WO2002016936A1 - Device and method for a local resolution study of cross-linked cells and/or cell systems and a use for the study of active ingredients using a microphysiometer - Google Patents

Device and method for a local resolution study of cross-linked cells and/or cell systems and a use for the study of active ingredients using a microphysiometer Download PDF

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Publication number
WO2002016936A1
WO2002016936A1 PCT/EP2001/009752 EP0109752W WO0216936A1 WO 2002016936 A1 WO2002016936 A1 WO 2002016936A1 EP 0109752 W EP0109752 W EP 0109752W WO 0216936 A1 WO0216936 A1 WO 0216936A1
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Prior art keywords
cells
light sources
cell
study
laps
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PCT/EP2001/009752
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German (de)
French (fr)
Inventor
Jan Behrends
Michael George
Christian Kirchner
Wolfgang Parak
Markus Seitz
Bernhard Stein
Rainer Metzger
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Jan Behrends
Michael George
Christian Kirchner
Wolfgang Parak
Markus Seitz
Bernhard Stein
Rainer Metzger
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Application filed by Jan Behrends, Michael George, Christian Kirchner, Wolfgang Parak, Markus Seitz, Bernhard Stein, Rainer Metzger filed Critical Jan Behrends
Priority to AU2001289829A priority Critical patent/AU2001289829A1/en
Publication of WO2002016936A1 publication Critical patent/WO2002016936A1/en

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    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28—Electrolytic cell components
    • G01N27/30—Electrodes, e.g. test electrodes; Half-cells
    • G01N27/305—Electrodes, e.g. test electrodes; Half-cells optically transparent or photoresponsive electrodes
    • 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
    • G01N33/4833—Physical analysis of biological material of solid biological material, e.g. tissue samples, cell cultures
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • 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
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5058—Neurological cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00—Screening for compounds of potential therapeutic value
    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the invention relates to a device and a method for the spatially resolved examination of networked cells and / or cell systems and their use for diagnostic applications and for the investigation of active substances.
  • LAPS light-addressable potentiometric sensor
  • Cytosensor ® microphysiometer In a known Cytosensor ® microphysiometer (see EP-A-0394406) are operated in parallel four or eight measuring chambers (LAPS). Each chamber is controlled by a separate light source and has an independent connection (channel) with which the photocurrent and thus the acidification or alkalization of the solution surrounding the cells is measured. This data is collected and individually converted into so-called 'raw data' by deriving the acidification / alkalization. Correspondingly continuously evaluable data is thus obtained for each chamber and each individual measuring channel. Each LAPS is illuminated by the light source from the bottom in its center. The spatial resolution in this configuration is approximately one millimeter (Nakao, M., S. Inoue, et al.
  • the object of the invention is to provide a device and a method for spatially resolved investigations of networked cells and / or cell compartments. This object is achieved with the features of the claims.
  • the invention is based on the basic idea of providing several light sources for a single measuring chamber of a microphysiometer.
  • the chip can be activated at several different (preferably four to eight or even 25 different) locations on the surface in this chamber, and thus on one LAPS each.
  • preferably four to eight or even 25 light sources, which serve to activate the chip are attached to the underside.
  • a reference electrode, a connection (channel) to the LAPS for measuring the photocurrent and a liquid supply or drainage system are provided for each chamber.
  • not all light sources are used or controlled simultaneously for the spatially resolved measurement.
  • the light sources are operated in a sequential cycle.
  • light source number 1 is activated first, so that the photocurrent at this time depends on the acidification or alkalization of the cells, which is on the LAPS within an area of approximately one square millimeter at the point illuminated by light source number 1.
  • Light source number 1 is operated until a complete photocurrent-voltage curve is recorded.
  • the switch is made to light source number 2, in which case the measured photocurrent depends on the acidification or alkalization of the cells which are located on the LAPS on the surface illuminated by light source number 2. This change continues until the last light source, preferably light source number 25. The cycle is then repeated, starting with the operation of light source number 1.
  • simultaneous or controlled activation of the light sources is provided (e.g. all light sources simultaneously or in individual groups (e.g. light sources No. 2, 4, 6, etc.)).
  • the photocurrent-voltage curves are recorded sequentially at all eight (or up to 25) points of the LAPS, under each of which one of the eight light sources is located.
  • the turning point that is to say a single data point, is then determined from each photocurrent voltage curve.
  • eight data points are determined in the individual channel used, which can be shown as so-called "raw data" on a display.
  • the first channel contains data points 1, 9, 17, ....
  • the second channel the Data points 2, 10, 18, ... etc., whereby data points 1 to 8 correspond to the first measurement cycle, data points 9 to 16 correspond to the next measurement cycle etc.
  • Each channel thus contains the time-dependent course of the inflection point of the photocurrent-voltage curve from a separately illuminated area of the LAPS.
  • the present invention thus enables the sequential examination at several (preferably up to 25) measuring points on the surface of a LAPS and thus of a channel, based on the configuration of the microphysiometer.
  • the present invention permits the specific observation of the metabolism of living cells, cell systems or cell compartments by determining the acidification or alkalization rates of the medium by the cells, which is achieved by time-resolved measurement of the pH with the technology of the light-addressable potentiometric sensor (LAPS) is.
  • the cells that can be used can be cell cultures (including bacterial or fungal cultures) or also fresh, animal, human (including biopsy material) or plant cell material.
  • the present invention is in a preferred embodiment with the known Cytosensor ® microphysiometer, so that thus the spatially resolved measurement of the acidification of the medium or Alkalleitersrate operable or coupled possible by for example cross-linked cell aggregates.
  • the spatially resolving measuring system has three aspects: 1) the integration of the multiple (for example eight) light sources in one measuring chamber; 2) the electronics for cyclical switching between the light sources; and 3) the software that splits and analyzes the measured data from one channel into several (preferably four to -25) channels.
  • the invention relates to a regulation for a) direct application of the cell cultures to the LAPS, b) possible structuring of the LAPS into sensitive and insensitive areas and c) an optimal stimulation system.
  • cells can be applied to specific carriers, which can have separated regions, in order to obtain a separation for the cells.
  • the cells can e.g. can be cultivated directly on the LAPS. This makes it possible to colonize different locations on the LAPS and the support with different cells.
  • Several different methods can be used to apply different spatially separated cultures:
  • the cell tissue pieces are applied to various points on the LAPS and, for example, fixed or allowed to grow there. It is crucial, for example, that the physiological function of the cells is full remain. In this way it can be ensured that the initially spatially separated cultures form contact points with one another by growth on the chip. In the case of isolated cell suspensions, these are applied in highly concentrated form at various points on the LAPS. To prevent mixing of the cell cultures during the adhesion phase (approx. 24 hours), the surface of the LAPS can be divided. For example, boundaries can be applied to the LAPS surface by thin lines made of a hydrophobic material. As a result, the locally applied cells remain in their respective compartments.
  • these lines have interruptions and are applied as thinly as possible so that cells or cell extensions can also grow over these lines after the adhesion.
  • the medium necessary for cultivation is added to the cells.
  • it is important that the ability of the cell to grow is maintained and that the cells from the different areas form contact points with one another or can interact in another way, for example via secretion of hormones or other signal substances.
  • the separation can e.g. B. be held so that it keeps the cells, which are applied selectively, completely and separately from one another over the duration of the measurement, or ensures that the cells grow together or migrate to one another.
  • a separating device which has several chambers can be placed on the chip during the adhesion phase and can be removed after the adhesion phase.
  • a device made of Teflon has proven particularly useful here.
  • cells can also be applied selectively by injecting them into a polymer gel (in particular agarose gel) by needles or pipettes, by reducing or restricting their mobility.
  • a polymer gel in particular agarose gel
  • the acidification is to be averaged over a particularly small area in order to examine particularly structured cell tissue associations, for example if only a small region of the cell association contains the cells of interest, this is possible with a pre-structured LAPS.
  • the LAPS can be divided into sensitive and non-sensitive areas in which no photocurrent flows by partial illumination with UV light.
  • a LAPS can be structured so that up to eight excellent areas of approximately one hundredth of a square millimeter can be represented. When one of the active areas is illuminated, the acidification rate averaged over the area of approximately one hundredth of a square millimeter can be determined.
  • the stimulation of the cells can be achieved in different ways. By adding an active ingredient to the supplied medium, global stimulation of all cells, e.g. possess a corresponding receptor, are triggered and determined.
