CA2334770C

CA2334770C - High throughput screen

Info

Publication number: CA2334770C
Application number: CA2334770A
Authority: CA
Inventors: David Geraint Owen; Nicholas Gerard Byrne
Original assignee: Xention Ltd
Current assignee: Xention Ltd
Priority date: 1998-06-12
Filing date: 1999-06-14
Publication date: 2011-04-12
Anticipated expiration: 2019-06-14
Also published as: EP1084410B3; PL201770B1; ATE428115T1; JP2002518678A; ATE302951T1; IL140199A0; US20110048939A1; US20150068925A1; US10006902B2; NO20006295D0; HUP0102915A3; US6936462B1; DK1084410T5; US20050221282A1; DE69940707D1; DK1621888T3; PL345272A1; US20160054298A9; US8759017B2; CA2334770A1

Abstract

The present invention relates to a structure comprising a biological membrane and a porous or perforated substrate, a biological membrane, a substrate, a high throughput screen, methods for production of the structure membrane and substrate, and a method for screening a large number of test compounds in a short period. More particularly it relates to a structure comprising a biological membrane adhered to a porous or perforated substrate, a biological membrane capable of adhering with high resistance seals to a substrate such as perforated glass and the ability to form sheets having predominantly an ion channel or transporter of interest, a high throughput screen for determining the effect of test compounds on ion channel or transporter activity, methods for manufacture of the structure, membrane and substrate, and a method for monitoring ion channel or transporter activity in a membrane.

Description

High Throughput Screen The present invention relates to a structure comprisng a biological membrane and a porous or perforated substrate, a biological membrane, a substrate, a high throughput screen, methods for production of the structure membrane and substrate, and a method for screening a large number of test compounds in a short period. More particularly it relates to a structure comprising a biological membrane adhered to a porous or perforated substrate, a biological membrane capable of adhering with high resistance seals to a substrate such as perforated glass and the ability to form sheets having predominantly an ion channel or transporter of interest, a high throughput screen for determining the effect of test compounds on ion channel or transporter activity, methods for manufacture of the structure, membrane and substrate, and a method for monitoring ion channel or transporter activity in a membrane.

Within the context of this specification the word "comprises" is taken to mean "includes" and is not intended o to mean "is limited to only".

Ion channels are transmemlbrane proteins which form pores in the membrane which allow ions ~io pass from one side to the other (Hille, 1992) The',' may show ion specificity, allowing specific ions to passively diffuse across a Membrane down their _lect_,CChemoCal g_ad-en ter. ~.-thong certain types of channels are on the average open all the time and at all physiological membrane potentials (so-called leak channels), many channels have `gates' which open -in response to a specific perturbation of the membrane.
Perturbations known to cause opening of ion channels include a change in the electric potential across the membrane (voltage-gated channels), mechanical stimulation (mechanically-gated. channels) or the binding of a signalling molecule (ligand-gated channels).

Transporters are proteins in the cell membrane which catalyse the movement of inorganic ions such as Na' and K` as well as organic molecules such as neurotransmitters as in the case of so-called re-uptake pumps, e.g. GABA, dopamine and glycine. Two distinguishing features of carriers versus pores are i) their kinetics-movement of ions via transporters is very much slower than the >106 ions per second that is encountered with ion channels and'.:,-ii) ion channels conduct down electrochemical gradients whereas transporters can 'pump' uphill i.e. against concentration gradients (Hille, 1992) The latter process is normally directly dependent upon energy being provided in a _tcichicmetric fashion.

The invention has application principally in the measurement of ion channel activity but also of transporters where these WO 99/66329 PCTiG B99/018 71 are e _ctr0 enic e ^ Na .. " ^?
y a i . a" glutamate transporter ;Brew & A.ttwe_l, _987) Ion channel activity has been studied using a technique referred to as "patch clamping" (Hamill et al, 1981).
According to this technique a small patch of cell membrane is generally isolated on the tip of a micropipette by pressing the tip against the membrane. It has been suggested that if a tight seal between the micropipette and the patch of membrane is established electric current may io pass through the micropipette only via ion channels in the patch of membrane. If this is achieved the activity of the ion channels and their effect on membrane 'potential, resistance and current may be monitored. If the electric potential across the membrane remains constant the current supplied to it is equal to the current flowing through ion channels in the membrane. If ion channels in the membrane close resistance of the membrane increases. If the current applied remains constant the increase of resistance is in direct proportion to an increase of electric potential across the membrane.

Many drugs are known to exert their effect by modulation of ion channels, but the development of novel compounds acting on them is hampered conside=abl'\,v by the difficulty of screening at h_.< h-thrcuahput rates for activity.

Conventional electronhysiological methods such as patch or voltage clamp technicues provide definitive mechanistic information but suffer from the problem that they are unsuited to the rapid screening of test compounds.

s W096/13721 describes apparatus for carrying out a patch clamp technique utilized in studying the effect of certain materials on ion transfer channels in biological tissue. It discloses patch clamp apparatus utilizing an autosampler, such as those utilized with HPLC apparatus, to provide a higher throughput than may be achieved by the conventional patch clamp technique. This apparatus suffers from the problems that it merely semi-automates the drug delivery system, not the patch clamp recording. It therefore suffers from the same limitations as traditional patch-clamping with respect to speed of processing compounds and can in no way be considered a high-throughput system. The system still requires linear processing (i.e. processing of data obtained for one cell after another). In direct contrast the invention described herein provides parallel processing and thus genuine high-throughput of compounds.

The term "biological membrane" used herein is taken to include artificial membranes such lipid bilayers and other membranes known to a person skilled in the arc.

The present invention addresses the problems associated the known screens and screer.'_nc methods.

