Qualification of Patch Ready Cells on a SyncroPatch 384PE
Recombinant cell lines that functionally express human cardiac ion channels are aCell LineGenesCHOKir2.1
KCNJ2
valuable tool for testing new drugs for potential side effects that induce
CHO KV4.3/KChip2
KCND3, KCNIP2
proarrhythmia. It can be difficult to maintain a constant quality of these cell lines inCHO KVLQT1/minKKCNQ1, KCNE1
a continually passaged culture making this process incompatible with routine
CHO hERG-DUO
KCNH2
screening in high-throughput mode. Here we demonstrate the preparation ofCHO NaV1.5-DUO
SCN5APatch Ready Cells prepared from five cell lines expressing recombinant ion chan- Tab. 1 CiPA Panel Cell Lines by B´SYSnels (B’SYS, Switzerland) which are recommended by the CiPA initiative for drug
safety testing. The Patch Ready Cells have been tested by automated patch-clamp played a high viability >90%, low amounton a SyncroPatch 384PE (Nanion, Germany) to demonstrate their applicability in of debris, and almost no aggregation(Fig. 1). The suspended cells display ahigh-throughput cardiotoxicity testing.
round shape, have a smooth surface andintroduction
adhere quickly within 48 hours (Fig. 2).
CHO Kir 2.1
CHO K 4.3/KChIP2The Comprehensive inautomated patch clamp
Vitro Proarrhythmia Assay
After thawing and resuspension in exter(CiPA) initiative employs
nal solution the Patch Ready Cells wereanalysis of a panel of cardiCHO Na 1.5-DUO
CHO K QLT1/mink
CHO hERG-DUO
directly tested on a SyncroPatch 384PE
ac ion channels known to(Nanion, Germany). Measurements werebe targeted by drugs reacquired either from single- or four-holesulting in heart failure.
chips in whole cell or perforated patch
The Swiss CRO B’SYS generclamp mode.
ated and validated recombi- Fig. 2: Patch Ready Cells after thawing (top left) and 48h.
After a good seal was established the ionnant cell lines which stably express ion hibit a strong and functional expression
channels were activated by individual
channels of the CiPA panel for safety of the ion channels and display a smoothvoltage protocols which were previously
pharmacology screening (Tab. 1). Opti- but durable cell membrane enabling audeveloped by Nanion. Specific ion chanmized for these cell lines, acCELLerate tomated patch clamp in high-throughput
nel blockers were added at different condeveloped a protocol to freeze the cells mode.
centrations simultaneously to individual
in a highly functional state. Instantly
preparation of cells
wells of the chip.
after thawing and without prior cultivaA vial of Patch Ready Cells wastion, these Patch Ready Cells (PRCs) exquickly thawed at 37°C in a water
bath: The cells were washed in 8ml
late
pre-warmed recovery buffer andpeak
centrifuged carefully at 80xg. The
lose cell pellet was resuspended instandard external solution and incubated for 30 minutes at room temperature. All cell lines recovered Fig. 3: Na 1.5-DUO Peak & late currents acVFig. 1: Viability of Patch Ready Cells
well from the frozen stock and dis- quired after blockage with Tetracaine.
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of the major bottlenecks is
the sufficient and on-time
supply of cells, which areclassically taken from a continuously passaged maintenance culture. Patch ReadyCells can be used reliably onautomated patch clamp devices designed for routine
high-throughput applications.
An average overall successrate of >80% was obtainedfrom all cell lines. The combiFig. 4: Concentration dependent blockage of currents acquired form hERG-DUO(A), KV4.3-KChip2 (B),nation of Patch Ready Cells
KVLQT7minK (C) and Kir2.1 (D) by specific inhibitors.
with the SyncroPatch 384PE
NaV1.5-DUO(Fig. 3)
rate of 86.5% >100MOhm. An average provide a versatile set-up to assess theThe CHO-NaV1.5 Patch Ready Cells dis- peak current of 2.5±1.5nA (n=269) was safety pharmacology of lead substances
played a good seal rate of 84.9% obtained.
early in the drug discovery process.
>500MOhm. An average peak current ofK LQT1/minK (Fig. 4C)-5.2±3.4nA (n=279) was obtained. Peak VChannel
Mode
Success Blocker / IC50
Measurements from the CHOand late currents could be acquired fromKvLQT1/minK Patch Ready NaV1.5 (peak) WC (1) 79.9 % Tetracaine: 7.7µM
the cells and blocked with Tetracaine
Cells were acquired in perfo- NaV1.5 (late) WC (1) 80.5 % Tetracaine: 0.52µM(IC50(peak) = 7.7µM / IC50(late) = 0.52µM).
rated mode using 4-hole chips. hERG-DUO
Perf. (4) 80.7 % Cisapride: 31.4nM
hERG-DUO(Fig. 4A)
The Patch Ready Cells disKV4.3-KChip2 WC (4) 84.1 % Quinidine: 36.9µM
The CHO-hERG-DUO Patch Ready Cells played a good seal rate ofKVLQT1/minK WC (4) 82.8 % HMR-1556: 101nM
were measured in perforated patch 82.8% >100MOhm. An average
WC (1) 81.0 % BaCl2: 5.0µM
mode to prevent current rundown. Be- current of 5.7±1.7nA (n=290) Kir2.1
Tab. 2: Overall success rate of blocking experiments
cause of the low average current ampli- was obtained.
performed with Patch Ready Cells.
tude of <100pA per cell, the cells were
CHO-Kir2.1 (Fig. 4D)
recorded on 4-hole chips where the sum
CHO-Kir2.1 Patch Ready Cells were meas- acknowledgementsof the currents of all four cells in eachured in whole cell mode using single hole • recombinant ion channel cell lines were
well is used. The Patch Ready Cells disprovided by B’SYS, Switzerland.
chips. The Patch Ready Cells displayed a • patch clamping experiments on the Synplayed a good seal rate of 81.2%
croPatch 384 PE were performed by Nanvery good seal rate of 85.2%>100MOhm. An average peak current ofion Technologies, Germany>500MOhm. An average current of
0.67±0.27nA (n=285) was obtained.
3.1±0.9nA (n=238) was obtained (Fig 4D). related products
PRC—Patch Ready Cells (5 million cells/vial)
KV4.3-KChip2 (Fig. 4B)
recovery & patch buffer included
discussion
Measurements from the CHO-KV4.3RE302
CHO Kir 2.1
Cost effective screening tests must be RE303
CHO KV4.3/KChip
KChip2 Patch Ready Cells were acquired
CHO KVLQT1/minK
developed to assess adverse effects of RE304
in whole cell mode using 4-hole chips.
RE305
CHO hERG-DUO
drug candidates as early as possible. One
The Patch Ready Cells displayed a seal
RE306
CHO NaV1.5-DUO