal Tubule Epithelial Cell Line SA7K Using Extracellular
Matrices Improved the Functionality of Key Drug Transporters
Toni Steiner, Michael Perez, Andrey Samsonov, Dmitry Malkov, Maureen Bourner and David Thompson
M1031
• Induction of key drug transporter genes was observed
using ECMs. However, the OAT expression was not
detected in either the absence or presence of ECMs.
• Extracellular matrices used to culture SA7K cells
induced morphological changes to a more tubule like
structure. This tubule like structure became more
advanced after 7 days in culture.
• Renal proximal tubule epithelial cells cultured in
optimized extracellular matrix conditions improved the
functionality of key drug transporters by creating a
more in-vivo like phenotype.
CONCLUSIONS
Use of extracellular matrices has been shown to create an in-vivo like environment. In
the presence of extracellular matrices, epithelial cells form cell to cell and cell to matrix
contacts. Formation of these contacts drives differentiation to a tubule like structure
and polarization of drug transporter proteins to the surface of apical and basolateral cell
membranes. The objective of this study was to optimize the functionality of drug
transporters by culturing renal proximal tubule epithelial cells in different extracellular
matrices.
PURPOSE
Gene Expression of Key Drug Transporters
Formation of tubule like structures was observed using optimized extracellular matrix conditions. Uptake and efflux of an OAT fluorescent substrate into the lumen of tubule like structure was demonstrated by fluorescent
imaging. Gene expression analysis by qRT-PCR of the following drug transporters showed a fold increase of approximately 20x MATE1, 50x MATE2K, 5x OCT2, 2x MRP2, and 1.5x MRP4 using optimized culture conditions. An
increase ≥ 50% in uptake of PAH and TEA substrates by OAT and OCT transporters, respectively, was observed when compared to cells cultured without optimized extracellular matrix conditions.
RESULTS
OCT Substrates OAT Substrates
Uptake of Lucifer Yellow
A B C D
40X images of tubule like structures formed after 10 days in optimized ECM cultures (A)
Brightfield images of SA7K cells (B) DAPI staining (C) Lucifer yellow substrate staining
(D) Merged DAPI and Lucifer Yellow images.
Drug Transporter Uptake Results
Morphology of SA7K Cells +/- ECM
No ECM Day 7 + ECM Day 1
+ ECM Day 4 + ECM Day 7
Special thanks to Jim Blasberg (LC-MS/MS), Kelly Keys
and Lillian Vickery (FACS).
ACKNOWLEDGMENTS