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WO2004104589A1 - Procede d'analyse de l'influence d'un principe actif sur des cellules biologiques - Google Patents

Procede d'analyse de l'influence d'un principe actif sur des cellules biologiques Download PDF

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Publication number
WO2004104589A1
WO2004104589A1 PCT/DE2004/000979 DE2004000979W WO2004104589A1 WO 2004104589 A1 WO2004104589 A1 WO 2004104589A1 DE 2004000979 W DE2004000979 W DE 2004000979W WO 2004104589 A1 WO2004104589 A1 WO 2004104589A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
electrical properties
cell
sample carrier
determined
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/DE2004/000979
Other languages
German (de)
English (en)
Inventor
Frank Arndt
Hendrik Rönsch
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Siemens AG
Siemens Corp
Original Assignee
Siemens AG
Siemens Corp
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 Siemens AG, Siemens Corp filed Critical Siemens AG
Publication of WO2004104589A1 publication Critical patent/WO2004104589A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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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

  • the invention relates to a method for examining the influence of an active ingredient on biological cells, in which the cells to be examined are immobilized on a sample carrier, the cells are acted upon with the active ingredient and the cells are examined for a reaction to the active ingredient.
  • a cell is immobilized on a sample carrier by means of a biocompatible adhesive layer.
  • the immobilized cell is loaded with active substance molecules that can be attached to different proteins in the cell membrane.
  • the cell is examined using near-field optical methods, and a change in the optical properties of the cell due to a reaction to the active substance can be determined. For example, an attachment of estrogens to the receptors of a cell can be determined by a shift in the fluorescence wavelength of bound estrogen compared to unbound estrogen.
  • the object of the invention is to provide a method for investigating the influence of an active substance on biological cells, which can be carried out comparatively easily.
  • the sample carrier is electrically conductive and a change in the electrical properties of the cells is determined with the aid of the sample carrier as a measure of a reaction of the cells to the active substance.
  • a change in the electrical properties of the cells which they have due to their cell-typical 'activities, also have a direct effect on the electrical properties of the sample carrier, so that a change in the electrical properties of the cells indirectly through a determination the electrical properties of the sample holder can be determined. Changes in the electrical properties of the sample carrier, which can be read out by means of a suitable electrical contact, thus allow a conclusion to be drawn about a change in the electrical
  • the electrical examination of the cells realized in this way has the advantage that an examination of the influence of the active substance on these cells is particularly simple since the active substances do not have to be prepared for an optical detection method such as fluorescent light excitation before the experiment is carried out, for example.
  • the labeling of the active substances with fluorescent markers can be omitted.
  • the active ingredient can be prepared by preparing for the test. implementation in the properties to be examined is changed.
  • a particularly favorable embodiment of the invention provides that the change in the electrical properties of the cells is determined by measuring the membrane potential of the cells.
  • the membrane potential can be measured capacitively, for example, the electrically conductive sample carrier being used here as a capacitor plate.
  • the fact that living cells build up an electrical potential due to their cell metabolism between the cell interior and their environment separated by the cell membrane is advantageously used in the measurement. Since this membrane potential is dependent metabolic, can be a change 'in the membrane potential as a result of a drug effect on the metabolism of the cell by means of the electrically conductive sample holder in a particularly simple manner determined.
  • toxic active substances e.g. B. a reduction in membrane potential due to a loss of vitality of the cells.
  • the membrane potential can, for. B. can be determined with a phase-sensitive electronics.
  • a multiplicity of electrically conductive regions which are electrically insulated from one another are provided on the sample carrier and with which a change in the electrical properties of the cells is determined independently of one another.
  • the large number of areas advantageously enables the electrical properties of the cells to be measured in a site-specific manner. In this way, for example, the cells can be individually examined for their electrical properties.
  • the electrically conductive areas can can also be made so small that several electrically conductive areas can be assigned to a cell. In this way, even a spatially resolved examination of the individual cells can advantageously be carried out.
  • the change in the electrical properties of the cells determined is compared with reference data which characterize specific reactions of the cells.
  • reference data which characterize specific reactions of the cells.
  • An examination method for the toxicity of active substances in which the animals to be examined are confronted with the active substance in order to determine the dose at which 50% of the animals examined die, can be replaced in a particularly simple manner by the method according to the invention.
  • the so-called LD50 test lethal dose 50%
  • the properties of the sample carrier are determined in the event that 50% of the cells immobilized on the sample carrier have died.
