WO2001094939A1 - Single cell auto patch - Google Patents

Single cell auto patch Download PDF

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Publication number
WO2001094939A1
WO2001094939A1 PCT/GB2001/002490 GB0102490W WO0194939A1 WO 2001094939 A1 WO2001094939 A1 WO 2001094939A1 GB 0102490 W GB0102490 W GB 0102490W WO 0194939 A1 WO0194939 A1 WO 0194939A1
Authority
WO
WIPO (PCT)
Prior art keywords
microchannel
cell
patch
access port
liquid
Prior art date
Application number
PCT/GB2001/002490
Other languages
French (fr)
Inventor
David Owen
Andrew Silverthorne
Original Assignee
Xention Discovery Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Xention Discovery Limited filed Critical Xention Discovery Limited
Priority to CA002411845A priority Critical patent/CA2411845A1/en
Priority to EP01936652A priority patent/EP1290441A1/en
Priority to AU6252101A priority patent/AU6252101A/en
Priority to AU2001262521A priority patent/AU2001262521B2/en
Publication of WO2001094939A1 publication Critical patent/WO2001094939A1/en
Priority to US10/313,060 priority patent/US20030129581A1/en

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/48707—Physical analysis of biological material of liquid biological material by electrical means
    • G01N33/48728—Investigating individual cells, e.g. by patch clamp, voltage clamp

Definitions

  • the system is based on delivery of cells via a system of microchannels to a patch clamp pipette.
  • Cells may be pre-sorted using a fluorescence activated cell-sorter (FACS) or other methods of sorting such as immunomagnetic selection.
  • FACS fluorescence activated cell-sorter
  • the system could also be used without pre-sorting for homogenous cell populations. Delivery of cells and events leading to and including a patch clamp recording are computer controlled as is subsequent drug delivery.
  • a patch-pipette accesses cells as they pass an access port in the microchannel.
  • High- resistance electrical seals between pipette and cell are achieved by applying suction to the pipette via a suction controller either on a continuous basis or triggered by the FACS detector which also diverts cells with an appropriate fluorescence signal or light scattering properties along the appropriate microchannel.
  • the minimal system would consist of a single microchannel with patch-clamp module. Cell suspensions are pumped (eg. using a peristaltic pump) from a cell incubator through the microchannel.
  • More sophisticated and also higher throughput devices would have a FACS with multi-wavelength capability to permit selection of several cell-types and also multiple patch-clamp modules to permit parallel recording from many cells which may be different or the same in respect to their fluorescence or cell-scattering 'signature'.
  • a perfusion flow controller switches the flow of solution through the microchannel from delivering cells to delivering drug solutions and the experiment is initiated.
  • Patch-clamp modules may be cascaded such that in the event more than one cell is detected by the FACS, multiple recordings may be made. Excess cells are simply recycled back to the cell incubator.
  • the FACS front end is not required although it would have the beneficial effect of eliminating debris from the system. In the case where cells come from a mixed background a FACS front end allows selection of even minor components of the overall cell suspension.
  • the system can be fully automated and because it also recycles cells and solutions, can run for extended periods of time without intervention.
  • a device for supplying conventional glass patch pipettes will be incorporated or alternatively a system for rejuvenating and hence re-using quartz glass pipettes will be used.
  • Data obtained can be automatically downloaded to a server for off-line analysis etc. without interrupting data acquisition.
  • FIG. 1 Flow Patch-Clamp System fluorescence-activated cell sorter (FACS)- optional cell incubator patch-clamp module control and data acquisition interface perfusion flow controller sorted cells: channel #1 sorted cells: channel #2 unsorted cells channel return channel to cell incubator
  • FACS Fluorescence-activated cell sorter

Abstract

The invention provides a method for obtaining a patch clamp recording from a cell, which includes the steps of providing a microchannel capable of an axial flow of a liquid, providing in the microchannel at least one access port to allow radial access from the exterior of the microchannel (air) to the interior of the microchannel (liquid); whereby liquid in the microchannel forms a meniscus at the port and produces an air/liquid interface, providing a patch-clamp pipette having a tip suitable for passing into the access port suitable for forming a high-resistance (giga-ohm) electrical seal between the tip and the cell, passing liquid carrying the cell axially along the microchannel, causing the cell to be carried to the access port, moving the patch-clamp pipette tip and the microchannel relative to each other radially to bring the tip into contact with the air/liquid interface in the access port, applying suction to the patch-clamp to draw the cell onto the tip to form the seal, and making a patch-clamp recording. The invention also provides an apparatus for carrying out the method.