In a first aspect the present invention provides a structur_ which comprises a biological membrane adhered with a high resistance seal to a porous or perforated substrate for use in a high through put screen wherein the biological membrane comprises an ion channel or transporter.

In a second aspect the invention provides a biological membrane for use in the structure which is capable of adhering to a substrate with a high resistance seal wherein each cell forms a tight junction with adjacent cells and expresses an ion channel which is localised in the cell membrane.

In a third aspect the invention provides a substrate for use in a high throughout screen which is perforated.

In a fourth aspect the invention provides a high throughput (HiT) screen for the detection and assay of test compounds with activity on voltage gated ion channels which comprises the biological meirnr_ane.

0 In a fifth aspect" the 1n.ventlon provides a method of manufacturing a structure comprising a biological membrane adhered with a high resistance seal to a perforated substrate which ccmprises the steps of selecting ,..
substrate, perforating it, introcucinc a biological membrane to the substrate and sealinc each pore with oiological membrane .

In a sixth aspect the invention provides a method of manufacturing the biological membrane which comprises the steps of selecting a cell type, evaluating it for ability to form contiguous layers of cells with tight junctions and for low to negligible numbers of voltage gated ion channels, culturing the cells on a substrate and ensuring that a contiguous layer of cells is grown.

In a seventh aspect the invention provides a method of manufacturing a perforated substrate which comprises the steps of shining a laser of preselected focal area, power or 1.5 time of exposure at a coverslip to perforate it. This method also may include the additional step of modification of the perforated area by exposure to plasma- and/or localised heating in order to attain the appropriate level of smoothness of the perforation(s) In an eighth aspect the_nvention provides a method of screening for the detection or assay of compounds with activity on ion channels which comprises the steps of placing a biological membrane which expresses ion channels of interest in contact with test compound in physiological solution or non-physioogical solution corr.--rising _ sc_vent such as d.met:hvl s,olphcxide and measuring the resistance õ-impedance of the biological membrane under the influence o:
test compound.

Preferably an embodiment of the biological membrane comprises cells having an ion channel or transporter which naturally resides in the cell membrane thereof, or it can be inserted by transfection with cDNA and/or cRNA encoding the ion channel or transporter. The invention thus has the to advantage that is permits studies of native channels or transporters where the precise subunit composition is unknown or indeed where the molecular identity is completely unknown (i.e. not yet cloned) but also heterologously-expressed cloned channels or transporters where the identity of the native channel or transporter is known or where precise knowledge is required of the interaction of compound structures and chemical moieties of the ion channel or transporter. Therefore the system is substantially more versatile then existing approaches which are insensitive and rely on getting h:_gh concentrations of cells (not always possible with neurones) and high signal to noise ratios which limits their use to only certain types of cells and ion channels.

Preferably an embodiment Or the biological membrane comprises a plurality of ion channels or transporters which are predominantly preselected' or. channe-s or transport _ of interest. This provides the invention with the advantace of permitting parallel screening of different channels potentially providing an even higher throughput of compounds.

More preferably an embodiment of the biological membrane comprises genetically engineered cells which have been engineered to predominantly express an ion channel or transporter.

io Preferably the ion channels are voltage gated ion channels.
Preferably an embodiment of the biological membrane comprises cells selected from the group which comprises HEK-293 cells, genetically modified Chinese hamster ovary (CHO) cells, primary neuronal tissue such as hippocampus, dorsal root ganglia, superior cervical ganglia etc.; skeletal muscle; smooth muscle; cardiac muscle; immune:_ cells;
epithelia; endothelia etc.

CHO cells and CHO sub-clones such as CHO-K1 and CHO-dhTr (also known as Dukx) have exceptionally low levels of endogenous ion channel exC= _ssion thus providing the a--'vantage o` having exc ilenc signal to noisy characteristics within a mam m alian cell environment.
Similarly, HEF:-293 (human embryonic kidney) cells exoress -ow levels of : a__'ve channels and provide a human expression `background'. Boti these expression systems are infinitely preferable to the well-used Xenopus oocvte technique where no: only are native channels and subunits abundant, but the amphibian cell environment differs in important ways from mammalian cells.

Preferably an embodiment of the biological membrane comprises ion channels having rapid activation and inactivation kinetics which existing methods of high-throughput screening can not resolve. Existing systems, therefore, average transient ion channel signals frequently over periods of many seconds. Channels inactivating with time-constants of the order of milliseconds and without a steady-state presence are effectively undetectable in such systems. The invention presented here however, has the advantage that it can easily resolve such kinetics just as traditional patch clamping does, but at high-throughput rates.

Preferably an embodiment of the biological membrane comprises ion channels which show specificity for ions selected from the grc'.:p which comprises sodium, potassium, calcium, chloride.

Preferably an em t) of the biological membrane comprises a contiguous laver of cells capable of adhering Wit a h;g.^. resistance S _al C substrates selected frcn ne group which comprises perfcrated class, elastcs, rubber, polytetraflurotethylene (PTc'E; / F/cass fabric and polyethylene tereohthalate (PET?) Preferably an em-todiment of the biological membrane comprises a pseudo-epithelium wherein one face of a contiguous layer of cells is permeabilized thereby providing access to the interior of the cells. This has the great advantage of providing the means for current and voltage-clamping which is not possible with any existing high-throughput screening system. Not only does this permit high time-resolution recording but it also provides the means to stimulate or activate voltage-gated ion channels in. a precise and controlled manner. For example, it is not necessary to alter the ionic composition e.g. by elevating K' to depolarize cells, which in itself can modulate the kinetics of ion channels (e.g. K- channels) and also obscure the activity of novel ligands by competition at ion~,.channel binding sites. This is a very great advantage over all existing systems. Permeabllization also allows the introduction to the cytosol of compounds that otherwise could not do so either by virtue of molecular weight or physicochemical characteristics.