  • local stimulation of certain cell areas is also possible in the invention. This can be done either by microinjection of an active ingredient, via a microcapillary at one point in the LAPS measuring chamber or by a voltage stimulus (ie a metallic stimulus microelectrode is integrated at one point of the LAPS) or by local application of mechanical pressure (e.g. by means of a pressure in the LAPS - Measuring chamber integrated micrometer screw) can be reached.
  • the device / method is used for the spatially resolved measurement of the metabolism of cells which are either functionally coupled or which have different properties (e.g. expression of different receptors) and remain functionally separate.
  • Coupled cell groups include synapses, gap and tight junctions, ie there is a flow of information (physical or chemical) between neighboring cells. Due to the coupling, there is the option of non-local stimulation.
  • the local electrical activity of coupled cell assemblies can be observed at the level of individual cells using the patch-clamp technique (Fitzsimonds, RM, H.-J. Song, et al. (1997), "Propagation of activity-dependent synaptic depression in simple neural networks ", Nature 388 (July 21): 439-448).
  • patch-clamp technique Fritzsimonds, RM, H.-J. Song, et al. (1997), “Propagation of activity-dependent synaptic depression in simple neural networks ", Nature 388 (July 21): 439-448).
  • extracellular techniques must be used for long-term stable measurements.
  • the extracellular electrical activity of brain sections can be monitored with the aid of extracellular microelectrodes in a spatially resolved manner (Egert, U., B. Schlosshauer, et al. (1998), "A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays ", Brain Research Protocols 2 (4): 229-242).
  • a corresponding system is commercially available, for example from the Natural Science Medical Institute (NMI) in Reutlingen.
  • NMI Natural Science Medical Institute
  • the acidification or alkalization of the medium surrounding the cells is measured by determining the pH.
  • this is possible for all types of cells.
  • the applications or examples described below can also be coupled with fields of field effect transistors.
  • the brain depicts highly complex compositions of coupled nerve cells that are divided into functional regions.
  • the nerve cells are networked with each other through synapses and can therefore communicate with each other.
  • Diseases such as epilepsy, have communicative dysfunctions on the cells.
  • This is investigated according to the invention as follows. Cuts or punch preparations from the affected regions are removed and adhered to various parts of the LAPS. After a few days, nerve cells from this area form cell processes (dendrites and axons) that spread along the LAPS. Through synapses, contacts are made between individual neurons, ie after several days the different regions are networked through synapses. For reasons of clarity, two stamping preparations (regions 1, 2) are used in this example.
  • the photocurrent caused by light source 1 or 2 analyzes the acidification of the cells from region 1 or 2. For example, Specifically stimulate cells from region 1. This can be either by adding an active ingredient in the supplied medium that acts specifically on the cells in region 1 or by local microinjection (micro perfusion) of an active ingredient in region 1 or by local electrical stimulation with a microelectrode from region 1.
  • the acidification rate or The metabolism of the cells from region 1 is then influenced within seconds to minutes and can then be read off. Since region 2 is coupled to region 1 via neuronal synapses, the altered metabolism in region 1 can also influence the metabolism of the cells in region 2. This can only take some time (minutes to seconds).
  • the device according to the invention enables the acidification rates of the two regions to be separated, measured in parallel and the complex interplay of the two networked regions to be examined in a long-term stable manner.
  • Example 2 Certain cell types such as Epithelial cells, cartilage and bone cells react to pressure.
  • the effect of non-local mechanical stress can be examined.
  • the piece of tissue to be examined can be cultivated on the surface of the LAPS.
  • part of the tissue pieces can be subjected to mechanical pressure, e.g. through a micrometer screw integrated locally in the chamber.
  • the possibility of spatially resolved measurement of the acidification rate enables long-term stability to be used to examine the metabolism of cells that are exposed to direct mechanical stress, as well as that of neighboring cells that are connected to them via gap or tight junctions.
  • Tumor cells react with a number of cells. This interaction can lead to cell proliferation, phagocytosis or cell death on the target cells.
  • Follicular cells etc.
  • the spatially resolved determination allows a direct comparison of the effectiveness of the medication.
  • the use of the invention is further preferred for studies for the spatially resolving detection of the secretion and secretion of messenger substances, ions and transmitters, for the detection of the interaction of the secreting cell and the receptive cell, i.e. the messenger receiving cell (e.g. insulin production), secretion and absorption by the recipient cell, or for studies on neuronal cells to demonstrate pre- and post-synaptic effects.
  • the secreting cell and the receptive cell i.e. the messenger receiving cell (e.g. insulin production), secretion and absorption by the recipient cell
  • neuronal cells e.g. the active substance post-synaptically - e.g. Dopamine.
  • control cells and recombinants e.g. cells equipped with a target protein for quick identification of an active substance.
  • the use of the invention is preferred for the spatially resolved, parallel investigation of effects of an active ingredient on cells which have different receptors (for example receptor proteins of a family with different subunit compositions) for the rapid determination of the receptor selectivity of the active ingredient. Furthermore, the use of the invention is preferred for spatially resolved studies for the detection of physiological effects which are triggered by the interaction of homogeneous or heterogeneous cell populations (eg cell type 1 acidifies medium, cell type 2 reacts to this acidification of cell type 1).
  • Also preferred is the use of the invention for determining the migration behavior of cells (determining the creep speed of the cells from a first position (i.e. above a first light source) to a second position 2 (e.g. formation of fruiting bodies, dictyostellium)).
  • position 1 is also preferred for the use of the invention for the examination of bacteria (position 1) to define the protective cover formed therefrom for spatially resolving detection by interaction with an adjacent cell in position 2 (for example Heliobacter pylori, determination of the width of the alkaline protective cover).
  • the use of the invention is preferred for (i) the determination of the amount of secretion and distance from substances which are released by a cell type 1 (position 1) and which can be interpreted in the further positions by using sensory reference cells (quality control); (ii) the investigation of neuronal primary cultures or neuronal thin tissue sections to demonstrate the interaction of neurons (normal state).
  • lesions By setting lesions (interrupting the neural connection) changes in the signal transmission in the spatially resolving area, for example from position 1 versus position 2, can be examined; (iii) the determination of differential nutrient media in terms of their cell specificity and adhesion specificity; (iv) determining the interaction of a specific substance with cells which have one (position 1), two (position 2), three or more (position 3) signal receivers for this substance, for example receptors, for determining the simultaneous and location-mediated specificity of the substance ; (v) determining cell-cell interaction of cells of the same type and cells of different types; (vi) the study of prokaryotes, eukaryotes and viruses; and (vii) use in drug screening, diagnostics, toxicity testing and quality control.
  • the invention will be explained with reference to the drawings. Show it:
  • Fig. 1 shows the basic structure of the device of a preferred one
  • FIG. 2 shows the sequential activation of the light sources; and FIG. 3 the electronic control of the device according to the invention. 4 shows a spatially resolved measurement of the reaction of Chinese Hamster Ovary (CHO).
  • FIG. 1 of the device 1 according to the invention four light sources LED 1 to LED 4 are shown. These are arranged below the measuring chamber 2 in order to generate the local photocurrent at the different positions.
  • the active ingredient is supplied or removed via inlet 6 or outlet 7.
  • a sawtooth-shaped voltage U (t) is applied between the bath electrode 3 and the back contact 10 and the photocurrent I is measured as a function of U (t).
  • U (t) serves as a trigger signal for the control device 8, which controls the light sources via corresponding connecting lines 9.
  • the device according to the invention illustrates the operation of the device according to the invention or the control of the light sources. As shown, the four LEDs arranged below the measuring chamber 2 are operated in sequence for the duration of a voltage ramp. After all four diodes have been activated in sequence, the measurement continues with the first diode. In this way, several measurement cycles are run through in succession.
  • the device according to the invention has a control circuit 8 for controlling the four LEDs as shown in FIG. 3.
  • FIG. 4 illustrates a spatially resolved measurement of the reaction of Chinese Hamster Ovary (CHO) cells to the active ingredient carbachol using an embodiment of the device according to the invention.
  • positions 1 and 3 cells of the wild type (without muscarinic receptor of type 1) and in positions 2 and 4 genetically manipulated cells (with muscarinic receptor of type 1) are applied Service. Only the latter react with an increase in the acidification rate when carbachol is added.