  • cell death can be determined, for example, by reducing the capacity of the system acting as a capacitor, consisting of cell membrane and sample carrier as capacitor plates in the broader sense.
  • the detected electrical measurement value for the sample carrier falls below the charac- teristic value for death of 50% of the cells.
  • the corresponding concentration can be determined of the 'toxic active ingredient in the nutrient solution for the cells as the LD 50. This can at least drastically reduce or even completely replace the administration of the toxic active substance to living organisms.
  • a special embodiment of the invention provides that the cells are examined with the electrically conductive areas in such a way that the measurement signals from areas assigned to a particular cell are evaluated together to determine the change in the electrical properties of this cell.
  • this advantageously enables a cell-specific evaluation of the measurement results.
  • the death of each individual cell can be determined, as a result of which a statistical value with a known number of cells can be derived directly.
  • each cell must have at least one electrically conductive area. In the event that several electrically conductive areas are used per cell, the electrical properties of the respective cell can also be locally resolved.
  • the determined change in the electrical properties of the respective cell is compared with reference data which characterize specific reactions of the respective cell.
  • the different reaction states of the individual cells can thus be determined, for example, when toxic agents are applied, since a superimposition of measurement signals from several cells can be avoided. .. ⁇ •
  • the electrical properties of the individual cells that are present when the respective cell has died are determined as reference data. These electrical properties can e.g. B. by lowering the membrane potential of a single cell below a critical value. The determination is carried out, in particular, indirectly by means of a capacitive measurement, as already described.
  • FIG. 1 shows schematically the section through an exemplary embodiment of an analysis device for carrying out the method according to the invention
  • FIG. 2 schematically shows a section of a cell immobilized on a sample carrier for carrying out the method according to the invention and
  • FIG. 3 shows a top view of the sample carrier according to FIG. 2.
  • An examination device for cells 11 has a sample chamber 12 in which the cells 11 are immobilized on a biocompatible layer 13.
  • the biocompatible layer 13 is applied to a sample carrier 14, which in turn carries the cells 11 as samples.
  • the reaction chamber 12 has a housing 15, from which an electrical connection 16 for the sample holder 14 is shown schematically.
  • An electrical signal generator 17 and a measuring device 18 are connected to the connection 16.
  • the signal generator 17 and the measuring device 18 can, for. B. consist of a phase-sensitive electronics. With such electronics, the measuring principle is based on the fact that a time shift .DELTA.t arises through capacitors in the AC circuit: the current maximum leads the voltage maximum. Each metabolically active cell has an electrical potential difference of approx. 50-100 mV on the cell membrane. This
  • Membrane potential allows the cell to act as a capacitor in an AC field. If the alternating current has a sinusoidal shape, this shift is measured in ° (degrees) and referred to as phase angle ⁇ (phi). Metaphorically speaking, vital cells with a stable membrane potential have a high phase angle, while cells with a low membrane potential with a reduced vitality have a correspondingly lower phase angle. The phase angle is most meaningful at a frequency of 50 Hz.
  • a lid 19 of the sample chamber 12 is made transparent, so that an optical examination of the cells 11 by means of a camera 20 in addition to the electrical according to the invention Examination of the cells can be done.
  • the electrical examination of the cells is carried out by the signal generator 17 generating an electrical signal with a known characteristic, the reaction of the sample carrier to this electrical signal being determined with the measuring device.
  • the condition of the cells 11 plays an important role here, because due to their electrical properties they directly influence the electrical properties of the sample carrier 14.
  • FIG. 2 shows a sample carrier 14a as a detail on which electrically conductive areas 21 are applied. These areas 21 are covered by the biocompatible layer 13, which on the one hand is responsible for the immobilization of the cell 11 and on the other hand can advantageously act as a dielectric. ⁇ • ⁇
  • An active ingredient 22, which penetrates into the cell according to FIG. 2, is also shown schematically.
  • the active substance could also accumulate on the cell membrane 23 of the cell 11.
  • An electrical examination of the cell 11 under the influence of the active substance takes place via the areas 21, which can be electrically contacted via a connection, not shown, as shown in FIG. 1.
  • These act as capacitor plates, with the aid of which - reinforced by the biocompatible layer 13 acting as a dielectric - the membrane potential of the membrane 23 can be measured indirectly in the area of immobilization. If the membrane potential, ie the electrical potential formed between the cell interior and the cell environment, is reduced, the capacitance of the capacitors formed from the cell membrane 23 and the regions 21 is also reduced.
  • An embodiment of the areas 21 as a supervision can be seen in FIG. These can be produced on the sample carrier 14a by structuring a conductive layer, the necessary connections 16 likewise being able to be formed from this layer and running in the spaces between the individual regions 21.