Preferably an embodiment of the biological membrane comprises a contiguous layer ci cells which is permeabil.i ed by an antibiotic selected from the group which comprises amphotericin and nystatin.; or detergent selected from the croup which comprises digltonin and saoonin; or physical disruption using a high voltage field; or by enzymatic digestion of a part of the membrane using an appropriate enzyme.

An advantage of using high voltage fields to permeabilize the membrane (electropermeabilisation) is that such a technique can permeabilize the plasmamembrane while sparing smaller intracellular structures such as mitochondria and endoplasmic reticulum. The technique can also be controlled very precisely and would not necessitate a facility to exchange solutions in a lower chamber of the recording apparatus.

Preferably an embodiment of the substrate comprises a perforated covers]-_p.

Preferably an embodiment of the substrate has pores of diameters between 0.5um and 10um. More preferably the pores are of diameters between 14m and 7pm. More preferably the diameter is i-2um.

Preferably an embodiment of the substrate comprises a arid of pores of greater number than 4 but less than 10. This provides the advantage of a statistically acceptable number of pa_alle _ recordings (-.e. >4) in each eatment but small enouch that the ratio of pores to ce11s can be mac_ van.--shingly small and thus the probability that a core =s sealed with and therefore occluded by a cell extremely high.

Preferably an embodiment of the substrate according to the invention is manufactured of a material selected from the group which comprises glass, plastics, rubber, polytetraflurotethylene (PTFE) , PTFE/glass fabric and polyethylene terephthalate (PETP).

io Preferably an embodiment of the screen comprises:

a plurality of chambers, each having a permeable peripheral surface providing a substrate for the biological membrane;

a plurality of wells each capable of receiving a >5 chamber and a test compound in a physiological solution or non-physiological solution comprising dimethyl sulfoxide (DMSO) or other solvent;

a pluraof reference electrodes, at least one having electrical contact with each well;

a movable recording head carrying at yeast one recording electrode thus providing the basic requirement for automated recording of ion channel activity in a multiwell plate format;

means for measuring electrical resistance or impedance between the recording and reference electrodes; wherein electrical current may pass between the recording and reference electrodes through the permeable peripheral surface of each chamber only via ion channels or io transporters in the biological membrane.

Preferably an embodiment of the screen comprises wells which are provided by a multiwell plate. The advantage of this being that high throughput can be achieved using industry-standard components which can be processed using commercially available automated equipment and robotics.
Users will have the possibility of using their''-existing plate processing equipment. thus containing costs in establishing a state-of-the-art high-throughput electrophysiology screen.

Preferably an embodiment o= r_he screen comprises a perforated substrate for the biological membrane.

preferably a further embodiment of the screen comprises a structure or bioio;ical membrane described above having ion channels of interest in an array of droplets on a porous substrate. Preferably an array of miniature recording chambers is created by placing a `lid' incorporating recording electrodes over the matrix of droplets such that a meniscus of the droplet solution is established.
Preferably a test compound in electrically conducting solution is placed in at least one of the droplets or io applied via access ports in the `lid' and the resistance/
impedance (in current-clamp configuration) of the biological membrane or conductance (in voltage-clamp configuration) is measured under the influence of the test compound. An advantage of this approach is that sheets of substrate can be designed without the need to transfer pieces of substrate (e.g. discs) to multiwell plates and also obviates complex chamber design with seals, `0'- rings and the like. The invention can still accommodate addition of solutions and has an additional advantage of using very small vol=umes and thus small quantities of reagents and cells. Excellent insulation is afforded by the air gaps between adjacent droplets.

Preferably an embod-ment of the recording head comprises a single recording electrode capable of being moved to visit each chamber segu_ntietly. Mere preferably an embodiment of the recording head comprises a plurality of recording e_e trades arranged in a i Even more preferably the recording head comprises a o1'~ra'=ty of reccrdng electrodes arranged in a matrix. The advantage of znis conf_c'ura-ion is that simultaneous recording from all wells is possible via a data-acquisition multiplexing system.

Preferably an embodiment of the screen is capable of multiplexing up to 384 recording elements to a data acquisition system utilizing multiple voltage-clamp amplifiers. This has the advantage of providing extremely to high time resolution and effectively simultaneous measurement from all wells. This has the advantage of providing the TERM system with the potential to achieve throughput rates similar to the best possible for conventional fluorescence-based ligand-receptor binding is assays (>150,000 compounds per week).

Preferably an embodiment of the method of manufacturing the structure comprises the steps of simultaneously perforating a coverslip and sealing the acres with biological membrane.
This embodiment provides the advantage of eliminating steps 20 in the establishment of the final configuration, namely procedures required to opt_mise the probability of a cell sealing with pores in the perforated substrate. This nas the advantage of simplifying :he final product.

Preferably an embodiment of ..: e method of manufacturing the biological membrane includes the step of permeabilizing one surface of the coat gUOU3 layer of cells thereby roviding access to the interior of the cells. This has the great advantage of providing the means for current and voltage-clamping which is not possible with any existing high-throughput screening system. Not only does this permit high time-resolution recording but is also provides the means to stimulate or activate voltage-gated ion channels in a io precise and controlled manner. For example, it is not necessary to alter the ionic composition e.g. by elevating K' to depolarize cells, which in itself can modulate the kinetics of ion channels (e.g. K' channels) and also obscure the activity of novel ligands by competition at ion channel is binding sites. This is a very great advantage over all existing systems. Permeabilization also allows the introduction to the cytosol of compounds that otherwise could not do so either by virtue of molecular weight or physicochemical characteristics.

