Note: Descriptions are shown in the official language in which they were submitted.
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IMPROVED INTERFACE PATCH CLAMPING
Introduction
The present invention provides a novel development of the
conventional patch clamp technique. This technique is
s referred to as the interface patch clamp method.
Voltage gated ion channels are potential targets for a
considerable range of novel treatments in a variety of
disease states. The development of the patch clamp technique
has provided a powerful method for the study of ion channel
to function and pharmacology in whole cells. However, while the
patch clamp technique provides a definitive method for the
investigation and screening of drugs with potential activity
on voltage gated ion channels, the technique is currently
highly dependent on the skill of the operator and tends to be
is very slow for drug screening. The present invention provides
a method for increasing the rate at which compounds may be
screened for ion channel blocking/agonist activity using the
patch clamp technique. The method can retain the essential
features of the conventional patch clamp recording system
2o while facilitating automation of the major time-consuming
components of the technique.
Background: Conventional Patch Clamp
The success of the patch clamp technique is derived from the
ability to form "tight" (i.e. high resistance: Giga Ohm)
2s electrical seals between an area of the cell membrane (the
Patch) and the tip of a pipette. The patch clamp pipette is
usually made from glass. The formation of the G-seal is
dependent on the profile of the top of the pipette, and is
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enhanced by the application of suction to the interior of the
pipette. The requirements for the formation of the G-seals
are well established and the process is usually monitored
electrically by display of the current pulse recorded in
response to a small voltage step applied throughout seal
formation. After formation of a G-seal, the area of membrane
under the pipette may be disrupted to obtain whole cell
voltage clamp recording mode.
The sequence of events leading to successful G-seal formation
to and whole cell recording mode using pre-formed patch pipettes
is as follows:
1. Selection of a suitable cell.
2. The patch pipette is positioned approximately 50 microns
above the cell.
3. The pipette is lowered until the cell surface is
deformed by the pipette tip.
4. Negative pressure is applied to the interior of the
pipette until a G-seal is formed between the pipette tip
and the cell membrane.
2o 5. Whole cell recording mode is established by the,
application of further negative pressure which disrupts
the cell membrane in the area under the pipette tip.
Steps two and three are slow and require considerable manual
dexterity and a high level of operator skill. Visualisation
of the cells and the patch pipette requires the use of a high
quality microscope and, in order to position the pipette, a
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high quality three axis micromanipulator with sub-micron
resolution in each axis is required.
Background: the Invention of PCT/GB99/04073 (WO 00/34776)
In its broadest terms WO 00/34776 provides for one or more
s cell or cells to be suspended in a liquid medium at a
liquid/air interface (by virtue of the effect of surface
tension at the interface) whereby the cell or cells are
accessible at the interface to a microstructure electrode
(such as a pipette tip) to which a cell can attach to form an
to electrical seal, for the purpose of whole cell voltage clamp
recording. According to the invention the electrode can be
caused to form a high resistance electrical seal with a cell
suspended in the liquid at the liquid/air interface without
the need to press the cell against a solid support surface.
15 Any body of liquid or column of liquid, which gives rise to a
situation in which a cell or cells are located in the liquid
at a liquid/air interface, can be used in the invention. For
example cells may be suspended in a column of liquid held by
surface tension in a capillary tube. Alternatively cells may
2o be suspended in a droplet of liquid, which droplet may itself
be suspended from or supported by a support.
It will readily be appreciate that the interface patch clamp
technique can be operated in "single cell mode", or could be
multiplexed to operate on a matrix of cells with multiple
2s electrodes.
According to one aspect of the invention, interface patching
can utilise a patch pipette of conventional type. Cells are
supported on a liquid/air interface at one end of a capillary
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tube (e. g. made of glass, polyethylene or other suitable
material). The axis of the patch pipette is in line with the
axis of the tube so that the pipette tip can be manipulated
into the opening of the tube where the cells are supported at
s the air/liquid interface. The capillary tube or the patch
pipette can be mounted onto a single axis manipulator. Only
one manipulator is required and this may be used to move
either the patch pipette or the capillary tube. Whole cell
recording mode is established as follows:
l0 6. A layer of cells is established at the interface between
the extracellular physiological solution (the liquid in
which the cells are suspended) and air by dipping the
capillary tube into a suspension of cells. The density
of cells in the suspension must be sufficient to provide
is a sufficient number of cells to form a layer of cells at
the interface.
