WO2018100206A1 - Dispositif d'amélioration de joint d'étanchéité - Google Patents

Dispositif d'amélioration de joint d'étanchéité Download PDF

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Publication number
WO2018100206A1
WO2018100206A1 PCT/EP2017/081388 EP2017081388W WO2018100206A1 WO 2018100206 A1 WO2018100206 A1 WO 2018100206A1 EP 2017081388 W EP2017081388 W EP 2017081388W WO 2018100206 A1 WO2018100206 A1 WO 2018100206A1
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WO
WIPO (PCT)
Prior art keywords
solution
patch clamp
ions
suitably
metal ions
Prior art date
Application number
PCT/EP2017/081388
Other languages
English (en)
Inventor
Lars Damgaard LØJKNER
Anders Lindqvist
Daniel Rafael SAUTER
Kadla Røskva ROSHOLM
Original Assignee
Sophion Bioscience A/S
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 Sophion Bioscience A/S filed Critical Sophion Bioscience A/S
Priority to JP2019529523A priority Critical patent/JP7086072B2/ja
Priority to US16/465,590 priority patent/US11215606B2/en
Priority to EP23155362.9A priority patent/EP4209783A1/fr
Priority to EP17818461.0A priority patent/EP3548888B1/fr
Publication of WO2018100206A1 publication Critical patent/WO2018100206A1/fr
Priority to US17/514,427 priority patent/US11692994B2/en

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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