2c Preferably the permeabilization is carried out by the step of contacting the surface with an antibiotic selected from the group which comprises amphotericin and nystatin; or detergent selected from the group which comprises digitonin and saponin; or physic al disru'ptlon using a high voltage field; or by en=ymatic digestion of a part of the cell membrane using an appropriate enzyme.

Ar. advantage of using high. voltage f i elds tt permeab lI ze the membrane (electronermeabilisaticn) is t: at such a technique can perme:ab-l-ze the plasmamembran.e while s_)ar-ng smaller intracellular structures such as mitochondria and endoplamic reticulum. The technique can also be controlled very precisely and would not necessitate a facility to exchange solutions in a lower chamber of the recording apparatus.

Preferably an embodiment of the method of manufacturing the io biological membrane includes the steps of transfecting cells with cDNA or cRNA encoding an ion channel of interest and cloning cells expressing the ion channel of interest. These steps provide the invention with the advantage of permitting studies of heterologously expressed cloned channels where the identity of the native channel is known or where precise knowledge is required of the interaction of compound structures and chemical moieties of the ion channel.
Preferably an embodiment of the method of manufacturing the perforated substrate comprises the steps of adjusting the profile, taper or diameter of the pore with a laser.
Preferably the laser source controlled by an automated stage under control of a computer and inverted phase-contrast microscope which provides the advantage of - _3 -oermitting visual examination of the pore characteristics e.g. profile, taper and diameter.

Preferably an embodiment of the method of manufacturing the perforated substrate comprises other non-laser methods such as photo-etching, casting and physical piercing of the substrate.

Preferably an embodiment of the screening method comprises the step of measuring ion channel activity by monitoring trans-epithelial resistance measurements (TERM) across an io intact cell layer.

In a further embodiment of the screening method a surface of the contiguous cell layer is preferably permeabilized thereby providing access to the interior of the cells. This has the great advantage of providing the means for current and voltage-clamping which is not possible with any existing high-throughput screening system. Not only does this permit high time-resolution recording but is also provides the means to stimulate or activate voltage-gated ion channels in a precise and controlled manner. For example, it is not necessary to alter the ionic composition e.g. by elevating K- to depolarize cells, which in itself can modulate the kinetics of ion channels (e.g. F" channels) and also obscure the activity of novel ligands by competition at ion channel binding sites. T_'his is a very great advantage over all ex -J sting systems . Perm eat_li --atior. also al_ows the introduction to the cytosc_ of compounds that ot:-er-,r:se could not do so either b,,., virtue of molecular weight or physicochemical characteristics.

Preferably a surface of the contiguous cell layer is permeabilized by antibiotics selected from the group which comprises amphotericin and nystatin; or detergents selected from the group which comprises digitonin and saponin; or physical disruption using a high voltage field; cr by enzymatic digestion of a part of the membrane using an appropriate enzyme thereby permitting intracellular voltage or current measurements to be made.

An advantage of using high voltage fields to permeabilize the membrane (electropermeabilisation) is that such a technique can permeabilize the plasmamembrane while sparing smaller intracellular structures such as mitochondria and endoplasmic reticulum. The technique can also be controlled very precisely and does not necessitate a facility to exchange solutions in a lower chamber of the recording apparatus.

Preferably an embodiment of the invention prove cgs a screening method which includes the step of multiplexing up to 384 recordinc elements a data acquisition system utilizing multiple voltage-c-amp amplifiers. This has the WO 99/66329 PCT/C;B99/01871 advantage of providing extremely hick :me resolution and effectively simultaneous measurement from all wells. This has the advantage of providing the TERN system with the potential to achieve throughput rates similar to the best possible for conventional fluorescence-bas=_d ligand-receptor binding assays (>150,000 compounds per week) Preferably an embodiment of the method of screening for the detection or assay of compounds with activity on ion channels of interest in an array of droplets on a porous substrate. An array of miniature recording chambers may be created by placing a `lid' incorporating recording electrodes over the matrix of droplets such that a meniscus of droplet solution is established. A test compound in conducting solution is placed in at least one of the droplets or applied via access ports in the `lid' and the resistance of the biological membrane or conductance (in voltage-clamp configuration) is measured under the influence of the test compound.

In an alternative embodiment of the screening method the biological membrane is placed in a plurality of chambers and test compound in physiological solution, or non-physiological solution comprising a solvent ea dimethvl sulphoxide, is added to the c:ambers.

Preferably an embodiment of t-e screening method comprises the steps of drug delivery and washing of the multi-wel_ plate.

Preferably an embodiment of the screening method incorporates a step of stimulation of cells involving the use of a photoactivatible `ion scavenger' eg of ions such as K. The active entity can be released by flashing the entire plate at once with a high intensity light source eg a laser or Xenon lamp. The advantage of this system is that membrane potential. can be altered by altering the ionic distribution in a non-invasive fashion and with fine temporal control.

It has surprisingly been found that a biological membrane can be adhered with a high resistance seal to a perforated substrate for use in a high throughput screen for test compounds having activity on ion channels. This was considered unobvious to a person skilled in the art at the outset in view Of the fact that achievement of a high resistance seal has not been possible without an undue ~o burden. Furthermore, perforated substrates having a biological- membrane sealed thereto have not been suggested for use in high throughput screens.

It has surprisingly been found that a biological membrane capable of adhering with a h=ah resistance seal to a substrate may be constructed for use in a high throughout screen. Surprisingly it has beer. found that the biological membrane may be constructed having ion cnannels which are predominantly the ion channels of interest. Furthermore, it has surprisingly been found that a high throughput screen may be constructed and used to detect and assay a throughput of test compounds which may be in excess of 30000 per week.
Surprisingly the screen may be used to obtain bona :fide electro physiological data relating to functional ion channel activity.