Abstract

The invention relates to a device and a method for a local resolution study of cross-linked cells and/or cell systems, a diagnostic use therefor, and a use for the study of active ingredients.

Description

VORRICHTUNG UND VERFAHREN ZUM ORTSAUFGELÖSTEN UNTERSUCHEN VERNETZTER ZELLEN UND /ODER ZELLSYSTEMEN UND VERWENDUNG FÜR DIE WIRKSTOFFUNTERSUCHUNG MITTELS EINES MICROPHYSIOMETERSDEVICE AND METHOD FOR LOCALLY EXAMINED NETWORKED CELLS AND / OR CELL SYSTEMS AND USE FOR THE ACTIVE SUBSTANCE EXAMINATION BY MEANS OF A MICROPHYSIOMETER
Die Erfindung betrifft eine Vorrichtung und ein Verfahren zum ortsaufgelösten Untersuchen vernetzter Zellen und/oder Zellsystemen und deren Einsatz für diagnostische Anwendungen und für die Wirkstoffuntersuchung.The invention relates to a device and a method for the spatially resolved examination of networked cells and / or cell systems and their use for diagnostic applications and for the investigation of active substances.
Zur Untersuchung des Stoffwechsels von lebenden Zellen, Zellsystemen oder Zellkompartimenten durch Bestimmung der Ansäuerung- oder Alkalisierungraten des Mediums durch die Zellen ist es bekannt, eine zeitaufgelöste Messung des pH-Wertes mit der Technik des Licht-adressierbaren potentiometrischen Sensors (LAPS) zu verwenden (Parce, J. W., H. M. McConnell, et al. (1990), Methods and apparatus for detecting the effect of cell affecting agents on living cells, Molecular devices Corporation; McConnell, H. M., J. C. Owicki, et al. (1992), "The Cytosensor Microphysiometer: Biological Applications of Silicon technology", 257:1906-1912). In einem bekannten Cytosensor® Mikrophysiometer (siehe EP-A-0 394 406) werden vier bzw. acht Messkammern (LAPS) parallel betrieben. Jede Kammer wird durch eine separate Lichtquelle angesteuert und hat einen unabhängigen Anschluss (Kanal), mit welchem der Photostrom und damit die Ansäuerung- oder Alkalisierung der die Zellen umgebenden Lösung gemessen wird. Diese Daten werden gesammelt und einzeln durch Ableitung der Ansäuerung/Alkalisierungin sogenannte ,raw data' umgewandelt. Somit erhält man für jede Kammer und jeden einzelnen Messkanal entsprechende kontinuierlich auswertbare Daten. Jeder LAPS wird dabei durch die Lichtquelle von der Unterseite in seinem Mittelpunkt beleuchtet. Die Ortsauflösung beträgt in dieser Konfiguration ca. einen Millimeter (Nakao, M., S. Inoue, et al. (1996), "High-resolution pH imaging sensor for microscopic observation of microorganisms", Sensors & Actuators B 34: 234-239). Dies bedeutet, dass der in jeder Kammer gemessene Photostrom von der Ansäuerung oder Alkalisierung der auf der Chipfläche verteilten Zellen abhängt, die sich auf dieser Fläche von ca. einem Quadratmillimeter befinden.To investigate the metabolism of living cells, cell systems or cell compartments by determining the acidification or alkalization rates of the medium by the cells, it is known to use a time-resolved measurement of the pH value using the technique of the light-addressable potentiometric sensor (LAPS) (Parce , JW, HM McConnell, et al. (1990), Methods and apparatus for detecting the effect of cell affecting agents on living cells, Molecular devices Corporation; McConnell, HM, JC Owicki, et al. (1992), "The Cytosensor Microphysiometer : Biological Applications of Silicon technology ", 257: 1906-1912). In a known Cytosensor ® microphysiometer (see EP-A-0394406) are operated in parallel four or eight measuring chambers (LAPS). Each chamber is controlled by a separate light source and has an independent connection (channel) with which the photocurrent and thus the acidification or alkalization of the solution surrounding the cells is measured. This data is collected and individually converted into so-called 'raw data' by deriving the acidification / alkalization. Correspondingly continuously evaluable data is thus obtained for each chamber and each individual measuring channel. Each LAPS is illuminated by the light source from the bottom in its center. The spatial resolution in this configuration is approximately one millimeter (Nakao, M., S. Inoue, et al. (1996), "High-resolution pH imaging sensor for microscopic observation of microorganisms", Sensors & Actuators B 34: 234-239 ). This means that the photocurrent measured in each chamber depends on the acidification or alkalization of the cells distributed on the chip area, which are located on this area of approximately one square millimeter.
In dem bekannten System wird bislang in jeder Messkammer die Ansäuerung oder Alkalisierung des gesamten Zellverbandes gemessen. Eine ortsaufgelöste Messung ist nicht möglich. Es ist ein LAPS bekannt, der eine parallele Messung der Ansäuerungs- oder Alkalisierungsrate an mehreren Punkten ermöglicht (Alajoki, M.L., G.T. Baxter, et al. (1997), High-Performance Microphysiometry in Drug Discovery (Kapitel 25), High Throughput Screening: The Discovery of Bioactive Substances, J.P. Devlin, Marcel Dekker (New York): 427-442). Bei dieser Anordnung werden beispielsweise vier Zelltypen mit acht Wirkstoffen in Verbindung gebracht. Dieser LAPS ist jedoch für eine Verwendung mit dem bekannten Cytosensor® Mikrophysiometer nicht geeignet. Ferner existieren Systeme für Messungen an Zellverbänden mittels Laserstrahlabtastung, (Nakao, M.S. Inoue, et al. (1995), "Observation of microorganism colonies using a scanning-laser-beam pH-sensing microscope", J. Ferm. Bioeng. 79(2): 163-166), die jedoch ebenfalls nicht mit dem bekannten Cytosensor® Mikrophysiometer kompatibel sind.In the known system, the acidification or alkalization of the entire cell structure has so far been measured in each measuring chamber. A spatially resolved measurement is not possible. A LAPS is known which enables a parallel measurement of the acidification or alkalization rate at several points (Alajoki, ML, GT Baxter, et al. (1997), High-Performance Microphysiometry in Drug Discovery (Chapter 25), High Throughput Screening: The Discovery of Bioactive Substances, JP Devlin, Marcel Dekker (New York): 427-442). With this arrangement, for example, four cell types are associated with eight active substances. However, this LAPS is not suitable for use with the known Cytosensor ® microphysiometer. There are also systems for measurements on cell assemblies using laser beam scanning, (Nakao, MS Inoue, et al. (1995), "Observation of microorganism colonies using a scanning-laser-beam pH-sensing microscope", J. Ferm. Bioeng. 79 (2 ): 163-166), which, however, are also not compatible with the known Cytosensor ® microphysiometer.
Der Erfindung liegt die Aufgabe zugrunde, eine Vorrichtung und ein Verfahren für ortsaufgelöste Untersuchungen vernetzter Zellen und/oder Zellkompartimente bereitzustellen. Diese Aufgabe wird mit den Merkmalen der Ansprüche gelöst.The object of the invention is to provide a device and a method for spatially resolved investigations of networked cells and / or cell compartments. This object is achieved with the features of the claims.
Die Erfindung geht von dem Grundgedanken aus, für eine einzige Messkammer eines Mikrophysiometers mehrere Lichtquellen vorzusehen. Dadurch kann in dieser Kammer, und somit an jeweils einem LAPS, der Chip an mehreren unterschiedlichen (vorzugsweise vier bis zu acht oder gar 25 verschiedenen) Stellen der Oberfläche aktiviert werden. Hierzu werden erfindungsgemäß entsprechend vorzugsweise vier bis zu acht oder gar 25 Lichtquellen, die zur Aktivierung des Chips dienen, an der Unterseite angebracht. Für je eine Kammer ist eine Referenzelektrode, ein Anschluss (Kanal) an den LAPS zur Messung des Photostroms und ein Flüssigkeitszuführungs bzw. -abflusssystem vorgesehen.The invention is based on the basic idea of providing several light sources for a single measuring chamber of a microphysiometer. As a result, the chip can be activated at several different (preferably four to eight or even 25 different) locations on the surface in this chamber, and thus on one LAPS each. For this purpose, according to the invention, preferably four to eight or even 25 light sources, which serve to activate the chip, are attached to the underside. A reference electrode, a connection (channel) to the LAPS for measuring the photocurrent and a liquid supply or drainage system are provided for each chamber.