  • a seal enhancer for improving the patch clamp seal in a patch clamp method or apparatus is provided.
  • a critical step in a planar patch clamp experiment is the seal formation i.e. formation of a tight bond between the cell membrane and the substrate of the planar patch clamp chip.
  • This seal poses a barrier for charge carriers such as ions and a good seal can therefore be measured as a high seal resistance.
  • the exact mechanism by which this connection occurs remains to be completely understood, however, it is known that small gaps between the cell and the substrate can be closed (sealed) by precipitation of a salt. To exploit this effect, it is important to have both components of this salt (i.e. anions and cations) separated in internal and external solution; otherwise, the salt would already start to precipitate before the cell is applied and this would result in an altered ionic strength of the solution.
  • Solubility of a solution is defined in the solubility product K sp .
  • K sp the solubility product
  • the smaller K sp the fewer ions are freely available and the more are present as solid salt.
  • R me m b r a n e was historically enhanced using fluoride in the internal solution and Ca 2+ in the external.
  • CaF 2 has a K sp of 3.58 x 10 "11 (McCann, 1968), thus CaF 2 precipitates at the interface between internal and external solution thus fostering seal formation (Vargas, Yeh, Blumenthal, & Lucero, 1999).
  • Ca 2+ in the internal solution and in the external solution e.g. Ca 2+ -activated K + and CI " channels
  • the use of F- in these solutions is not feasible due to the low K sp .
  • the concentration of metal ions in the external solution is in the region of 50- lOOmM, cf. EP1775586.
  • too metal ion concentrations may possibly affect ion channel function in an adverse manner leading to incorrect responses and inaccuracies in drug target responses.
  • a patch clamp system comprising a patch clamp device, an internal solution and an external solution.
  • the internal solution comprises one or more anions selected from phosphate (P0 4 3 ⁇ ) ions, sulfate (S0 4 2 ⁇ ) ions and fluoride (F ) ions
  • the external solution comprises one or more metal ions selected from Ca 2+ , Sr 2+ and Ba 2+ , or combinations thereof.
  • the concentration of said metal ions in the external solution is between 1 and 20mmol/L.
  • anions selected from phosphate (P0 4 3 ⁇ ), sulfate (S0 4 2 ⁇ ) and fluoride (F ) anions in combination with one or more metal ions selected from Ca 2+ , Sr 2+ and Ba 2+ , or combinations thereof as a seal enhancer in a patch clamp system is also provided, wherein the anions are present in an internal solution, and the metal ions are present in an external solution of the patch clamp system.
  • the concentration of said metal ions in the external solution is between 1 and 20mmol/L
  • a method for providing a gigaseal in a patch clamp system in which the patch clamp system comprises a patch clamp device, an internal solution and an external solution.
  • the method comprising the steps of: a introducing a solution of one or more anions selected from phosphate (P0 4 3 ⁇ ) sulfate (S0 4 2 ⁇ ) and fluoride (F ) anions into the internal solution; b introducing a solution of cells into the external solution, a nd capturing the cells within the patch clamp device; c introducing a solution of one or more metal ions selected from Ca 2+ , Sr 2+ and
  • steps a and b. can be performed in any order.
  • Fig. 1 shows a simplified view of a patch clamp device 102 for use in patch clamp analysis of a cell 120 sealed to the chip 102 to form a gigaseal.
  • the device 102 separates an external solution 122 on a first side of the device from an internal solution 124 on an opposite side thereof.
  • the diameter a of the cell is approximately 5-20 ⁇ , such as approximately 10 ⁇ .
  • a hole 108 is formed in the device 102, through which suction may be applied to secure the cell 120 in place over the hole 108.
  • Electrodes (not shown) may be provided in both sides of the device in order to determine an electrical resistance, a flow of ions or a voltage difference across the cell and through the hole 108.
  • external solution refers to "extracellular physiological solution", external meaning outside the cell.
  • intermediate solution refers to "intracellular physiological solution", internal meaning inside the cell or the solution intended to provide electrical and fluidic contact with the inside of the cell.
  • a patch clamp system is provided, as illustrated schematically in Figure 1.
  • the patch clamp system comprises a patch clamp device 102, an internal solution 124 and an external solution 122.
  • the patch clamp system may be useful for determining or monitoring current flow through ion channel-containing structures such as cell membranes, with a high throughput and reliability and under conditions that are realistic with respect to the influences to which the cells or cell membranes are subjected.
  • the results determined e.g., variations in ion channel activity as a result of influencing the cell membrane with, e.g., various test compounds, can be relied upon as true manifestations of the proper influences and not of artefacts introduced by the measuring system, and can be used as a valid basis for studying electrophysiological phenomena related to the conductivity or capacitance of cell membranes under given conditions.
  • the current through one or more ion channels is directly measured using reversible electrodes as characterized below, typically silver/silver halide electrodes such as silver chloride electrodes, as both measuring electrodes and reference electrodes.
  • the patch clamp device may comprise an array of a plurality of chips on a single carrier. It may be is used in a method for determining and/or monitoring electrophysiological properties of ion channels in ion channel-containing structures, typically lipid membrane-containing structures such as cells, by establishing an electrophysiological measuring configuration in which a cell membrane forms a high resistive seal around a measuring electrode, making it possible to determine and monitor a current flow through the cell membrane.
  • the patch clamp system is for example useful in a method for analysing the electrophysiological properties of a cell membrane comprising a glycocalyx.
  • the patch clamp system may be used in or form part of an apparatus for studying electrical events in cell membranes, such as an apparatus for carrying out patch clamp techniques utilised to study ion transfer channels in biological membranes.