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  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Food Science & Technology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Hematology (AREA)
  • Biophysics (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Immunology (AREA)
  • Pathology (AREA)
  • Investigating Or Analyzing Materials By The Use Of Electric Means (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé d'analyse de l'influence d'un principe actif sur des cellules. Ce procédé consiste à immobiliser les cellules sur un support d'échantillon (13, 14) que l'on place dans une chambre de réaction (12). On alimente la chambre de réaction d'un principe actif (contenu, par exemple, dans la solution nutritive destinée aux cellules), ce qui permet d'extraire des signaux électriques par le support d'échantillon (14) électroconducteur et ces signaux permettent de tirer une conclusion sur les propriétés électriques des cellules (11). On peut ainsi déterminer notamment la mort des cellules et donc analyser l'action toxique du principe actif. Cette analyse permet de limiter, voire de supprimer les essais sur les animaux sinon indispensables. (test de toxicité LD50 usuel).
PCT/DE2004/000979 2003-05-16 2004-05-03 Procede d'analyse de l'influence d'un principe actif sur des cellules biologiques Ceased WO2004104589A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE2003123096 DE10323096A1 (de) 2003-05-16 2003-05-16 Verfahren zur Untersuchung eines Wirkstoffeinflusses auf biologische Zellen
DE10323096.3 2003-05-16

Publications (1)

Publication Number Publication Date
WO2004104589A1 true WO2004104589A1 (fr) 2004-12-02

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/DE2004/000979 Ceased WO2004104589A1 (fr) 2003-05-16 2004-05-03 Procede d'analyse de l'influence d'un principe actif sur des cellules biologiques

Country Status (2)

Country Link
DE (1) DE10323096A1 (fr)
WO (1) WO2004104589A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6051422A (en) * 1997-05-30 2000-04-18 Board Of Trustees, Leland Stanford, Jr., University Hybrid biosensors
WO2000071742A2 (fr) * 1999-05-21 2000-11-30 Hickman James J Genomique fonctionnelle a fort rendement
DE19961445A1 (de) * 1999-12-20 2001-07-05 Forschungszentrum Juelich Gmbh Messverfahren unter Einsatz mindestens einer biologischen Rezeptorzelle sowie eine zur Durchführung des Messverfahrens geeignete Vorrichtung
WO2001070002A2 (fr) * 2000-03-22 2001-09-27 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Enregistrement du signal d'un systeme recepteur-effecteur par une electrode extracellulaire plane sensible au potentiel

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6051422A (en) * 1997-05-30 2000-04-18 Board Of Trustees, Leland Stanford, Jr., University Hybrid biosensors
WO2000071742A2 (fr) * 1999-05-21 2000-11-30 Hickman James J Genomique fonctionnelle a fort rendement
DE19961445A1 (de) * 1999-12-20 2001-07-05 Forschungszentrum Juelich Gmbh Messverfahren unter Einsatz mindestens einer biologischen Rezeptorzelle sowie eine zur Durchführung des Messverfahrens geeignete Vorrichtung
WO2001070002A2 (fr) * 2000-03-22 2001-09-27 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Enregistrement du signal d'un systeme recepteur-effecteur par une electrode extracellulaire plane sensible au potentiel

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
BAUMANN W H ET AL: "Microelectronic sensor system for microphysiological application on living cells", SENSORS AND ACTUATORS B, ELSEVIER SEQUOIA S.A., LAUSANNE, CH, vol. 55, no. 1, 25 April 1999 (1999-04-25), pages 77 - 89, XP004175066, ISSN: 0925-4005 *

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