DATA QUALITY IS NOT AFFECTED BY C E L L
S U S P E N S I O N D E N S I T Y O N Q PAT C H 1 6
l
HERVØR L. OLSEN
l
M I C H A E L S T O LT E N B O R G
|
MORTEN SUNESEN
WHOLE-CELL CURRENT TRACES
For the tests we used two expression systems: (1)
CHO cells expressing hERG potassium channels, and
(2) HEK cells expressing Nav1.2a sodium channels.
We used test compounds with very different oilwater partition coefficient (log(P)): astemizole
and verapamil with log(P) values of 6.43 and 3.45,
respectively, (source: www.syrres.com/esc/physdemo) for hERG assays, and tetrodotoxin (TTX) with
log(P) of -6.21 for the Nav1.2a assay.
Cell positioned
on patch clamp
site
MEAS
Silicon chip
Capillary
stop
Figure 1.
Schematic transection of
the QPlateTM The targeted
cell is positioned on top of
the patch clamp hole in the
silicon chip. Throughout
the extracellular (upper)
flow channel cells are shown
sticking to the floor of the
flow channel. Electrodes
are indicated in red.
7
16
TTX log(P) = -6.21.
N = 4-13 (average: 8.4)
400
300
200
100
0
1
2
4
8
14
x 106 cells
5
4
3
2
1
0
1
2
4
8
14
12
Figure 5.
The cell positioning time
was reduced similarly at
higher cell suspension
densities when using CHO
(left) and HEK (right) cells
in suspension.
25
20
15
10
80
70
60
50
40
30
20
10
0
4
6
x 10 cells
6
4
2
0,5
1,5
3
7
16
8
14
10
Astemizole log(P)=6.43.
N = 6-16 (average: 9.7)
CONCLUSION
100
90
80
70
60
50
40
30
20
10
0
0,5
6,5
x 106 cells
x 106 cells
90
2
8
3
0
E X P E R I M E N TA L S U C C E S S R AT E S
1
10
0
5
x 106 cells
100
Verapamil log(P) = 3.45.
N = 4-10 (average: 7.0)
14
CELL POSITIONING TIMES
Surplus cell attached
to flow channel floor
Figure 2.
Graphic representation of
voltage protocols used for
hERG (left) and Nav1.2a
(right) as viewed with the
QPatch assay software.
3
600
Data analysis. Recorded ion channel whole-cell currents were stored
in an integrated database (Oracle). Drug effects (IC50) were analysed
as a function of concentration with the QPatch assay software.
Waste
reservoir
1,5
x 106 cells
Compounds. Astemizole (Sigma, Buchs, Switzerland) was employed
in four concentrations from 1.9 to 50 nM in three-fold increments.
Verapamil (Sigma) was employed in four concentrations from 0.03
to 30 µM in ten-fold increments. Tetrodotoxin (Alomone Labs.,
Jerusalem, Israel) was employed in concentrations from 0.5 to
500 nM in ten-fold increments.
REF
Intracellular
flow channel
10
500
Average positioning time (s)
Extracellular
flow channel
15
0,5
Figure 4.
Concentration-response
relationships for TTX (top
left), verapamil (top right)
and astemizole (bottom)
as viewed with the QPatch
assay software. Green
vertical lines indicate IC50.
Completed experiments (%)
Inlet to
extracellular
flow channel
20
C O N C E N T R AT I O N - R E S P O N S E R E L AT I O N S H I P S
Completed experiments (%)
Inlet to
intracellular
flow channel
25
0
Average positioning time (s)
Ringer’s solutions.
For CHO-hERG the following solutions were used:
Extracellular (mM): 2 CaCl2, 1 MgCl2, 10 HEPES, 4 KCl, 145 NaCl, 10
Glucose, pH=7.4 (NaOH), ~305 mOsm.
Intracellular (mM): 5.4 CaCl2, 1.75 MgCl2, KOH/EGTA 31.3/10, 10
HEPES, 120 KCl, 4 Na2-ATP, pH=7.2 (KOH), ~290 mOsm.
For HEK293-Nav1.2a the following solutions were used:
Extracellular (mM): 1 CaCl2, 1 MgCl2, 5 HEPES, 5 MES, 3 KCl, 140
NaCl, 0.1 CdCl2, 20 TEA-Cl, pH=7.3 (NaOH), ~320 mOsm.
Intracellular (mM): 135 CsF, 1/5 EGTA/CsOH, 10 HEPES, 10 NaCl,
pH=7.3 (CsOH), ~320 mOsm.
Figure 7.
No effect of cell suspension
density was observed for any of
the three compounds tested.
The figure depicts the results
for the compounds arranged
after increasing degree of
hydrophobicity.
5
M AT E R I A L S A N D M E T H O D S
Cells. Cultured HEK293 cells stably expressing Nav1.2a and CHO
cells stably expressing hERG potassium channels were used.
NIELS J. WILLUMSEN
30
IC50 (nM)
We have tested whether IC50 values determined
with the QPatch 16 automated patch-clamp system
are dependent on the density of the applied cell
suspension. We assume that cell density determines
the number of cells present in the extracellular flow
channel.
It is predicted that increasing the cell suspension
density may reduce the time it takes to position a
cell on the patch-clamp site. The cell positioning
step is accomplished by suction. We tested the
effect of the cell suspension density using a wide
range of densities (from 0.5 to 16 million cells per
ml) on the cell positioning time.
SOPHION BIOSCIENCE, INC.
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USA
Phone: +1 732 745 0221
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I C 5 0 5 V A L U E D E T E R M I N AT I O N S
IC50 (nM)
When determining the potencies of drugs in singlecell assays it is a major concern that the employed
assay is affected by factors such as the density of
the applied cell suspension. It is envisaged that a
high density of cells may lead to titration of test
compound molecules (by receptor-binding and
adsorption) by cells in the vicinity of the targeted
cell. This would cause a rightward shift in the concentration-response relationship, and consequently,
incorrectly increased IC50 values.
Figure 3.
Characteristic hERG (left)
and Nav1.2a (right) wholecell currents as viewed with
the QPatch assay software.
Twelve individual current
traces are superimposed in
each panel.
|
IC50 (nM)
C H R I S M AT H E S
SOPHION BIOSCIENCE A/S
Baltorpvej 154
DK-2750 Ballerup
DENMARK
[email protected]
w w w. s o p h i o n . c o m
1,5
3
6
x 10 cells
7
16
Figure 6.
Variations in the cell suspension density affected
the number of successfully
completed experiments
similarly for CHO-hERG (left)
as well as for HEK-Nav1.2a
(right). The figures demonstrate that a cell density of
3-8 million cells per ml is
optimal.
The QPatch experiments presented here demonstrate that cell suspension density has a clear effect on
cell positioning time which is reduced with increasing density. The experimental success rate is also
affected, and it appears that the optimal density is 3-8 million cells per ml. It can be estimated that at
such densities, sedimented cells do not cover the flow channel floor area completely. This likely explains
why the experimental success rate is optimal in this density range. Finally, and most importantly, we
observed no effect of cell suspension density on the obtained IC50 values neither for very hydrophilic
(TTX) nor for very hydrophobic compounds (astemizole). Thus, no right-shift of the concentration-response
relationship is introduced by using high density cell suspensions (10-16 million cells per ml) in the
QPatch. This is likely a consequence of the small volume of the extracellular flow channel which leads
to sedimentation of few cells in the vicinity of the patch clamp site as compared to open-well systems