The biological membrane of the invention was unobvious at the outset to a person skilled in the art. Construction of a biological membrane having high resistance seals with a substrate such as perforated glass had not been achieved and was not considered possible without an undue burden. In addition construction of a membrane having ion channels which are predominantly an ion channel of interest-had not been considered possible without an undue burden.

The high throughput screens and methods of the invention were unobvious at the outset to a person skilled in the art in view of the fact that it was not considered possible without an undue burden to screen the high throughput of test compounds which may be achieved by the invention.

In addition to the advantage of a high-throughput of test compounds, embodiments of the screen and method of the invention may provide functional assays (cf. say ligand binding) in which the mode of action (eg. blocking or enhancing) of the test compound on voltage gated ion channels is measured via changes in membrane resistance or by recording the current flowing through ion channels in the membrane directly.

The invention will now be described by reference to the following examples of preferred embodiments and accompanying drawings in which:

Figures 1A and 1B show an epithelial cell version of a screen according to an embodiment of the invention.
Figures 2A and 2B show an embodiment of the screen of the invention having a perforated substrate.

Figure 3 shows adaption of a commercially available multiwell plate for use in a screen according to an embodiment of the invention. The figure shows an integral multi-recording electrode head cluster.

Figure 4 shows an embodiment using a movable recording head wherein a single recording head reads single wells sequentially.

Figures 5a to 5e show an embodiment of a fluid matrix system wherein an array of miniature recording chambers are created by dispensing droplets on to the recording substrate in a pre-determined pattern and density. Figure (f) shows the full sandwich (recording configuration) of the system.

Figures 6a to 6d show a further embodiment of a fluid matrix system wherein multiple arrays of droplets are sandwiched together.

Figure 7 shows a pore formed in a substrate, according to the invention. The light micrograph shows a pore in a thin glass substrate. The pore, which was approximately 2 micrometers in diameter, was manufactured by using pulses of focussed laser energy followed by a combination of fire polishing and plasma modification. The scale bar is micrometers across.

Examples An embodiment of the screen of the invention comprises a multi well plate with integrated recording devices, by which means a massively parallel voltage clamp (MPVC) can be performed on a plurality of wells in a plate within short periods of time (ca. 1-60s). Preferably commercially available 96 or 384 well plates are employed, or the 96 or 384 position array format is adopted.

An embodiment of the screen of the invention preferably provides a throughout of test compounds in excess of 30,000 per week with bona =1de electrophvsiological 'read-out' of functional ion channel activity. An embodiment of the screen may provide high resolution both in terms of time;
for a 96 well plate, lms/point/voltage clamp setup. The sensitivity to modulation of channel activity is >10.

An embodiment of the present invention provides a method which comprises the steps of measuring transepithelial electrical resistance across a contiguous layer of cells expressing predominantly an ion channel of interest. It will be apparent to a person skilled in the art that the method depends on adherence of the cells to a substrate that allows formation of tight junctions between adjacent cells such that a high-resistance sheet is formed. Changes in activity of the ion channels expressed in the membranes of individual cells are reflected by changes in the resistance across the sheet as a whole. In a refinement ` of this embodiment of the invention, access to the interior of the cells comprising the sheet is obtained by means which permit population current and voltage clamp recording to be made.
ransepichellal resistance meal cements have been carried out as follows:

a) Epithelial/ endothelial type cells.

'J -The overall transecithelial resistance is composed of two principal components, the Sum of the whole-ce'll' conductances of the individual cells making-up the pseudo-epithelium and the sum of the intercellular conductance pathways.

Naturally-occurring epithelial (and endothelial) cells form tight junctions with one another. This tight packing reduces the leakage of ions/ solutes between cells, resulting in relatively low numbers of intercellular conductance pathways io across the membrane as a whole. Thus, where conditions permit tight junctions to form, changes in the cell transmembrane conductance are measureable. One approach to this method was to select a host cell with appropriate growth properties. Cell-types which also express native ion channels at low levels are considered suitable for expression of cloned ion channels. A large hurdle for a person skilled in the art in this approach lay in obtaining cells in which the latter is true.

b) Transepithelial resistance measurement in non-epithelial cells.

An alternative to the approach described above is to use non-epithelial cells trial are known to express negligible numbers of ion channels of their own as a basic expression system for cloned cells which express ion channels o-, WO 99/66329 PCT/C;B99/01871 choice. There a: several cell-types tact fulL_1-tI:_S
Cr--ter.Lon. Howeve:-, a large hurdle to a person skilled _n the ort was presented in that they do not rm conticuous layers of cells in culture with tight junctions. In an s embodiment of the invention there is provided a nigh resistance `epithelial' laver in which this hurdle has been overcome.

Transepithelial current measurement (massively parallel voltage clamp (MPVC) An embodiment of the invention was obtained by gaining access to the interior of cells in the `epithelium' by disrupting or permeabilizing one face of each cell contributing to the cell layer. This has been carried out by chemical methods, for example by allowing certain types of antibiotics (eg. amphotericin) or detergents (digitonin) to come into contact with one face of the cell surface or through physical disruption eg. using high voltage- fields (electroporation/ integral zapper).

Electrical recording Systems A number of svicei:.s have been developed and they are outlined below:

- 2L, -Pilot test systems For pilot testing of the integrity of pseudo-epithelial lavers, transepithelial resistance was measured using a chamber into which permeable tissue culture supports were inserted (Fig 1) . Cells were grown. in culture to confluencv on this support. In the case of perforated substrates, the material (eg. coverslip) was inserted in a purpose-built test rig which permitted variation in the pressure in the lower compartment and/ or upper chamber and at the same time allowed resistance measurements to be made (Fig 2) . To avoid polarization of the electrodes, an oscillating voltage signal was passed across the cell-layer via linear or circular electrodes positioned above and below the permeable support on which the laver of cells was growing and the impedance of the cell-layer was measured. In the case of permeabilized cell--layers (Figures lb and 2b) , voltage and current-clamp recording was carried out using a voltage-clamp amplifier interfaced with a compUte:r to control experimental protocols and sample the data.

