Assay Ready TRP-Channel Expressing Cells - a Flexible Tool toScreen for New Drug Candidates
Summary
Looking for new targets in pain and cancer therapy, the transient receptor potential chan-nels (TRP-channels) gained a lot of interest during the last decade. The ligand-gated calcium
channels play an important role in the perception of pain and temperature and are often
dysregulated in tumor tissues. They have become appealing targets for Drug Discovery. Recombinant cell lines which stably express different TRP-channels have been successfully
used for lead identification and compound profiling.
Assay ready cryopreserved aliquots prepared from these cell lines can be used instantly afterthawing without prior cultivation. Here, we demonstrate that Assay Ready Cells preparedfrom TRP-channel expressing cell lines resemble the pharmacology of cells from continuous
culture in different end-point assays. The cells were successfully qualified for plate-based
fluorescent calcium-flux assays and for recording of activated ion channel currents using
automated patch clamping.
Preparation of Assay Ready Cells

into cryovials at 10 million cells per vial.

Recombinant cell lines stably expressing

Cryopreservation was performed in a Cryomed 7452 (Thermo Scientific) controlled

TRPA1, TRPV1, TRPV3, TRPV4, and TRPM8
had been kindly provided by Assay.WorksGmbH (Regensburg).
The cells were expanded in T-Flasks andscaled-up to 10-layer CellSTACKs (Corning).
For the final harvest the cells were carefully
detached by Accutase (Innovative Cell Technologies) and resuspended in cryopreservation medium containing 5% DMSO. By using
a XSD-Biofill (Brooks Life Sciences) the cellsuspension was automatically dispensed

rate freezer using an optimized freezing
protocol.
Assay Ready Cells thawed for quality control
displayed a viability of greater than 95% andre-entered proliferation without a signifi-cant lag phase. 24 hours after seeding, themorphology of the Assay Ready Cells wascomparable to cells from a continuously
passaged culture (Fig. 1). No elevated levelsof debris or dead cells were observed.

Fig. 1: CHO-huTRPA1 Assay Ready Cells 24 hours
after seeding from a frozen aliquot.

Tab. 1: AC50 Values of Calcium Flux experiments
recorded on a FLIPR Tetra®, shown in figure 2.
AC50 [M]Channel
TRPV1
TRPV3

TRPV4
TRPA1
TRPM8

Compound

ARC

fresh

Capsaicine

3.23E-09

4.63E-09

BCTC

1.61E-09

3.70E-09

2-APB

5.34E-06

1.61E-05

Nimodipine

8.38E-06

2.97E-06

GSK1016790A

6.14E-10

1.21E-09

HC067047

5.72E-08

1.85E-07

AITC

1.01E-06

1.56E-06

A967079

4.80E-08

4.32E-08

Icilin1.04E-08

2.86E-08

AMTB

2.13E-06

1.72E-06

A1

B1

C1

D1

E1

A2

B2

C2

D2

E2Fig. 2: Dose dependent Calcium flux measured in TRP-channels. Assay Ready Cells (blue) and cells from a continuous culture (light blue) after activation(A1-E1) and subsequent inhibition (A2-E2). A: TRPV1, B: TRPV3, C: TRPV4, D: TRPA1, E: TRPM8EUROPEAN OFFICEUS OFFICE+49 (160) 987 577 56

please@accellerate.me+1 (732) 698 3404

acCELLerate GmbH
Osterfeldstraße 12-14
22529 Hamburg - Germany

www.accellerate.me

acCELLerate GmbH
1 Jill Court, Bldg. 16/10
Hillsborough, NJ 08844 - USA

To demonstrate that Assay Ready Cells canbe functionally used, immediately afterthawing, the cells were tested in different
applications and were compared with cells
from a continuous culture.

Calcium Flux in Assay Ready Cells
For a plate-based fluorescence assay detecting intracellular calcium flux, Assay Ready
Cells and cultured cells were seeded into
384-well plates at 10.000 cells per well andincubated at 37°C for 24 hours. Serial dilu-tions of pharmacological reference compounds were added to the cells and thecalcium signal was measured in a FLIPR Tetra® (Molecular Devices). All cell lines wereactivated by their respective ligand and theresponse was blocked by selective inhibitors
in a dose-depended manner. No significant
difference in potency or efficacy was observed between Assay Ready Cells and continuously cultured cells (Fig. 2). The experiments were conducted by Assay.Works.
Cation Currents in Assay Ready Cells
TRP-channel expressing Assay Ready Cells
were furthermore tested in automatedpatch clamp experiments.
CHO-huTRPA1 Assay Ready Cells were activated with increasing concentrations of
Carvacrol and subsequently blocked withthe specific inhibitor AM0902. Current traces of the activated calcium ion channel wereFig. 4: Cation current traces (A1-C1) and time courses of currents (A2-C2) for TRPV1 (A), TRPV3 (B) andTRPV4 (C) recorded on a Patchliner.

mode using 4-hole high resistance chips
applying a holding potential of -20mV. A

can become a bottleneck in the process.
Assay Ready Cells from recombinant cell

success rate of >90% was achieved on 4lines expressing different TRP-channels haveconsecutive chips. Carvacrol activated thebeen validated for the use in plate-based

cells with an EC50 of 308µM (n=355). The
response was inhibited by AM0902 with an
IC50 of 48nM (n=349) (Fig. 3).

Calcium flux assays as well as in automatedpatch clamp assays. No significant difference in the response to the receptor ligands

TRPV expressing Assay Ready Cells wereor specific inhibitors was observed whichassessed on a Patchliner (Nanion Technologies GmbH). The recordings were performed
in whole-cell mode using single-hole, medi-

makes the Assay Ready Cells to a reliable
and flexible tool in drug discovery.
Since Assay Ready Cells can be prepared andum resistance chips, applying a holding potential of -60mV and a voltage ramp from-100 to 100mV over 200ms. TRPV1 was activated with Capsaicin, TRPV3 with 2-APB andTRPV4 with GSK1016790A. TRPV1 wasstored in large homogeneous batches andeach batch can be prequalified for the particular application, cell culture dependent
variances can be substantially excluded fromthe assay which makes a screening more

blocked with Capsazepine, TRPV3 with Ruthenium Red and TRPV4 with GSK2193874.

precise and less prone to failures.

recorded on the SyncroPatch 384i (NanionAll Assay Ready Cells could be specificallyTechnologies GmbH) in perforated patch

activated, blocked, and patched with a success rate between 65% for TRPV3 and 89%AB

for TRPV1 (Fig 4). All patch clamp assays
were performed by Nanion Technologies.

Discussion
For the cell based discovery and profiling of
new drug candidates, the on-time supply of
Fig 3: Dose-dependent activation and blockage of
TRPA1 measured by automated patch clamping ona SyncroPatch 384i in CHO-huTRPA1 Assay ReadyCells.

Related Products
CHO-huTRPA1 Assay Ready Cells (Cat. AW210)
CHO-huTRPM8 Assay Ready Cells (Cat. AW220)

robust and reliable cells is of the essence.

CHO-huTRPV1 Assay Ready Cells (Cat. AW230)

Classically cells from a continuously pas-

CHO-huTRPV3 Assay Ready Cells (Cat. AW240)

saged maintenance culture are used which

CHO-huTRPV4 Assay Ready Cells (Cat. AW250)