  • the internal solution comprises one or more anions selected from phosphate (P0 4 3 ⁇ ), sulfate (S0 4 2 ⁇ ) and fluoride (F ) anions, preferably sulfate (S0 4 2 ⁇ ) ions.
  • concentration of anions, such as sulfate (S0 4 2 ⁇ ) ions, in the internal solution is between 20 and 200mmol/L, suitably between 45 and 175mmol/L, more suitably between 50 and 150mmol/L, even more suitably between 75 and 125mmol/L, inclusive, most suitably around lOOmmol/L.
  • S0 4 2 does not inhibit any ion channel or relevant up-stream pathway.
  • sulfate salts are highly soluble in water, under physiological conditions. This allows a relatively broad spectrum of anions to be incorporated in the internal solution.
  • sulfate ions are provided in the internal solution in the form of Mg 2+ , Ca 2+ , K + or Na + salts, preferably K + or Na + salts.
  • the internal solution is suitably substantially free from ions which may cause sulfate precipitation.
  • the internal solution is preferably substantially free from Ba 2+ , Fe 3+ or Be 2 ions, and is preferably completely free from Ba 2+ , Fe 3+ or Be 2 ions.
  • the external solution comprises one or more metal ions selected from Ca 2+ , Sr 2+ and Ba 2+ , or combinations thereof.
  • the metal ions are Ba 2+ .
  • the concentration of the metal ions in the external solution is between 1 and 20mmol/L, suitably between 4 and 17mmol/L, more suitably between 5 and 15mmol/L, even more suitably between 7.5 and 12.5mmol/L, inclusive, most suitably around lOmmol/L.
  • Ba 2+ is known to inhibit some K + channels in the ⁇ to low mM - range (Wulff & Zhorov, 2008) . However, to the best of our knowledge, Ba 2+ does not have any effect on any of the CI " channels.
  • the here described seal enhancer will also be useful for patch clamp applications where Ba 2+ functions as an inhibitor (e.g. inward rectifier K + channels).
  • the external solution is suitably substantially free of ions which may cause metal ion precipitation.
  • the external solution is preferably substantially free from sulfate, and is preferably completely free from sulfate ions.
  • the one or more metal ions are suitably added in the form of their CI " salt.
  • all cations in the external solution are CI " .
  • the internal solution may have a concentration of fluoride (F ) ions which is less than 5mmol/L, suitably less than lmmol/L, and is more preferably zero. Fluoride ions can therefore be reduced and/or eliminated.
  • F fluoride
  • the use of one or more anions selected from phosphate (P0 4 3 ), sulfate (S0 4 2 ) and fluoride (F ) anions in combination with one or more metal ions selected from Ca 2+ , Sr 2+ and Ba 2+ , or combinations thereof as a seal enhancer in a patch clamp system is provided.
  • the use of sulfate (S0 4 2 ) ions in combination with metal ion selected from Ba 2+ as a seal enhancer in a patch clamp system is provided.
  • the anions are present in an internal solution, and the metal ions are present in an external solution of the patch clamp system.
  • the concentration of said metal ions in the external solution is between 1 and 20mmol/L.
  • a method for providing a gigaseal in a patch clamp system comprises a patch clamp device, an internal solution and an external solution.
  • the method comprises the steps of: a. introducing a solution of one or more anions selected from phosphate (P0 4 3 ), sulfate (S0 4 2 ) and fluoride (F ) anions into the internal solution; b. introducing a solution of cells into the external solution, and capturing the cells within the patch clamp device; c.
  • the method comprises the steps of: a. introducing a solution of sulfate (S0 4 2 ⁇ ) ions into the internal solution;
  • concentration of the Ba 2+ metal ions in the external solution is between 1 and 20mmol/L.
  • steps a and b. can be performed in any order; i.e. a solution of cells can be introduced to the external solution, and captured within the patch clamp device before sulfate solution is introduced in the external solution. However, it is preferred that step a. is performed before step b.
  • the method may further comprise the step of (d) performing patch clamp measurements on the cell(s) in the patch clamp device.
  • the concentration of anions, in particular sulfate (S0 4 2 ⁇ ) anions, in the internal solution is between 20 and 200mmol/L, suitably between 45 and 175mmol/L, more suitably between 50 and 150mmol/L, inclusive.
  • the metal ions are suitably Ba 2+ .
  • the concentration of the metal ions in the external solution is suitably between 4 and 17mmol/L, more suitably between 5 and 15mmol/L, inclusive.
  • Cells for use in the method may be selected from CHO or HEK293.
  • S0 4 2 ⁇ has a relatively high solubility with Ca 2+ .
  • the solubility constant (Ksp) of CaS0 4 is 4.93xl0 "5 .
  • the present technology is useful for applications where Ca 2+ should have a precise internal concentration over the entire course of the experiment.
  • the internal solution and/or the external solution may additionally comprise calcium (Ca 2+ ) ions.
  • EXAMPLE 1 Test of ion pairs as seal enhancers in Qube experiment
  • Cell line TE671 expressing the Na v 1.4 Channel Tested ion pairs: CaF 2 (reference), SrF 2 , BaS0 4 , Ca 3 (P0 4 ) 2
  • the cation in the extracellular (EC) solutions were applied at low concentration (2 mM) during cell addition and positioning and at high concentration (10 mM) before sealing and current measurement.
  • the osmolality of the internal solutions is adjusted to 350mOsm with KCI .
  • the pH of the internal solutions is adjusted to pH 7.0 using KOH. Test procedure
  • HEK293 cells stably expressing the hTMEM16A ion channel General method: The internal solutions (at various concentrations of sulfate) are provided to the internal side of the Qube device. A priming solution, containing cells as specified, is then provided to the external side of the Qube device, and cells are captured. The external solutions (at various Ba 2+ concentrations) are then provided to the external side of the device.
  • Table 3 Summary of R m embrane from 5 individual experiments (QChips) measured on CHO- hERG cells. Statistical significance was assessed using an ANOVA test. * depicts significance with respect to the next lower concentration of Barium and # refers to significance to the next lower sulfate concentration. * or # indicate p ⁇ 0.05; ** or ## indicate p ⁇ 0.01; *** or ### indicate p ⁇ 0.001. *
  • Table 4 Summary of Rmembrane from 1 experiment (QChips) measured on HEK-hTMEM16A cells. Statistical significance was assessed using an ANOVA test. * depicts significance with respect to the next lower concentration of Barium and # refers to significance to the next lower sulfate concentration. * or # indicate p ⁇ 0.05; ** or ## indicate p ⁇ 0.01 ; *** or ### indicate p ⁇ 0.001. *