Scale-ups and operational systems In either TEFNI or ?'-1PVC commercial screens ut_'1_:_e a multiwell plate measuring system (Fig 3) or equivalent (eg.
using a droplet matrix generated using a nano litre pieco-dispenser). 1'1:is was derived to some extent from the l t w eam s pi of t ric tut r u:~re.. ~:, e d_eigr. of an in oegral reccrdinc head of which embodimenos of the invention include a number of possl.bilities. They are described below.

i) single recording head which reads single wells sequentially (Figure 4).

ii) moveable linear row of recording heads (eg. 12 for a 96 well plate system; 24 for a 384 well system) which are moved across the plate in 8 (96 well) or 16 (384 well) steps.

iii) electrode matrix built into the plate with multiplexing for recording headstage & acquisition system. For larger density plates multiple voltage-clamps were used to maintain sampling frequency and therefore time resolution (Figure 3) iv) droplet system (Figure 5) Multiwell plate adaptation ?5 Embodiments of the screen of the present invention preferably include an integral automated pipettor in the working versions cf TERM and MPVC.

Preferably embodiments o the screen of the invention, include a facIls:v for washing recordlna heads between use in separate rows.

According to an e:nbodiment o 11vent_on the method manufacture of the biological me.. rane Com.prises the stems of obtaining a hog: resistance seal with per:orated glass substrate (or other support) and/ or the step of obtaining a cell-line having the ability to form sheets and having low or negligible numbers of native ion channels.

Epithelial cell approach Naturally occurring cell-lines and engineered cell-lines have been developed. They are described below:

Naturally-occurring cell-lines Cell-lines referred to in the literature have been evaluated for 'off the shelf' suitability. Initial candidates included ECV-304, RBE4 and C6 glioma cells. Criteria for use were:
a) ability to form contiguous lavers of cells with tight is junctions; transecithelial resistance of >-125S2cm-2.

b) low to negligible numbers of background voltage-gated ion channels as assessed by whole cell patch clamp by standard methods. Pre_`erab' the c ndu an, -'eve-11 or _2nS per cell.

Engineered cell-lines A suitable cell-line was prepared by molecular engineer.
~ng.
If background conductances of the naturally-occurring cell-lines were above the threshold given in (b) above, the cell-line was assessed for possible gene knock-out to engineer a novel host cell.

Artificial epithelia Perforated substrates Perforated substrates have been developed as set out below:
Laser-generated substrates a) Prototypes Glass coverslips were perforated in a sequential fashion (1 hole at a time) using a laser energy source in conjunction with automated stage under computer control and an inverted optics microscope. This permitted prototypes to be constructed with fine control of parameters such as focal area, laser cower and time c'_ exposure. The ability to achieve hick resistance sealing between cells and substrate was tested using coverslips created in this way.

WO 99/66329 PCTiG:B99/01871 Grid patterns we-T_ reproducibly generated in variable formats by means of a computer'-controlled Stage which was interfaced with the laser via the compute'. Coverslips of various materials :_ncluding glass (as well as plastics and other biocompatible materials) were used. Their diameters were 10mm (95 well plate) or 5mm (384 well plate); and of variable thickness (ca.1-20pm). Pores were generated with diameters varying between 0.5p and 10p. The profile of the pore, its taper and internal and external diameters were evaluated to optimise sealing with the zest cells. It, is important to establish the appropriate level of smoothness of the pore. Pore density was optimized for signal--to-noise characteristics, fidelity of voltage-clamp recording and statistical considerations.

To encourage sealing between cell and pore, a number of approaches were taken (Fig 1). They are outlined below:

i) negative pressure in lower liquid compartment e,g. using a venturi effect caused by flowing solution across the ventral orifice and/or by supplying the flowing solution at a reduced overall pressure.

_i) positive pressure in the upcer liquid compartment iii) coating of tr.e coverslip with anti-adhesion material that is burned off in the pore region during the pore manufacturing process (le. laser induced core formation) iv) facility to jog or vibrate coverslip to encourage cells to 'find' pores before adhering to the substrate at non-pore locations v) either a coverslip carousel or multiwell plate carousel to permit centrifugation.

vi) application of voltage field across pores to move cells io into pore mouth.

Surprisingly, laser induced pore formation provided remarkable results.

Figure 7 shows a typical pore produced by this method. When physiological solutions were added to either side of the pore, trans-substrate resistances, typically in the range 200 kOhms to 400 kOhms, were routinely observed. With the addition of cells, the observed resistance was approximately double this figure. With the addition al a Dllcatlo'] or one or more of _ the aczDroa:.nes outl'ned above, resistance measurements approaching the clgaohm range were observed.

b) Scale-ups Bulk perforation. and simultaneous recording (sealing) were evaluated. The approach comprised ' f l ash_ngg' the whole bottom surface of a multiwell plate (or equivalent matrix) with a high energy laser source. With appropriate well structure, the precise location of the required pores was known and with appropriate titration of cell density, a high probability of a having a cell 'in residence' was achieved.
The plate was perforated and the ventral cell surface to breached almost simultaneously. This required a much higher energy laser than that used in protoypes (above) c) Cell-types Although Chinese hamster ovary (CHO) cells have been used to develop the invention, it will be apparent to a person of ordinary skill in the art that a wide variety of cell-lines and primary cells such as neurones isolated from- intact tissues may be employed.