Erfindungsgemäß werden zur ortsaufgelösten Messung nicht alle Lichtquellen simultan verwendet bzw. angesteuert. Um die Ansäuerung oder Alkalisierung an jedem der mehreren (vorzugsweise bis zu 25) Punkte separat bestimmen zu können, werden die Lichtquellen in einem sequenziellen Zyklus betrieben. Dabei wird zu einem Zeitpunkt T1 zuerst Lichtquelle Nummer 1 aktiviert, womit der Photostrom zu dieser Zeit von der Ansäuerung oder Alkalisierung der Zellen abhängt, welche sich auf dem LAPS innerhalb einer Fläche von ca. einem Quadratmillimeter an der von Lichtquelle Nummer 1 beleuchteten Stelle befinden. Lichtquelle Nummer 1 wird so lange betrieben, bis eine vollständige Photostrom-Spannungs-Kurve aufgenommen ist. Danach wird zum Zeitpunkt T2 zur Lichtquelle Nummer 2 umgeschaltet, wobei dann der gemessene Photostrom abhängig von der Ansäuerung oder Alkalisierung der Zellen ist, die sich auf dem LAPS an der von Lichtquelle Nummer 2 beleuchteten Fläche befinden. Dieser Wechsel wird bis zur letzten Lichtquelle, vorzugsweise Lichtquelle Nummer 25, fortgesetzt. Danach wird der Zyklus wiederholt, beginnend mit dem Betrieb von Lichtquelle Nummer 1.According to the invention, not all light sources are used or controlled simultaneously for the spatially resolved measurement. In order to be able to separately determine the acidification or alkalization at each of the several (preferably up to 25) points, the light sources are operated in a sequential cycle. At a time T1, light source number 1 is activated first, so that the photocurrent at this time depends on the acidification or alkalization of the cells, which is on the LAPS within an area of approximately one square millimeter at the point illuminated by light source number 1. Light source number 1 is operated until a complete photocurrent-voltage curve is recorded. Then, at time T2, the switch is made to light source number 2, in which case the measured photocurrent depends on the acidification or alkalization of the cells which are located on the LAPS on the surface illuminated by light source number 2. This change continues until the last light source, preferably light source number 25. The cycle is then repeated, starting with the operation of light source number 1.
Alternativ ist eine simultane bzw. eine gesteuerte Aktivierung der Lichtquellen vorgesehen (z.B. alle Lichtquellen gleichzeitig oder in einzelnen Gruppen (etwa Lichtquellen Nr. 2, 4, 6, usw.)).Alternatively, simultaneous or controlled activation of the light sources is provided (e.g. all light sources simultaneously or in individual groups (e.g. light sources No. 2, 4, 6, etc.)).
Für einen Messaufbau mit beispielsweise acht Lichtquellen werden innerhalb eines Messzyklus sequentiell die Photostrom-Spannungs-Kurven an allen acht (bzw. bis zu 25) Punkten des LAPS aufgenommen, unter denen sich je eine der acht Lichtquellen befindet. Es wird dann von jeder Photostrom-Spannungskurve der Wendepunkt, also ein einziger Datenpunkt bestimmt. Während eines Zyklus werden in dem einzelnen verwendeten Kanal damit acht Datenpunkte ermittelt, die als sogenannte „raw data" über ein Display angezeigt werden können. Dabei entspricht der i-te (i = 1 , 2, ..., 8) Datenpunkt dem Wendepunkt der Photostrom-Spannungs-Kurve an der von der i-ten Lichtquelle beleuchteten Fläche des LAPS. Diese Daten werden anschließend unter Zuordnung zu Messkanälen ausgewertet. Der erste Kanal enthält die Datenpunkte 1 , 9, 17,...., der zweite Kanal die Datenpunkte 2, 10, 18,... usw., wobei die Datenpunkte 1 bis 8 dem ersten Messzyklus, die Datenpunkte 9 bis 16 dem nächsten Messzyklus entsprechen usw. Damit enthält jeder Kanal den zeitabhängigen Verlauf des Wendepunktes der Photostrom-Spannungs-Kurve von einer separat beleuchteten Stelle des LAPS.For a measurement setup with, for example, eight light sources, the photocurrent-voltage curves are recorded sequentially at all eight (or up to 25) points of the LAPS, under each of which one of the eight light sources is located. The turning point, that is to say a single data point, is then determined from each photocurrent voltage curve. During a cycle, eight data points are determined in the individual channel used, which can be shown as so-called "raw data" on a display. The i-th (i = 1, 2, ..., 8) data point corresponds to the turning point the photocurrent-voltage curve on the surface of the LAPS illuminated by the i-th light source. This data is then evaluated with assignment to measurement channels. The first channel contains data points 1, 9, 17, ...., the second channel the Data points 2, 10, 18, ... etc., whereby data points 1 to 8 correspond to the first measurement cycle, data points 9 to 16 correspond to the next measurement cycle etc. Each channel thus contains the time-dependent course of the inflection point of the photocurrent-voltage curve from a separately illuminated area of the LAPS.
Somit ermöglicht die vorliegende Erfindung die sequentielle Untersuchung an mehreren (vorzugsweise bis zu 25) Messstellen auf der Oberfläche eines LAPS und damit eines Kanals, bezogen auf die Konfiguration des Mikrophysiometers. Die vorliegende Erfindung erlaubt die spezifische Beobachtung des Stoffwechsels von lebenden Zellen, Zellsystemen oder Zellkompartimente durch Bestimmung der Ansäuerungs- oder Alkalisierungsraten des Mediums durch die Zellen, was durch zeitaufgelöste Messung des pH-Wertes mit der Technik des Licht-adressierbaren potentiometrischen Sensors (LAPS) realisiert ist. Bei den verwendbaren Zellen kann es sich um Zellkulturen (einschließlich Bakterien- oder Pilzkulturen) oder auch um frisches, tierisches, menschliches (inklusive Biopsiematerial) oder pflanzliches Zellmaterial, handeln.The present invention thus enables the sequential examination at several (preferably up to 25) measuring points on the surface of a LAPS and thus of a channel, based on the configuration of the microphysiometer. The present invention permits the specific observation of the metabolism of living cells, cell systems or cell compartments by determining the acidification or alkalization rates of the medium by the cells, which is achieved by time-resolved measurement of the pH with the technology of the light-addressable potentiometric sensor (LAPS) is. The cells that can be used can be cell cultures (including bacterial or fungal cultures) or also fresh, animal, human (including biopsy material) or plant cell material.
Die vorliegende Erfindung ist in einer bevorzugten Ausführungsform mit dem bekannten Cytosensor® Mikrophysiometer betreibbar bzw. koppelbar, so dass damit die ortsaufgelöste Messung der Ansäuerungs- oder Alkalisierungsrate des Mediums durch zum Beispiel vernetzte Zellverbände möglich ist.The present invention is in a preferred embodiment with the known Cytosensor ® microphysiometer, so that thus the spatially resolved measurement of the acidification of the medium or Alkalisierungsrate operable or coupled possible by for example cross-linked cell aggregates.
Das erfindungsgemäße ortsauflösende Messsystem weist drei Aspekte auf: 1) Die Integration der mehreren (beispielsweise acht) Lichtquellen in eine Messkammer; 2) die Elektronik zum zyklischen Umschalten zwischen den Lichtquellen; und 3) die Software, die die gemessenen Daten aus einem Kanal in mehrere (vorzugsweise vier bis zu -25) Kanäle aufspaltet und analysiert.The spatially resolving measuring system according to the invention has three aspects: 1) the integration of the multiple (for example eight) light sources in one measuring chamber; 2) the electronics for cyclical switching between the light sources; and 3) the software that splits and analyzes the measured data from one channel into several (preferably four to -25) channels.
Weiterhin betrifft die Erfindung eine Vorschrift zum a) direkten Aufbringen der Zellkulturen auf den LAPS, b) eine mögliche Strukturierung des LAPS in sensitive und insensitive Bereiche und c) ein optimales Stimulationssystem.Furthermore, the invention relates to a regulation for a) direct application of the cell cultures to the LAPS, b) possible structuring of the LAPS into sensitive and insensitive areas and c) an optimal stimulation system.