7. Electrical contact with the extracellular solution is
established via a non-polarizable electrode (e.g. an
Ag/AgCl wire) and the tube is mounted either to a fixed
2o clamp or single axis manipulator.
8. A patch pipette is provided which can be filled with
electrolyte solution.
9. The patch pipette is mounted concentrically with the
capillary tube either via a single axis manipulator or
2s fixed clamp (if the capillary tube is to be moved). The
pipette filling solution is connected via the non-
polarizable electrode to the headstage of a conventional
patch clamp amplifier. The pipette holder allows
suction to be applied to the pipette interior.
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10. Cell attached patch mode of recording is established by
bringing the pipette tip in contact with the interface
by moving the pipette and the capillary tube
respectively together along the single mounting axis
(e.g. either by moving the pipette towards the tube and
interface or vice versa). On entry into the interface
the movement of the pipette and capillary tube together
is stopped and the pipette current is offset to zero on
the patch clamp amplifier. The resistance of the
to pipette increases when the pipette contacts one of the
cells at the air/liquid interface. Suction is then
applied to the interior of the pipette and the pipette
and capillary tube are moved closer together until the
pipette tip is located inside the capillary tube.
Initial seal formation between the pipette tip and
the cell may also~be assisted by the application of gentle
suction during entry of the pipette into the interface.
A G-seal is formed between the patch pipette tip and
the cell membrane by the application of further suction
zo to the interior of the pipette and monitoring the pipette
resistance.
11. Following the formation of cell attached patch mode, the
suction is released, pipette current is offset to zero
and a holding voltage applied to the pipette (e.g. -
2s 60mV) .
12. A whole cell recording is obtained by the application of
further suction to the pipette interior until the whole
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cell recording mode is established in conventional
manner.
According to this invention it is preferred that the
capillary tube should be mounted in an upright orientation
s (i.e. essentially vertically) with the air/liquid interface
at the downward end of the tube.
This has the advantage that suspended cells will tend to
"sediment" naturally to the downward end of the tube and be
collected there in a layer. The layer will preferably be
to several cells deep and loosely packed. Thus according to the
invention the pipette tip may be moved upwardly relative to
the air/liquid interface at the tube end (either by moving
the pipette or the tube along the single axis) so as to come
into contact with a cell in the layer at the interface. The
is relative density or concentration of cells at the interface
compared to the density in the bulk of the liquid in the tube
ensures a high probability that a cell can be collected on
the tip without the need for visualisation of the operation
and without the need for multidirectional manipulation of the
2o tip/cell positional relationship. Surprisingly it has been
found that G-seal formation between the cell and the pipette
can occur without pressing the cell against a solid
substrate.
Where the arrangement is intended to operate with the pipette
25 in an upright orientation (i.e. essentially vertically) with
the tip uppermost and pointing upwardly, the pipette should
be constructed so as to prevent the filling electrolyte
solution flowing out and being lost. This may be achieved
for example by use of a custom-made mounting assembly and/or
3o by shaping the pipette body to prevent loss of filling
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solution (e.g by bending the pipette shaft into a U- or J-
shape).
The invention also provides methods and apparatus employing
control logic to allow automation of a patch clamp system
s employing the Interface Patch Clamp technique described
herein. The logic described will control one or more
electromechanical micromanipulators/translators holding one
or more patch clamp pipettes and/or capillary tubes in order
to patch clamp cells and apply drugs/compounds in order to
to screen for activity on membrane ion channels. A major
advantage of the logic described is that automation is
achieved in this system by the use of feedback from signals
from the patch clamp amplifier and no image recognition
software is required.