Other perforation methods Alternative methods of perforating glass cove--slips and zo other materials have been evaluated such as etching, casting glass or plastics sheets.

Porous rubber Porous rubber substrates are commercially available for growing cells in cell-culture. The porosity has been evaluated in the context of the resistance and current-measuring applications described herein.

Other materials It will be apparent to a person skilled in the art that additional materials such as PTFE, PETP etc. may be employed in accordance with. the present invention. These have the io advantage of having high dielectric constants but also of being manufactured in extremely thin sheets. This has the advantage of reducing the minimum series resistance in the whole system and also facilitating the introduction of exogenous substances to the cell cytosol.

15-- Multi-well plate recording apparatus The basal multi-well plate recording apparatus preferably accommodates a 96-well/ location format. Preferably multiples of the 96-well format are constructed with a minimal expansion to 3114 well format. An advantage of 20 increasing well-dens:'.%- is that the amount of test compound used is reduced and fewer elates are used with concomitant reductions in ancillary running costs per compound. tested.

The following two approaches have been evaluated:-a) A Tr2RM wor}atation designed to interface with commercially-availaable robots and plate processors.

b) Fully integrated stand-alone system which provides plate handling, solution changes and recording headstages.

Fluid Matrix System An array of miniature recording chambers were created by dispensing droplets containing a suspension of cells onto the recording substrate in a pre-determined pattern and density (see Figure 5) . The substrate can be of any of the types described above eg. perforated glass, plastic, rubber, etc. The complete recording configuration is accomplished by placing a `lid', or moveable recording head, incorporating recording electrodes over the matrix of droplets such that a meniscus of the droplet solution. is established as exemplified in Fig S. An array of droplets may also be generated on the reverse of the porous substrate to provide a conducting pathway to at least one reference electrode and also the means by which substances may be applied to the lower surface of the substrate and hence cell membranes. Similarly, reagents can be applied via a further droplet matrix applied to the `recording plate' as shown in Figure 6. Drug solutions may be dispensed onto a recording WO 99/66329 PCT'GB99/01871 late, .he p-ate may the. be lnue_ted, and e "head"

inverted plate may then be docked with the 0211 matrix. to bring the drug into contact with the cells. The advantage of this approach is that sheets of substrate can be designed without the need to transfer substrate discs to multiwell plates and also obviates complex chamber design with seals, `0' -rings and the like. The invention can still accommodate addition of solutions and has the additional advantage of using very small volumes and thus small quantities of reagents and cells.

References Brew, H and Attwell, D. (1987). Electrocenic glutamate uptake is a major current carrier in the membrane of axolotl retinal glial cells. Nature, 327, '707-9 Hamill, O.P., Marty, A., Neher, E., Sakmann, B. & Sigworth, F.J. (1981). Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pfluger's Archives, 391, 85-100.

Hille, B.(ed). Ionic channels of excitable membranes. 1992.
Sinauer Associates, Sunderland. Sakmann, B. &Neher, E.
(eds). Single-channel recording. 1995. Plenum Press, New York & London.

Margolskee, R.F., McHendry-Rinde, B. And Horn, R. (199:3).
Panning transfected cells for electrophysiologicalstudies.
Biotechniques, 15, 906-911 Sheng, M. (1996) . PD 7S and receptor/ channel clustering:
rounding up the latest suspects. Neuron, 17.

WO 99/66329 PCT/G;B99/01871 Figure Legends 1 cells 2 permeabilized cell surface 3 voltage clamp 4 porous substrate 5 electrode 6 well wall 7 solution perfusion channel 8 cell line or primary cell 9 permeabilized cell 10 multiwell plate (e.g. 96 wells) 11 integral recording head cluster 12 multiplexer 13 ADC/computer 14 fluid level 15 pore 16 O-ring 17 recording assembly 18 cell plate 19 reference plate 20 recording plate 21 reference electrode 22 recording electrode -3 "demi --sandwich"

24 full sandwich (recording configuration)

Claims

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A structure for use in a high throughput screening process, which comprises a biological membrane adhered with a high resistance seal to a porous or perforated substrate wherein:
(i) the biological membrane comprises an ion channel or transporter;
(ii) the substrate has pores of diameters between 0.5µm and 10µm; and (iii) the structure comprises genetically engineered cells which have been engineered to predominantly express an ion channel or transporter.

2. A structure for use in a high throughput screening process, which comprises a biological membrane adhered with a high resistance seal to a porous or perforated substrate, wherein:
(i) the structure comprises voltage gated ion channels; and (ii) the substrate has pores of diameters between 0.5µm and 10µm.

3. A structure for use in a high throughput screening process, which comprises a biological membrane adhered with a high resistance seal to a porous or perforated substrate, wherein:
(i) the structure comprises an ion channel which shows specificity for an ion selected from the group consisting of sodium, potassium, calcium, and chloride;
and (ii) the substrate has pores of diameters between 0.5µm and 10µm.

4. A structure according to any one of claims 1 to 3 wherein the biological membrane comprises a contiguous layer of cells which is capable of adhering to a substrate with a high resistance seal wherein each cell forms a tight junction with adjacent cells and expresses an ion channel or transporter which is localised in the cell membrane.

5. A structure according to any one of claims 1 to 4 which comprises cells having an ion channel or transporter which naturally resides in the cell membrane thereof or, it can be inserted by transfection with cDNA
and/or cRNA encoding the ion channel or transporter.

6. A structure according to any one of claims 1 to 5 which comprises a plurality of ions channels or transporters which are predominantly pre-selected ion channels or transporters of interest.

7. A structure according to claim 2 or 3 which comprises genetically engineered cells which have been engineered to predominantly express an ion channel or transporter.