Das Aufbringen von Zellen kann erfindungsgemäß auf spezifischen Trägern erfolgen, die separierte Bereiche besitzen können, um damit eine Abtrennung für die Zellen zu erhalten. Die Zellen können z.B. direkt auf dem LAPS kultiviert werden. Dadurch ist es möglich, verschiedene Stellen des LAPS und des Trägers mit unterschiedlichen Zellen zu besiedeln. Für das Aufbringen verschiedener räumlich getrennter Kulturen können dabei mehrere verschiedene Verfahren angewendet werden:According to the invention, cells can be applied to specific carriers, which can have separated regions, in order to obtain a separation for the cells. The cells can e.g. can be cultivated directly on the LAPS. This makes it possible to colonize different locations on the LAPS and the support with different cells. Several different methods can be used to apply different spatially separated cultures:
Im Falle von Gewebeschnitten/ -Stanzpräparaten werden die Zellgewebestücke auf verschiedene Stellen des LAPS aufgebracht und z.B. fixiert oder dort anwachsen gelassen. Entscheidend ist z.B., dass die physiologische Funktion der Zellen dabei voll erhalten bleiben. So kann gewährleistet werden, dass die zunächst räumlich getrennten Kulturen durch Wachstum auf dem Chip Kontaktstellen untereinander ausbilden. Im Falle von vereinzelten Zellsuspensionen werden diese hochkonzentriert an verschiedenen Stellen des LAPS aufgebracht. Um eine Vermischung der Zellkulturen während der Adhäsionsphase (ca. eine-24 Stunden) zu verhindern, kann dazu die Oberfläche des LAPS unterteilt werden. Beispielsweise können Abgrenzungen durch dünne Linien, die aus einem hydrophoben Material bestehen, auf der LAPS Oberfläche aufgebracht werden. Dadurch verbleiben die lokal aufgebrachten Zellen in ihrem jeweiligen Kompartiment. Eine solche Strukturierung ist entweder durch Stempeln oder mittels photolithographischer Verfahren möglich. In einer besonderen Ausführungsform weisen diese Linien Unterbrechungen auf, und werden möglichst dünn aufgebracht, damit nach der Adhäsion auch Zellen oder Zellfortsätze über diese Linien hinwegwachsen können. Nach der Adhäsion wird den Zellen das zur Kultivierung nötige Medium zugegeben. Auch hier ist es wichtig, dass die Fähigkeit des Zellwachstums erhalten bleibt und die Zellen aus den unterschiedlichen Bereichen Kontaktstellen untereinander ausbilden oder in anderer Weise, zum Beispiel über Sekretion von Hormonen oder anderen Signalstoffen, wechselwirken können.In the case of tissue sections / punch preparations, the cell tissue pieces are applied to various points on the LAPS and, for example, fixed or allowed to grow there. It is crucial, for example, that the physiological function of the cells is full remain. In this way it can be ensured that the initially spatially separated cultures form contact points with one another by growth on the chip. In the case of isolated cell suspensions, these are applied in highly concentrated form at various points on the LAPS. To prevent mixing of the cell cultures during the adhesion phase (approx. 24 hours), the surface of the LAPS can be divided. For example, boundaries can be applied to the LAPS surface by thin lines made of a hydrophobic material. As a result, the locally applied cells remain in their respective compartments. Such structuring is possible either by stamping or by means of photolithographic processes. In a special embodiment, these lines have interruptions and are applied as thinly as possible so that cells or cell extensions can also grow over these lines after the adhesion. After the adhesion, the medium necessary for cultivation is added to the cells. Here, too, it is important that the ability of the cell to grow is maintained and that the cells from the different areas form contact points with one another or can interact in another way, for example via secretion of hormones or other signal substances.
Die Abtrennung kann z. B. so gehalten sein, dass sie die Zellen, die punktuell aufgetragen werden, komplett und über die Zeitdauer der Messung voneinander getrennt hält, oder ein Zusammenwachsen bzw. die Migration der Zellen zueinander gewährleistet.The separation can e.g. B. be held so that it keeps the cells, which are applied selectively, completely and separately from one another over the duration of the measurement, or ensures that the cells grow together or migrate to one another.
Alternativ kann auf den Chip während der Adhäsionsphase eine Trennvorrichtung, die mehrere Kammern aufweist, aufgelegt werden, welche nach der Adhäsionphase abgenommen werden kann. Hier hat sich insbesondere eine Vorrichtung aus Teflon bewährt.Alternatively, a separating device which has several chambers can be placed on the chip during the adhesion phase and can be removed after the adhesion phase. A device made of Teflon has proven particularly useful here.
Alternativ können Zellen (Zellsuspensionen) können ebenfalls punktuell aufgebracht werden, indem sie durch Nadeln oder Pipetten in ein Polymergel (insbesondere Agarosegel) injiziert werden, indem ihre Beweglichkeit aufgehoben oder eingeschränkt ist. Für bestimmte Anwendungen kann es wünschenswert sein, eine höhere Ortsauflösung zu benutzen. Soll beispielsweise zur Untersuchung besonders strukturierter Zellgewebeverbände die Ansäuerung über einen besonders kleinen Bereich gemittelt werden, etwa wenn nur eine kleine Region des Zellverbandes die Zellen von Interesse enthält, ist dies mit einem vorstrukturierten LAPS möglich. Der LAPS kann durch partielle Beleuchtung mit UV-Licht in sensitive und nichtsensitive Bereiche, in denen kein Photostrom fließt, unterteilt werden. Beispielsweise ist damit eine Strukturierung eines LAPS durchführbar, so dass bis zu acht ausgezeichnete ca. ein hundertstel Quadratmillimeter große Bereiche darstellbar sind. Bei Beleuchtung eines der aktiven Bereiche kann die über den Bereich von ca. einem hundertstel Quadratmillimeter gemittelte Ansäuerungsrate bestimmt werden.Alternatively, cells (cell suspensions) can also be applied selectively by injecting them into a polymer gel (in particular agarose gel) by needles or pipettes, by reducing or restricting their mobility. For certain applications it may be desirable to use a higher spatial resolution. If, for example, the acidification is to be averaged over a particularly small area in order to examine particularly structured cell tissue associations, for example if only a small region of the cell association contains the cells of interest, this is possible with a pre-structured LAPS. The LAPS can be divided into sensitive and non-sensitive areas in which no photocurrent flows by partial illumination with UV light. For example, a LAPS can be structured so that up to eight excellent areas of approximately one hundredth of a square millimeter can be represented. When one of the active areas is illuminated, the acidification rate averaged over the area of approximately one hundredth of a square millimeter can be determined.
Die Stimulation der Zellen kann auf verschiedene Weise erzielt werden. Durch Zugabe eines Wirkstoffes in das zugeführte Medium kann eine globale Stimulation aller Zellen, die z.B. einen entsprechenden Rezeptor besitzen, ausgelöst und bestimmt werden. Optional ist in der Erfindung auch die lokale Stimulation von bestimmten Zellbereichen möglich. Dies kann entweder durch Mikroinjektion eines Wirkstoffes, über eine Mikrokapillare an einer Stelle der LAPS - Messkammer erfolgen oder durch einen Spannungsreiz (d.h. an einer Stelle des LAPS ist eine metallische Reizmikroelektrode integriert) oder durch lokale Applikation von mechanischem Druck (z.B. durch eine in der LAPS - Messkammer integrierte Mikrometerschraube) erreicht werden.The stimulation of the cells can be achieved in different ways. By adding an active ingredient to the supplied medium, global stimulation of all cells, e.g. possess a corresponding receptor, are triggered and determined. Optionally, local stimulation of certain cell areas is also possible in the invention. This can be done either by microinjection of an active ingredient, via a microcapillary at one point in the LAPS measuring chamber or by a voltage stimulus (ie a metallic stimulus microelectrode is integrated at one point of the LAPS) or by local application of mechanical pressure (e.g. by means of a pressure in the LAPS - Measuring chamber integrated micrometer screw) can be reached.
Erfindungsgemäß wird die Vorrichtung/das Verfahren zur ortsaufgelösten Messung des Stoffwechsels von Zellen verwendet, die entweder funktionell gekoppelt sind, oder die verschiedene Eigenschaften (z.B. Expression unterschiedlicher Rezeptoren) aufweisen und funktioneil getrennt bleiben.According to the invention, the device / method is used for the spatially resolved measurement of the metabolism of cells which are either functionally coupled or which have different properties (e.g. expression of different receptors) and remain functionally separate.