15 It will be readily appreciated by those skilled in the art
from the teaching of WO 00/34776 that:
1. The stability of recording using the interface patch
clamp technique may be superior to that of conventional
patch clamping. The greater stability of interface
2o patch clamping is because the cell is held by the patch
pipette alone. In conventional patch clamp recordings
the cell is held by the path pipette and a solid
substrate and vibration tends to move the pipette
relative to the substrate causing loss of the G-seal.
2s The interface patch clamp is, in contrast to
conventional patch clamp apparatus, relatively
insensitive to vibration during drug application.
2. This method of drug application could be applied to a
plurality of recording pipettes/capillaries and form the
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basis for a high throughput electrophysiological assay
system. It will readily be appreciated that the
Interface Patch Clamp technique could be used with
multiple pipettes and multiple capillaries in a manner
s in which each pipette enters its respective aligned
capillary either individually in sequence or all
together. Although not currently preferred, a single
pipette could be used which is caused to enter more than
one capillary sequentially. Multiple patch clamp
to recordings could be made either sequentially or
simultaneously, depending on the application.
As was mentioned above, it is not essential to the general
principle of the invention to use a capillary in order to
create a column of liquid which gives rise to a liquid/air
is interface at which cells can be located. Other ways can be
envisaged in which the same effect can be achieved. For
example, a droplet or "blob" of liquid may be provided on a
support surface. The surface has a hole through it and the
droplet covers the hole. Surface tension prevents the liquid
2o from the droplet dropping through the hol3. Within the
droplet cells are suspended. This allows access to the
droplet and the cells contained therein by a suitable
electrode such as a patch pipette. Means may be provided for
flow of other liquids in to and out of a dish or other
zs container of which the support surface with the hole in it
forms a wall. Once a cell has been attached to the
electrode, other liquids may be introduced into the container
either in batch mode or in flow-through mode in order to
result in the cell being exposed at its external surface to
3o the surrounding liquid. Clearly in such an arrangement, the
original liquid and the remaining un-attached cells will tend
to be washed away from the area of the electrode/pipette.
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Droplets might be provided on non-perforate support surfaces.
The effect of surface tension may be to allow droplets of a
suitable liquid to adhere automatically to the underside of a
suitable support surface. The support surface might for
s example be a cover slip of glass or other material. Droplets
in which cells are suspended provide the air/liquid interface
and consequently may be used in a method of interface pathc
clamping as described above.
The arrangement allows for the formation of a matrix of cell
to suspensions so that multiple electrodes can be multiplexed to
take readings either simultaneously or sequentially (as well
as singly).
Tt will be appreciated by those skilled in the art that a
conventional glass "patch pipette" could be replaced by an
is equivalent electrode. The electrode might be either a single
region or a.matrix of regions on a sheet of material (such as
a silicon wafer) which incorporates a microstructure to which
a cell can be attached and which would provide the necessary
electrical connection. Microstructures could be etched on to
2o a silicon wafer (e. g. an oxidised silicon wafer), which
microstructures would be designed and adapted to be able to
capture a cell from the liquid/air interface of an
arrangement according to the present invention. Thus, the
performance and advantage of the invention is not limited to
z5 the currently preferred conventional glass patch pipette but
would include functionally equivalent means.
A drug in liquid solution can be applied to the cell in a
number of ways. For example the drug could be applied via
the capillary if the air interface is formed in a capillary
30 tube. Alternatively the drug can be applied by perfusion
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into a dish. Furthermore, perfusion could be achieved by
flowing the drug-containing liquid through a dish or
container.
A further arrangement for drug application is described in WO
s 00/34776. In this case the electrode (for example the patch
pipette) penetrates through the lower wall of a well. A
suspension of cells is loaded in to a capillary tube as
previously described. Attachment of a single cell to each
pipette tip follows, as described before. Once cells are
to attached to the pipette tips the capillary tubes containing
the remainder of the cells in suspension can be removed.
Subsequently, a drug solution is dispensed into each well and
patch clamp measurements can then be carried out on the cell
in the environment of the surrounding drug solution.
is Optimisation of Patch Clamping Conditions
Those skilled in the art will appreciate that within the
general teaching for the interface patch clamping method and
apparatus, it may be necessary to optimise certain conditions
for patch clamp measurements. For example the concentration
zo and packing density of cells in the suspension may need to be
optimised. Furthermore, the cells and/or solutions may be
temperature sensitive and an optimum temperature of operation
may need to be determined. Since the technique relies on the
formation of a liquid/air interface at which the cells are
z5 located, it may be necessary to optimise the osmolarity of
the suspending liquid medium in order to achieve the optimum
level of surface tension etc.