Description

SINGLE CELL AUTO PATCH
The system is based on delivery of cells via a system of microchannels to a patch clamp pipette. Cells may be pre-sorted using a fluorescence activated cell-sorter (FACS) or other methods of sorting such as immunomagnetic selection. However the system could also be used without pre-sorting for homogenous cell populations. Delivery of cells and events leading to and including a patch clamp recording are computer controlled as is subsequent drug delivery.
A patch-pipette accesses cells as they pass an access port in the microchannel. High- resistance electrical seals between pipette and cell (in the order of G or more) are achieved by applying suction to the pipette via a suction controller either on a continuous basis or triggered by the FACS detector which also diverts cells with an appropriate fluorescence signal or light scattering properties along the appropriate microchannel. The minimal system would consist of a single microchannel with patch-clamp module. Cell suspensions are pumped (eg. using a peristaltic pump) from a cell incubator through the microchannel. More sophisticated and also higher throughput devices would have a FACS with multi-wavelength capability to permit selection of several cell-types and also multiple patch-clamp modules to permit parallel recording from many cells which may be different or the same in respect to their fluorescence or cell-scattering 'signature'.
The process in essence consists of:-
1) Cells scanned by FACS (or not as the case may be) and a cell or cells having appropriate fluorescence signal diverted along microchannel toward a patch- clamp module.
2) Suction is applied to the patch-pipette located in the patch clamp module either at the same time or according to some fixed predetermined interval such that suction occurs as the selected cell passes an access port in the microchannel whereby the cell is drawn to the pipette tip.
3) Seal resistance is monitored automatically and suction controlled by feedback mechanism under control of a computer. Subsequent steps involved in standard patch-clamping are also determined under software control.
4) Once the desired patch-clamp configuration has been achieved, a perfusion flow controller switches the flow of solution through the microchannel from delivering cells to delivering drug solutions and the experiment is initiated.
Patch-clamp modules may be cascaded such that in the event more than one cell is detected by the FACS, multiple recordings may be made. Excess cells are simply recycled back to the cell incubator.
In the case of a homogeneous source of cells, the FACS front end is not required although it would have the beneficial effect of eliminating debris from the system. In the case where cells come from a mixed background a FACS front end allows selection of even minor components of the overall cell suspension.
The system can be fully automated and because it also recycles cells and solutions, can run for extended periods of time without intervention. A device for supplying conventional glass patch pipettes will be incorporated or alternatively a system for rejuvenating and hence re-using quartz glass pipettes will be used.
Data obtained can be automatically downloaded to a server for off-line analysis etc. without interrupting data acquisition. Legends
Figure 1. Flow Patch-Clamp System fluorescence-activated cell sorter (FACS)- optional cell incubator patch-clamp module control and data acquisition interface perfusion flow controller sorted cells: channel #1 sorted cells: channel #2 unsorted cells channel return channel to cell incubator
10. waste
11. computer workstation to control system
Figure 2. Patch-Clamp Module
1. Patch-clamp module
2. Cell
3. Patch-clamp pipette
4. Pipette filling solution (electrolyte)
5. Earth connection
6. Bathing solution
7. Patch-clamp output (membrane current)
8. Patch-clamp amplifier
9. Suction control system
10. Control and data acquisition line
11. MicroChannel outflow
12. MicroChannel inflow Figure 3. Perfusion Flow Controller
1. Perfusion flow controller
Inflow from FACS
Inflow from drug application system
Manifold and controller to switch between drug solutions
Multiwell plate containing drug solutions