Die im folgenden beschriebenen Anwendungen basieren auf Untersuchungen gekoppelter Zellverbände. Hierzu zählen Synapsen, gap- und tight-junctions, d.h. es findet ein Informationsfluss (physikalischer oder chemischer Art) zwischen benachbarten Zellen statt. Dabei besteht aufgrund der Kopplung optional die Möglichkeit zu nicht-lokaler Stimulation. Die lokale elektrische Aktivität gekoppelter Zellverbände kann auf dem Niveau einzelner Zellen mit Hilfe der patch-clamp Technik beobachtet werden (Fitzsimonds, R.M., H.-J. Song, et al. (1997), "Propagation of activity-dependent synaptic depression in simple neural networks", Nature 388 (July 21): 439-448). Allerdings sind solche Messungen nur über Stunden stabil. Für langzeitstabile Messungen müssen hingegen extrazellulare Techniken verwendet werden. So kann z.B. die extrazelluläre elektrische Aktivität von Hirnschnitten mit Hilfe extrazellulärer Mikroelektroden ortsaufgelöst verfolgt werden (Egert, U., B. Schlosshauer, et al. (1998), "A novel organotypic long- term culture of the rat hippocampus on substrate-integrated multielectrode arrays", Brain Research Protocols 2(4): 229-242). Ein entsprechendes System ist kommerziell, z.B. vom Naturwissenschaftlich Medizinischen Institut (NMI) in Reutlingen erhältlich. Derartige Anordnungen sind aber nur auf elektrisch aktive Zellen begrenzt anwendbar.The applications described below are based on studies of coupled cell groups. These include synapses, gap and tight junctions, ie there is a flow of information (physical or chemical) between neighboring cells. Due to the coupling, there is the option of non-local stimulation. The local electrical activity of coupled cell assemblies can be observed at the level of individual cells using the patch-clamp technique (Fitzsimonds, RM, H.-J. Song, et al. (1997), "Propagation of activity-dependent synaptic depression in simple neural networks ", Nature 388 (July 21): 439-448). However, such measurements are only stable for hours. In contrast, extracellular techniques must be used for long-term stable measurements. For example, the extracellular electrical activity of brain sections can be monitored with the aid of extracellular microelectrodes in a spatially resolved manner (Egert, U., B. Schlosshauer, et al. (1998), "A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays ", Brain Research Protocols 2 (4): 229-242). A corresponding system is commercially available, for example from the Natural Science Medical Institute (NMI) in Reutlingen. Such arrangements can only be used to a limited extent on electrically active cells.
In dem erfindungsgemäßen System wird anstelle des extrazellulären Potentials die Ansäuerung oder Alkalisierung des die Zellen umgebenden Mediums durch Bestimmung des pH-Wertes gemessen. Dies ist prinzipiell für alle Arten von Zellen möglich. Die im folgenden beschriebenen Anwendungen bzw. Beispiele lassen sich auch mit Feldern von Feldeffekt-Transistoren koppeln.In the system according to the invention, instead of the extracellular potential, the acidification or alkalization of the medium surrounding the cells is measured by determining the pH. In principle, this is possible for all types of cells. The applications or examples described below can also be coupled with fields of field effect transistors.
Beispiel 1 :Example 1 :
Das Gehirn bildet hochkomplexe Zusammensetzungen von gekoppelten Nervenzellen ab, die in funktioneile Regionen unterteilt sind. Durch Synapsen sind die Nervenzellen untereinander vernetzt und können dadurch miteinander kommunizieren. Erkrankungen, wie z.B. Epilepsie, weisen kommunikative Dysfunktionen an den Zellen auf. Dies wird erfindungsgemäß folgendermaßen untersucht. Es werden Schnitte oder Stanzpräparate der betroffenen Regionen entnommen und an verschiedenen Stellen des LAPS adhaeriert. Nervenzellen aus diesem Bereich bilden nach einigen Tagen Zellfortsätze (Dendriten und Axone) aus, die sich entlang des LAPS ausbreiten. Durch Synapsen werden dabei Kontakte zwischen individuellen Neuronen hergestellt, d.h. nach mehreren Tagen sind die unterschiedlichen Regionen durch Synapsen vernetzt. In diesem Beispiel werden aus Gründen der Übersichtlichkeit zwei Stanzpräparate (Regionen 1, 2) verwendet. Diese werden über zwei verschiedene Lichtquellen Nummer 1 und 2 aktiviert. Der durch Lichtquelle 1 bzw. 2 verursachte Photostrom analysiert die Ansäuerung der Zellen aus Region 1 bzw. 2. Beispielsweise lassen sich Zellen aus Region 1 spezifisch stimulieren. Dies kann entweder durch Zugabe eines Wirkstoffes in das zugeführte Medium sein, welcher spezifisch auf die Zellen in Region 1 wirkt oder durch lokale Mikroinjektion (Mikroperfusion) eines Wirkstoffes in Region 1 oder durch lokale elektrische Stimulation mit einer Mikroelektrode von Region 1. Die Ansäuern ngsrate bzw. der Metabolismus der Zellen aus Region 1 wird dann innerhalb von Sekunden bis Minuten beeinflusst und kann dann abgelesen werden. Da Region 2 mit Region 1 über neuronale Synapsen gekoppelt ist, kann der veränderte Metabolismus in Region 1 auch den Metabolismus der Zellen in Region 2 beeinflussen. Dies kann erst nach einiger Zeit (Minute bis Sekunden) erfolgen. In dieser Konfiguration ermöglicht die erfindungsgemäße Vorrichtung die Ansäuerungsraten beider Regionen zu trennen, parallel zu messen und das komplexe Zusammenspiel beider vernetzter Regionen langzeitstabil zu untersuchen.The brain depicts highly complex compositions of coupled nerve cells that are divided into functional regions. The nerve cells are networked with each other through synapses and can therefore communicate with each other. Diseases, such as epilepsy, have communicative dysfunctions on the cells. This is investigated according to the invention as follows. Cuts or punch preparations from the affected regions are removed and adhered to various parts of the LAPS. After a few days, nerve cells from this area form cell processes (dendrites and axons) that spread along the LAPS. Through synapses, contacts are made between individual neurons, ie after several days the different regions are networked through synapses. For reasons of clarity, two stamping preparations (regions 1, 2) are used in this example. These are activated by two different light sources number 1 and 2. The photocurrent caused by light source 1 or 2 analyzes the acidification of the cells from region 1 or 2. For example, Specifically stimulate cells from region 1. This can be either by adding an active ingredient in the supplied medium that acts specifically on the cells in region 1 or by local microinjection (micro perfusion) of an active ingredient in region 1 or by local electrical stimulation with a microelectrode from region 1. The acidification rate or The metabolism of the cells from region 1 is then influenced within seconds to minutes and can then be read off. Since region 2 is coupled to region 1 via neuronal synapses, the altered metabolism in region 1 can also influence the metabolism of the cells in region 2. This can only take some time (minutes to seconds). In this configuration, the device according to the invention enables the acidification rates of the two regions to be separated, measured in parallel and the complex interplay of the two networked regions to be examined in a long-term stable manner.
Beispiel 2: Bestimmte Zelltypen wie z.B. Epithelzellen, Knorpel- und Knochenzellen reagieren auf Druck. Mit Hilfe der Erfindung kann dabei die Auswirkung nicht-lokalen mechanischen Stresses untersucht werden. Dazu kann das zu untersuchende Gewebestück auf der Oberfläche des LAPS kultiviert werden. Während des Experiments kann ein Teil der Gewebestücke mechanischem Druck ausgesetzt werden, z.B. durch eine lokal in der Kammer integrierte Mikrometerschraube. Die Möglichkeit ortaufgelöster Messung der Ansäuerungsrate ermöglicht langzeitstabil sowohl den Stoffwechsel von Zellen zu untersuchen, die direktem mechanischem Stress ausgesetzt sind, als auch den von benachbarten Zellen, welche über gap- bzw. tight-junctions mit diesen verbunden sind.Example 2: Certain cell types such as Epithelial cells, cartilage and bone cells react to pressure. With the help of the invention, the effect of non-local mechanical stress can be examined. For this purpose, the piece of tissue to be examined can be cultivated on the surface of the LAPS. During the experiment, part of the tissue pieces can be subjected to mechanical pressure, e.g. through a micrometer screw integrated locally in the chamber. The possibility of spatially resolved measurement of the acidification rate enables long-term stability to be used to examine the metabolism of cells that are exposed to direct mechanical stress, as well as that of neighboring cells that are connected to them via gap or tight junctions.