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Summary of the Invention
Where legally permissible the content of PCT/GB99/04073 (WO
00/34776) is explicitly incorporated herein by way of
reference.
The present invention can provide an improved method for
operating the Interface Patch Clamp technique generally
described in PCT/GB99/04073.
According to the present invention the Interface Patch Clamp
technique is modified in that the step of bringing the
to microstructure electrode (pipette tip) into contact with the
interface is achieved not by relative movement of the parts
of the equipment, but by applying a differential pressure
across the liquid/air interface to cause the meniscus to be
lowered and so to cause the surface of the liquid/air
interface to "bulge" towards and into contact with the
electrode.
The term "lower the meniscus" means that the radius of
curvature of the surface of the liquid droplet becomes
greater, and the droplet expands.
2o It will be appreciated that one or more mechanical
manipulator may be employed, according to this invention, to
bring the respective parts of the patch clamp equipment into
close proximity, provided that the final relative movement of
the electrode and the interface are cause by the applied
pressure differential. Alternatively, according to the
invention, a part of the equipment holding the electrode may
be mated with, attached to or held together with a part of
the equipment comprising the interface in any suitable
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arrangement allowing a small gap between the electrode and
interface in the absence of an applied pressure differential.
The term "pressure differential across the interface" means
that the hydrostatic pressure in the liquid phase (inside the
s droplet) differs from the air pressure ambient at the outer
surface of the meniscus. In order to cause a lowering of the
meniscus the internal liquid phase pressure is raised above
the external ambient air pressure. Alternatively the same
effect can be achieved by a relative lowering of the ambient
to air pressure. The meniscus surface level can be caused to
move towards and come into contact with the electrode pipette
tip by applying a (small) increase in air pressure (by any
suitable means) above the liquid in the capillary. The same
effect could be achieved by increasing the volume of liquid
is in the capillary or by driving the liquid down the tube (e. g.
by use of a piston or plunger).
It will be apparent that the invention could be performed by
a relative reduction in pressure around the capillary tip;
for example by mounting the capillary tip region in a sealed
2o housing having a controllable interior housing pressure.
As with the invention of the Interface Patch Clamp technique
described in PCT/GB99/04073, the present invention may be
employed to make single cell recordings, or may be applied to
arrays in which multiple single cells are attached to
2s multiple electrodes.
An advantage of the present invention is that once the
equipment has been set up with the interface near to the
electrode (e.g. by mechanical or physical manipulation and
positioning), the actual step of making contact with the
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interface (and hence with a cell) by the electrode involves
no moving parts and can be sensitively pressure-controlled.
This is especially useful for patch clamping in large arrays
for High Throughput Screening. Clearly, the technique can
s employ a means for overall pressure-control and may be
designed to allow pressure-control of individual elements of
an array.
It will be a matter for optimisation of any particular patch
clamp arrangement to set up the equipment so that the
to meniscus can be lowered enough to come into contact with the
electrode, without breaking the surface tension. Tt is
important that the meniscus stays intact during the initial
contact with the interface, up to the point at which a giga-
ohm-seal is formed with a cell. After that stage has been
15 successfully reached it is possible (as an additional
advantageous feature of the invention) that the relative
differential pressure may be maintained or further increased
so as to cause the remaining liquid (and remaining cells) in
the liquid phase to be ejected from the tube. This may
zo permit an easy way of replacing the first cell-containing
liquid with another liquid (e. g. containing an active
substance for testing/screening).
The invention is illustrated by way of example with reference
to the figures, in which:
25 Figure +1
Aerial view of plexiglass multiwell plate (207.) showing the
position of 1 of the 18 wells (202) .