Claims

Claims
1. A method for obtaining a patch clamp recording from a cell, which includes the steps of:
(i) providing a microchannel capable of carrying an axial flow of a liquid; (ii) providing in the microchannel at least one access port to allow radial access from the exterior of the microchannel (air) to the interior of the microchannel
(liquid); whereby liquid in the microchannel forms a meniscus at the port and produces an air/liquid interface at the port; (iii) providing a patch-clamp pipette having a pipette tip suitable for passing into the access port suitable for forming a high-resistance (giga-ohm) electrical seal between the tip and the cell; (iv) passing liquid carrying the cell axially along the microchannel, causing the cell to be carried to the access port; (v) moving the patch-clamp pipette tip and the microchannel relative to each other radially to bring the tip into contact with the air/liquid interface in the access port; (vi) applying suction to the patch-clamp pipette to draw the cell onto the tip to form the seal; and (vii) making a patch-clamp recording.
2. A method according to claim 1 in which the cell has been sorted or selected from a heterogeneous source of cells.
3. A method according to claim 2 in which the cell has been sorted and selected using a Fluorescence Activated Cell-Sorter (FACS).
4. A method according to any preceding claim in which a plurality of cells are carried to the access port singly in a sequential flow.
5. Apparatus for carrying out the method of any of claims 1 to 4, comprising:
(i) a microchannel capable of carrying an axial flow of a liquid; the microchannel having an access port to allow radial access from the exterior of the microchannel to the interior; and
(ii) a patch-clamp pipette having a pipette tip suitable for passing into the access port.
6. Apparatus according to claim 5 where the cross-sectional microchannel dimension permits only one cell to pass the access port at a time.
7. Apparatus according to claim 5 or 6 wherein the microchannel is tubular.
8. Apparatus according to claim 7 wherein the diameter of the tubular microchannel is between 1 and 2 times the diameter of a cell.
9. Apparatus according to any of claims 5 to 8 wherein the microchannel has more than one access port spaced axially.
10. Apparatus according to any of claims 5 to 9 wherein there is more than one microchannel.
PCT/GB2001/002490 2000-06-06 2001-06-06 Single cell auto patch WO2001094939A1 (en)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CA002411845A CA2411845A1 (en) 2000-06-06 2001-06-06 Single cell auto patch
EP01936652A EP1290441A1 (en) 2000-06-06 2001-06-06 Single cell auto patch
AU6252101A AU6252101A (en) 2000-06-06 2001-06-06 Single cell auto patch
AU2001262521A AU2001262521B2 (en) 2000-06-06 2001-06-06 Single cell auto patch
US10/313,060 US20030129581A1 (en) 2000-06-06 2002-12-06 Patch-clamping method and apparatus

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB0013584A GB0013584D0 (en) 2000-06-06 2000-06-06 Automated flow patch-clamp system
GB0013584.8 2000-06-06

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US10/313,060 Continuation-In-Part US20030129581A1 (en) 2000-06-06 2002-12-06 Patch-clamping method and apparatus

Publications (1)

Publication Number Publication Date
WO2001094939A1 true WO2001094939A1 (en) 2001-12-13

Family

ID=9892963

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2001/002490 WO2001094939A1 (en) 2000-06-06 2001-06-06 Single cell auto patch

Country Status (5)