8. A structure according to claim 1 or 3 which comprises voltage gated ion channels.

9. A structure according to any one of claims 4 to 7 wherein the cells are selected from the group consisting of HEK-293 cells; genetically modified Chinese hamster ovary (CHO) cells; primary neuronal tissue; skeletal muscle cells; smooth muscle cells; cardiac muscle cells;
immune cells; epithelial cells; and endothelial cells.

10. A structure according to claim 1 or 2 having an ion channel which shows specificity for an ion selected from the group consisting of sodium, potassium, calcium and chloride.

11. A structure according to any one of claims 4 to 10 wherein the contiguous layer of cells is capable of adhering with a high resistance seal to a substrate selected from the group which comprises glass, plastics, rubber, polytetraflurotethylene (PTFE), PTFE/glass fabric and polyethylene terephthalate (PETP).

12. A structure according to any one of claims 1 or 3 and to 11 which comprises a pseudo-epithelium wherein one face of a contiguous layer of cells is permeabilized thereby providing access to the interior of the cells.

13. A structure according to any one of claims 1 to 4 which comprises a pseudo-epithelium wherein one face of a contiguous layer of cells is permeabilized thereby providing access to the interior of the cells.

14. A structure according to claim 11 which comprises a pseudo-epithelium wherein one face of the contiguous layer of cells is permeabilized by an antibiotic selected from the group which comprises amphotericin and nystatin;
or detergent selected from the group which comprises digitonin and saponin; or physical disruption using a high voltage field; or by enzymatic digestion of a part of the membrane using an enzyme.

15. A structure according to any one of claims 1 to 14 wherein the substrate is perforated.

16. A structure according to any one of claims 1 to 15 which comprises a perforated coverslip.

17. A structure according to any one of claims 1 to 16 wherein the pores are of diameter between 1 µm and 7 µm.

18. A structure according to claim 17 wherein the pores are of diameter 1 to 2 µm.

19. A structure according to any one of claims 1 to 18 which comprises a coverslip having a grid of pores.

20. A structure according to any one of claims 1 to 16 wherein the perforated substrate is manufactured of a material selected from the group consisting of glass, plastics, rubber, polytetraflurotethylene (PTFE), PTFE/glass fabric and polyethylene terephthalate (PETP.

21. A high throughput screening apparatus for detecting and assaying compounds with activity on voltage gated ions channels which comprises a structure according to any one of claims 1 to 20.

22. A high throughput screening apparatus according to claim 21 which comprises:
a plurality of chambers, each having a permeable peripheral surface providing a substrate for the biological membrane;
a plurality of wells each capable of receiving a chamber and a test compound in a physiological solution or non-physiological solution comprising dimethyl sulphoxide;
a plurality of reference electrodes, having electrical contact with each well;

a movable recording head carrying at least one recording electrode;
means for measuring electrical resistance or impedance between the recording and reference electrodes;
wherein electrical current may pass between the recording and reference electrodes through the permeable peripheral surface of each chamber only via ion channels or transporters in the biological membrane.

23. A high throughput screening apparatus according to claim 22 wherein the wells are provided by a multiwell plate.

24. A high throughput screening apparatus according to claim 21 which comprises an array of droplets on the porous substrate.

25. A high throughput screening apparatus according to any one of claims 21 to 24 which comprises a recording head having a single recording electrode capable of being moved to visit each chamber sequentially.

26. A high throughput screening apparatus according to any one of claims 21 to 24 which comprises a recording head having a plurality of recording electrodes arranged in a line.

27. A high throughput screening apparatus according to any one of claims 21 to 24 which comprises a recording head having a plurality of recording electrodes arranged in a matrix.

28. A method of manufacturing the structure of any one of claims 1 to 20 which comprises the steps of selecting the substrate, perforating it, introducing a biological membrane to the substrate and sealing each pore with biological membrane.

29. A method according to claim 28 which comprises the steps of simultaneously perforating a substrate and sealing the pores with the biological membrane.

30. A method of screening for the detection or assay of compounds with activity on ion channels or transporters which comprises use of the structure of any one of claims 1 to 20, wherein the method comprises the steps of placing a biological membrane which expresses ion channels or transporters of interest in contact with test compound in physiological solution, or non-physiological solution comprising dimethyl sulphoxide, and measuring the resistance or impedance of the biological membrane under the influence of test compound.

31. A method according to claim 30 wherein ion channel activity is monitored by trans-epithelial resistance measurements (TERM) across an intact cell layer.

32. A method according to claim 30 which comprises the step of permeabilizing a contiguous cell layer to provide access to the interior of the cells permitting intracellular voltage and current measurements to be made.

33. A method according to claim 32 wherein a contiguous cell layer is permeabilized by antibiotic selected from the group which comprises amphotericin and nystatin; or detergent selected from the group which comprises digitonin and saponin; or physical disruption using a high voltage field; or by enzymatic digestion of a part of the cell membrane using an appropriate enzyme thereby permitting intracellular voltage or current measurements to made.

34. A method according to any one of claims 30 to 33 which includes the step of multiplexing up to 384 recording elements to a data acquisition system utilizing multiple voltage-clamp amplifiers.

35. A method according to any one of claims 30 to 34 which comprises the step of placing an array of droplets having ion channels or transporters of interest therein on a porous substrate and screening test compounds for activity on the ion channels or transporters.

36. A method according to any one of claims 30 to 34 which comprises the step of placing biological membrane having ion channels or transporters of interest and test compounds in physiological solution, or non physiological solution comprising dimethyl sulphoxide, in a plurality of chambers and screening the test compounds for activity on the ion channels or transporters.

37. A method according to claim 28 or 29 wherein, subsequent to pore formation, the substrate is exposed to localised heat and/or to electrical plasma in order to impart an appropriate raised level of smoothness to the pore(s).