Beispiel 3:Example 3:
Tumorzellen reagieren mit einer Reihe von Zellen. Diese Interaktion kann an den Zielzellen zur Zellvermehrung, Phagozytose oder zum Zelltod führen.Tumor cells react with a number of cells. This interaction can lead to cell proliferation, phagocytosis or cell death on the target cells.
Folgende Verwendungen der Erfindung sind hierzu bevorzugt:The following uses of the invention are preferred for this:
a.) Darstellung der Interaktion von Tumorzellen mit Zellgemischen aus Blutzellen zur Bestimmung der Immunantwort der Blutzellen b.) Darstellung der Interaktion von Tumorzellen mit spezifischen Immunzellen (z.B.a.) Representation of the interaction of tumor cells with cell mixtures from blood cells to determine the immune response of the blood cells b.) Representation of the interaction of tumor cells with specific immune cells (e.g.
Makrophagen, T-Zellen, NK (Natural Killerzellen) usw. zur Bestimmung derMacrophages, T cells, NK (Natural Killer cells) etc. to determine the
Immunantwort der Blutzellen c.) Darstellung der Interaktion Tumorzelle und normale Zelle aus dem Gewebe, aus dem die Tumorzelle gewonnen wurde. Diese Bestimmung dient der Evaluierung der Nebenwirkung von Wirkstoffen, die möglichst gezielt an der Tumorzelle reagieren sollen und begleitende Nebeneffekte an den normalen Zellen aus dem gleichen Gewebegebiet gering halten sollen, d.) Darstellung der Interaktion Tumorzelle und normale teilungsaktive Zelle aus einem anderen Gewebe. Diese Untersuchung dient dem Nachweis unspezifischerImmune response of the blood cells c.) Representation of the interaction of the tumor cell and normal cell from the tissue from which the tumor cell was obtained. This determination serves to evaluate the side effects of active substances that should react as specifically as possible to the tumor cell and to minimize accompanying side effects on normal cells from the same tissue area, d.) Representation of the interaction of tumor cell and normal dividing cell from another tissue. This examination serves to prove non-specific
Wirkungen von Wirkstoffen an teilungsaktivem Gewebe, wie z.B. Keimzellen,Effects of active substances on dividing tissue, e.g. Germ cells,
Follikelzellen etc. Die erfindungsgemäße ortsaufgelöste Bestimmung lässt einen direkten Vergleich der Wirksamkeit der Medikamente zu. e.) Darstellung der Interaktion Tumorzelle und Leberzelle unter Inkubation mit einem Wirkstoff, der im Körper in der Leber metabolisiert wird (Metabolisierungs- undFollicular cells etc. The spatially resolved determination allows a direct comparison of the effectiveness of the medication. e.) Representation of the interaction of tumor cell and liver cell under incubation with an active substance that is metabolized in the body in the liver (metabolism and
Aktivierungsnachweis)Activation Detection)
Weiter bevorzugt ist die Verwendung der Erfindung für Studien zum ortsauflösenden Nachweis der Sekretion und Sezemierung von Botenstoffen, Ionen und Transmittern, zum Nachweis der Interaktion von sezernierender Zelle und rezeptiver, also den Botenstoff empfangender Zelle (z.B. Insulinproduktion), Sekretion und Absorption durch Empfängerzelle, oder für Studien an neuronalen Zellen zum Nachweis prä- und postsynaptischer Effekte. Zellen sitzen in Position 1 und wirken präsynaptisch, Zellen auf Position 2 empfangen den Wirkstoff postsynaptisch - z.B. Dopamin.The use of the invention is further preferred for studies for the spatially resolving detection of the secretion and secretion of messenger substances, ions and transmitters, for the detection of the interaction of the secreting cell and the receptive cell, i.e. the messenger receiving cell (e.g. insulin production), secretion and absorption by the recipient cell, or for studies on neuronal cells to demonstrate pre- and post-synaptic effects. Cells sit in position 1 and act presynaptically, cells in position 2 receive the active substance post-synaptically - e.g. Dopamine.
Ferner bevorzugt ist die Verwendung der Erfindung für die ortsaufgelöste Untersuchung von Kontrollzellen und Rekombinanten, z.B. mit einem Targetprotein ausgestatteten Zellen zur schnellen Identifizierung eines Wirkstoffes.Also preferred is the use of the invention for the spatially resolved examination of control cells and recombinants, e.g. cells equipped with a target protein for quick identification of an active substance.
Ferner bevorzugt ist die Verwendung der Erfindung für die ortaufgelöste, parallele Untersuchung von Wirkungen eines Wirkstoffs auf Zellen, welche verschiedene Rezeptoren (z.B. Rezeptoreproteinen einer Familie mit verschiedener Untereinheitenzusammensetzung), zur schnellen Bestimmung der Rezeptorselektivität des Wirkstoffs. Ferner bevorzugt ist die Verwendung der Erfindung für ortsaufgelöste Studien zum Nachweis physiologischer Effekte, die durch die Interaktion homogener bzw. heterogener Zellpopulationen ausgelöst werden (z.B. Zelltyp 1 säuert Medium an, Zelltyp 2 reagiert auf diese Ansäuerung von Zelltyp 1).Furthermore, the use of the invention is preferred for the spatially resolved, parallel investigation of effects of an active ingredient on cells which have different receptors (for example receptor proteins of a family with different subunit compositions) for the rapid determination of the receptor selectivity of the active ingredient. Furthermore, the use of the invention is preferred for spatially resolved studies for the detection of physiological effects which are triggered by the interaction of homogeneous or heterogeneous cell populations (eg cell type 1 acidifies medium, cell type 2 reacts to this acidification of cell type 1).
Ferner bevorzugt ist die Verwendung der Erfindung für die Bestimmung des Migrationsverhaltens von Zellen (Bestimmung der Kriechgeschwindigkeit der Zellen von einer ersten Position (d.h. über einer ersten Lichtquelle) zu einer zweiten Position 2 (z.B. Ausbildung von Fruchtkörpern, Dictyostellium)).Also preferred is the use of the invention for determining the migration behavior of cells (determining the creep speed of the cells from a first position (i.e. above a first light source) to a second position 2 (e.g. formation of fruiting bodies, dictyostellium)).
Ferner bevorzugt ist die Verwendung der Erfindung für die Untersuchung von Bakterien (Position 1) zur Definition der davon ausgebildeten Schutzhülle zum ortsauflösenden Nachweis durch Interaktion mit benachbarter Zelle in Position 2 (Beispielsweise Heliobacter pylori, Bestimmung der Weite der alkalischen Schutzhülle).Also preferred is the use of the invention for the examination of bacteria (position 1) to define the protective cover formed therefrom for spatially resolving detection by interaction with an adjacent cell in position 2 (for example Heliobacter pylori, determination of the width of the alkaline protective cover).