Country Link
EP (1) EP1290441A1 (en)
AU (2) AU6252101A (en)
CA (1) CA2411845A1 (en)
GB (1) GB0013584D0 (en)
WO (1) WO2001094939A1 (en)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004034052A1 (en) * 2002-10-09 2004-04-22 St. Boniface General Hospital High throughput assay system
ES2208082A1 (en) * 2002-05-29 2004-06-01 Jose Luis Bardasano Rubio Device is for measuring potential of transmembrane action in cellular preparations under action of electromagnetic fields of variable frequency and intensity
US6936462B1 (en) 1998-06-12 2005-08-30 Xention Discovery Limited High throughput screen
US7384733B1 (en) 1998-12-05 2008-06-10 Xention Discovery Limited Interface patch clamping
US7563614B2 (en) * 2002-02-12 2009-07-21 Cellectricon Ab Systems and methods for rapidly changing the solution environment around sensors
US8232074B2 (en) 2002-10-16 2012-07-31 Cellectricon Ab Nanoelectrodes and nanotips for recording transmembrane currents in a plurality of cells
WO2012155973A1 (en) 2011-05-19 2012-11-22 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Method and device for automatically determining the position of a microsystem for manipulating a spherical microobject
US8338150B2 (en) 1997-11-06 2012-12-25 Cellectricon Ab Method for combined parallel agent delivery and electroporation for cell structures an use thereof

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998055870A1 (en) * 1997-06-04 1998-12-10 A+ Science Invest Ab Detection of biologically active molecules by use of pre-activated cell-based biosensors in liquid-based separation systems
US6063260A (en) * 1994-10-28 2000-05-16 Neurosearch A/S Patch clamp apparatus and technique having high throughput and low fluid volume requirements
WO2000034776A1 (en) * 1998-12-05 2000-06-15 Cenes Limited Interface patch clamping

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6063260A (en) * 1994-10-28 2000-05-16 Neurosearch A/S Patch clamp apparatus and technique having high throughput and low fluid volume requirements
WO1998055870A1 (en) * 1997-06-04 1998-12-10 A+ Science Invest Ab Detection of biologically active molecules by use of pre-activated cell-based biosensors in liquid-based separation systems
WO2000034776A1 (en) * 1998-12-05 2000-06-15 Cenes Limited Interface patch clamping

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
HAMILL O P ET AL: "IMPROVED PATCH-CLAMP TECHNIQUES FOR HIGH-RESOLUTION CURRENT RECORDING FROM CELLS AND CELL-FREE MEMBRANE PATCHES", PFLUEGERS ARCHIV, SPRINGER VERLAG, BERLIN, DE, vol. 391, 1981, pages 85 - 100, XP000196663, ISSN: 0031-6768 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8338150B2 (en) 1997-11-06 2012-12-25 Cellectricon Ab Method for combined parallel agent delivery and electroporation for cell structures an use thereof
US6936462B1 (en) 1998-06-12 2005-08-30 Xention Discovery Limited High throughput screen
US10006902B2 (en) 1998-06-12 2018-06-26 Sophion Bioscience A/S High throughput screen
US7384733B1 (en) 1998-12-05 2008-06-10 Xention Discovery Limited Interface patch clamping
US7563614B2 (en) * 2002-02-12 2009-07-21 Cellectricon Ab Systems and methods for rapidly changing the solution environment around sensors
ES2208082A1 (en) * 2002-05-29 2004-06-01 Jose Luis Bardasano Rubio Device is for measuring potential of transmembrane action in cellular preparations under action of electromagnetic fields of variable frequency and intensity
WO2004034052A1 (en) * 2002-10-09 2004-04-22 St. Boniface General Hospital High throughput assay system
US8232074B2 (en) 2002-10-16 2012-07-31 Cellectricon Ab Nanoelectrodes and nanotips for recording transmembrane currents in a plurality of cells
WO2012155973A1 (en) 2011-05-19 2012-11-22 NMI Naturwissenschaftliches und Medizinisches Institut an der Universität Tübingen Method and device for automatically determining the position of a microsystem for manipulating a spherical microobject
US11054409B2 (en) 2011-05-19 2021-07-06 Multichannel Systems Mcs Gmbh Method and device for automatically determining the position of a microsystem for manipulating a spherical microobject

Also Published As

Publication number Publication date
EP1290441A1 (en) 2003-03-12
CA2411845A1 (en) 2001-12-13
AU2001262521B2 (en) 2005-07-14
GB0013584D0 (en) 2000-07-26
AU6252101A (en) 2001-12-17

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