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Figure +2
Side view of recording assembly showing positions of
multiwell plate (201), support stand (203), patch-clamp
headstage (204) and cell application assembly (205).
s Figure +3
Cross section of cell application system wherein cells are
applied via a disposable pipette tip (1) and recording
chamber (2) also showing position of recording pipette (3),
pipette holder (4), overflow channel (5) and earth wire
to access port ( 6 ) .
Figure +4
Cross section of an alternative cell application system where
cells are pipetted directly into chamber (7). Recording
chamber (2), recording pipette (3), pipette holder (4),
is overflow channel (5) and earth wire access port (6).
Figure +5
A) Cell applicator consisting of pressure line (8), earth
wire (9), suspension of cells (10), driven syringe in
starting position (11), syringe in active position (12),
2o stepper motor (13), serial communication line to computer
(14). B) Illustration of movement of meniscus from (15) to
(16) as pressure (P) is increased due to movement of driven
syringe from starting position (11) to active position (12).
Figure +6
25 Suction control system showing starting position of driven
syringe (11), active position of driven syringe (12), stepper
motor (13), serial communication line to computer (14) and
pressure/vacuum line to patch-pipette holder (8).
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Fiaure +7
Exemplary recording made using the pressure controlled
interface patch-clamp illustrated in Figures +1-6. The
configuration used for the cell applicator was as illustrated
in Figure +3. Kvl.l potassium channels were expressed in a
Chinese hamster ovary cells and recordings made using
standard patch-pipette filled with (in mM): 100Kgluconate,
20KC1, lCaCl2, lMgCl2, lOHEPES, 11EGTA-KOH, 5ATP-Naz, 2GSH, pH
7.2 and a cell bathing solution consisting of (in mM):
l0 140NaCl, 2.5KC1, 2MgClz, 2CaClz, 10HEPES, l0glucose, sucrose
to 320mOsm, pH7.4. Top: superimposed series of voltage steps
used to activate the Kvl.1 channel. Bottom: superimposed
whole-cell Kvl.1 currents recorded in response to voltage
steps.
Example
Mode of operation of Cell Applicator
1. The cell applicator comprises a suitably shaped adaptor
containing a length of small-bore tubing and a silver/silver
chloride reference electrode fashioned into an integrated
leakproof assembly. The other end of the tubing is connected
to a gas tight syringe which can be driven by a computer
controlled motor such as a stepper motor (Figure +5)
2. The tip of the cell applicator is placed into a suspension
of cells and a sample of said cells is aspirated by
withdrawal of the piston.
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3. The cell applicator is placed into position above and
concentric with the tip of the patch pipette (Figure +3).
Cell suspension can also be applied directly into a channel
in the top plate as in Figure +4.
4. The suction device is commanded to provide a small amount
of suction to the interior of the patch electrode.
Concomitantly, the piston in the cell applicator is advanced
manually or automatically in such a manner that the interface
of the cell-containing saline solution approaches slowly
to towards the patch pipette (Figure +5B). Automatic operation
of the cell applicator would be analogous to the suction
control operation below.
5. When contact between the patch pipette and the interface
is established (measured by the passage of current in the nA
range on passing a small voltage pulse), movement of the
interface is continued for a small distance to ensure that
the patch pipette is immersed in the cell suspension.
6. When the resistance measured between pipette and cell
suspension falls into the region 200M to 3G the whole cell
2o mode of recording is established by the application of a
greater degree of suction to the interior of the patch
pipette.
Mode of operation of suction control device
1. The suction device is computer controlled and utilises an
RS-232 serial communications protocol to operate the linear
motion stepper motor. Solenoid valve speed and distance of
stepper motor movements are established by sending
appropriate command strings from the computer to the
interface electronics assembly (Figure +6).
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2. On first use, the suction device is initialised by sending
commands to obtain the following sequence:
a) open the solenoid valve and to drive the stepper
motor until the end of travel of the piston air
s displacement system is reached.
b) close the solenoid valve and drive the stepper motor
to the first (lowest suction) position.
3. The degree of suction may subsequently be varied by
sending the appropriate command strings from the computer to
to the suction control device such that the air displacement
piston is moved to varying degrees from the position reached
at the end of the initialisation process.