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Abstract

L'invention concerne un dispositif d'amélioration de joint d'étanchéité destiné à améliorer le joint de fixation de pièce de réparation dans un procédé ou un appareil de fixation de pièce de réparation. La solution interne comprend des anions particuliers et la solution externe comprend un ou plusieurs ions métalliques à faible concentration.
PCT/EP2017/081388 2016-12-02 2017-12-04 Dispositif d'amélioration de joint d'étanchéité WO2018100206A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2019529523A JP7086072B2 (ja) 2016-12-02 2017-12-04 シールエンハンサー
US16/465,590 US11215606B2 (en) 2016-12-02 2017-12-04 Seal enhancer
EP23155362.9A EP4209783A1 (fr) 2016-12-02 2017-12-04 Dispositif d'amélioration de joint
EP17818461.0A EP3548888B1 (fr) 2016-12-02 2017-12-04 Dispositif d'amélioration de joint d'étanchéité
US17/514,427 US11692994B2 (en) 2016-12-02 2021-10-29 Seal enhancer

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
DKPA201670957 2016-12-02
DKPA201670957 2016-12-02
US201762449278P 2017-01-23 2017-01-23
US62/449,278 2017-01-23

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US16/465,590 A-371-Of-International US11215606B2 (en) 2016-12-02 2017-12-04 Seal enhancer
US17/514,427 Continuation US11692994B2 (en) 2016-12-02 2021-10-29 Seal enhancer

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WO2018100206A1 true WO2018100206A1 (fr) 2018-06-07

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1042468A2 (fr) * 1997-12-03 2000-10-11 Merck & Co., Inc. Compositions a canaux calcium actives a basse tension et procedes correspondants
US20040168912A1 (en) * 2000-02-11 2004-09-02 James Klemic Planar patch clamp electrodes
EP1775586A1 (fr) 2005-10-17 2007-04-18 Nanion Technologies GmbH Procédé d'examen électrophysiologique d'une membrane comprenant un canal ionique
US20090098546A1 (en) * 2000-11-13 2009-04-16 The Queen's Medical Center Methods of Screening for LTRPC7 Modulators
US20100087446A1 (en) * 2007-04-26 2010-04-08 Chakravarty Prasun K 2-substituted indole derivatives as calcium channel blockers
US20110166136A1 (en) * 2008-07-03 2011-07-07 Lectus Therapeutics Limited Calcium Ion Channel Modulators & Uses Thereof

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1042468A2 (fr) * 1997-12-03 2000-10-11 Merck & Co., Inc. Compositions a canaux calcium actives a basse tension et procedes correspondants
US20040168912A1 (en) * 2000-02-11 2004-09-02 James Klemic Planar patch clamp electrodes
US20090098546A1 (en) * 2000-11-13 2009-04-16 The Queen's Medical Center Methods of Screening for LTRPC7 Modulators
EP1775586A1 (fr) 2005-10-17 2007-04-18 Nanion Technologies GmbH Procédé d'examen électrophysiologique d'une membrane comprenant un canal ionique
US20070087327A1 (en) * 2005-10-17 2007-04-19 Niels Fertig Method and kit-of-parts for the electrophysiological examination of a membrane comprising an ion channel
US20100087446A1 (en) * 2007-04-26 2010-04-08 Chakravarty Prasun K 2-substituted indole derivatives as calcium channel blockers
US20110166136A1 (en) * 2008-07-03 2011-07-07 Lectus Therapeutics Limited Calcium Ion Channel Modulators & Uses Thereof

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