Ferner bevorzugt ist die Verwendung der Erfindung für (i) die Bestimmung der Sekretionsmenge und Wegstrecke von Substanzen, die von einem Zelltyp 1 (Position 1) abgegeben werden und durch Einsatz von sensorischen Referenzzellen auf den weiteren Positionen interpretiert werden können (Qualitätskontrolle); (ii) die Untersuchung von neuronalen Primärkulturen oder neuronalen dünnen Gewebeschnitten zum Nachweis der Interaktion von Neuronen (Normalzustand). Durch Setzen von Läsionen (Unterbrechung der neuronalen Verbindung) können Veränderungen bei der Signalübertragung im ortsauflösenden Bereich z.B. von Position 1 versus Position 2 untersucht werden; (iii) die Bestimmung von differentiellen Nährböden in Bezug auf deren Zellspezifität und Adhäsionsspezifität; (iv) die Bestimmung der Interaktion einer spezifischen Substanz mit Zellen, die für diese Substanz einen (Position 1), zwei (Position 2), drei oder mehr (Position 3) Signalempfänger, z.B. Rezeptoren besitzen zur Bestimmung der gleichzeitigen und ortsvermittelten Spezifität der Substanz; (v) die Bestimmung der Zell-Zellinteraktion von Zellen gleichen Typs und Zellen unterschiedlichen Typs; (vi) die Untersuchung von Prokaryonten, Eukaryonten und Viren; und (vii) den Einsatz im Wirkstoffscreening, im Diagnostikbereich, zur Toxizitätsüberprüfung und zur Qualitätskontrolle. Die Erfindung wird mit Bezug auf die Zeichnungen erläutert. Es zeigen:Furthermore, the use of the invention is preferred for (i) the determination of the amount of secretion and distance from substances which are released by a cell type 1 (position 1) and which can be interpreted in the further positions by using sensory reference cells (quality control); (ii) the investigation of neuronal primary cultures or neuronal thin tissue sections to demonstrate the interaction of neurons (normal state). By setting lesions (interrupting the neural connection) changes in the signal transmission in the spatially resolving area, for example from position 1 versus position 2, can be examined; (iii) the determination of differential nutrient media in terms of their cell specificity and adhesion specificity; (iv) determining the interaction of a specific substance with cells which have one (position 1), two (position 2), three or more (position 3) signal receivers for this substance, for example receptors, for determining the simultaneous and location-mediated specificity of the substance ; (v) determining cell-cell interaction of cells of the same type and cells of different types; (vi) the study of prokaryotes, eukaryotes and viruses; and (vii) use in drug screening, diagnostics, toxicity testing and quality control. The invention will be explained with reference to the drawings. Show it:
Fig. 1 den prinzipiellen Aufbau der erfindungsgemäßen Vorrichtung einer bevorzugtenFig. 1 shows the basic structure of the device of a preferred one
Ausführungsform; Fig. 2 die sequentielle Ansteuerung der Lichtquellen; und Fig. 3 die elektronische Steuerung der erfindungsgemäßen Vorrichtung. Fig. 4 eine ortsaufgelöste Messung der Reaktion von Chinese Hamster Ovary (CHO)-embodiment; 2 shows the sequential activation of the light sources; and FIG. 3 the electronic control of the device according to the invention. 4 shows a spatially resolved measurement of the reaction of Chinese Hamster Ovary (CHO).
Zellen auf den Wirkstoff Carbachol mit Hilfe einer Ausführungsform der erfindungsgemäßen Vorrichtung.Cells on the active ingredient carbachol with the help of an embodiment of the device according to the invention.
In der Ausführungsform gemäß Fig. 1 der erfindungsgemäßen Vorrichtung 1 sind vier Lichtquellen LED 1 bis LED 4 gezeigt. Diese sind unterhalb der Messkammer 2 angeordnet, um den lokalen Photostrom an den verschiedenen Positionen zu erzeugen. Der Wirkstoff wird über den Einlass 6 bzw. Auslass 7 zugeführt bzw. abgeführt. Eine sägezahnförmige Spannung U(t) wird zwischen der Badelektrode 3 und dem Rückkontakt 10 angelegt und der Photostrom I abhängig von U(t) gemessen. Gleichzeitig dient U(t) als Triggersignal für die Steuereinrichtung 8, die die Lichtquellen über entsprechende Verbindungsleitungen 9 angesteuert.In the embodiment according to FIG. 1 of the device 1 according to the invention, four light sources LED 1 to LED 4 are shown. These are arranged below the measuring chamber 2 in order to generate the local photocurrent at the different positions. The active ingredient is supplied or removed via inlet 6 or outlet 7. A sawtooth-shaped voltage U (t) is applied between the bath electrode 3 and the back contact 10 and the photocurrent I is measured as a function of U (t). At the same time, U (t) serves as a trigger signal for the control device 8, which controls the light sources via corresponding connecting lines 9.
Fig. 2 veranschaulicht den Betrieb der erfindungsgemäßen Vorrichtung bzw. die Ansteuerung der Lichtquellen. Wie gezeigt, werden die unterhalb der Messkammer 2 angeordneten vier LEDs der Reihe nach für die Dauer einer Spannungsrampe betrieben. Nachdem alle vier Dioden der Reihe nach angesteuert wurden fährt die Messung erneut mit der ersten Diode fort. Auf diese Weise werden nacheinander mehrere Messzyklen durchlaufen.2 illustrates the operation of the device according to the invention or the control of the light sources. As shown, the four LEDs arranged below the measuring chamber 2 are operated in sequence for the duration of a voltage ramp. After all four diodes have been activated in sequence, the measurement continues with the first diode. In this way, several measurement cycles are run through in succession.
In einer bevorzugten Ausführungsform weist die erfindungsgemäße Vorrichtung eine Steuerschaltung 8 zur Ansteuerung der vier LEDs wie in Fig. 3 gezeigt auf.In a preferred embodiment, the device according to the invention has a control circuit 8 for controlling the four LEDs as shown in FIG. 3.
Fig. 4 veranschaulicht eine ortsaufgelöste Messung der Reaktion von Chinese Hamster Ovary (CHO)-Zellen auf den Wirkstoff Carbachol mit Hilfe einer Ausführungsform der erfindungsgemäßen Vorrichtung. Dabei sind in Position 1 und 3 Zellen des Wildtyps (ohne muskarinischen Rezeptor vom Typ 1) und in Position 2 und 4 genetisch manipulierte Zellen (mit muskarinischen Rezeptor vom Typ 1) aufgebracht worden. Nur letztere reagieren bei Zugabe von Carbachol mit einer Erhöhung der Ansäuerunggsrate. FIG. 4 illustrates a spatially resolved measurement of the reaction of Chinese Hamster Ovary (CHO) cells to the active ingredient carbachol using an embodiment of the device according to the invention. In positions 1 and 3 cells of the wild type (without muscarinic receptor of type 1) and in positions 2 and 4 genetically manipulated cells (with muscarinic receptor of type 1) are applied Service. Only the latter react with an increase in the acidification rate when carbachol is added.

Claims

Patentansprüche claims
1. Vorrichtung zur ortsaufgelösten Untersuchung von Zellen, insbesondere vernetzter Zellen oder Zellsystemen, durch ortsaufgelöste Messung der Ansäuerung der die Zellen umgebenden Lösung, mit mindestens einer Meßkammer (2) für die zu untersuchenden Zellen mit einem Chip (4), dadurch gekennzeichnet, daß für die mindestens eine Meßkammer (2) mehrere Lichtquellen zum ortsaufgelösten Aktivieren des Chips (4) vorgesehen sind.1. Device for spatially resolved examination of cells, in particular networked cells or cell systems, by spatially resolved measurement of the acidification of the solution surrounding the cells, with at least one measuring chamber (2) for the cells to be examined with a chip (4), characterized in that for the at least one measuring chamber (2) is provided with a plurality of light sources for activating the chip (4) in a spatially resolved manner.
2. Vorrichtung nach Anspruch 1, wobei mindestens vier Lichtquellen vorgesehen sind.2. Device according to claim 1, wherein at least four light sources are provided.
3. Vorrichtung nach Anspruch 2, wobei vier bis acht, insbesondere 25 Lichtquellen vorgesehen sind.3. Device according to claim 2, wherein four to eight, in particular 25 light sources are provided.
4. Vorrichtung nach Anspruch 1 , 2 oder 3, ferner mit einer Steuereinrichtung (8) mit der die mehreren Lichtquellen sequentiell ansteuerbar sind.4. The device according to claim 1, 2 or 3, further comprising a control device (8) with which the plurality of light sources can be controlled sequentially.
5. Verfahren zum ortsaufgelösten Untersuchen vernetzter Zellen und/oder Zellsystemen in einer Meßkammer (2) mit einem Chip (4), mit den Schritten:5. A method for the spatially resolved examination of networked cells and / or cell systems in a measuring chamber (2) with a chip (4), with the steps:
Bereitstellen mehrerer Lichtquellen zum Aktivieren des Chips (4); Sequentielles Ansteuern der mehreren Lichtquellen;Providing a plurality of light sources for activating the chip (4); Sequential control of the multiple light sources;
Aufnehmen einer Photostrom-Spannungskurve abhängig von der Ansäuerung und/oder Alkalisierung der Zellen für jede Lichtquelle.Record a photocurrent voltage curve depending on the acidification and / or alkalization of the cells for each light source.
6. Verfahren nach Anspruch 5, ferner mit dem Schritt Ermitteln des Wendepunktes einer jeden Photostrom-Spannungskurve.6. The method of claim 5, further comprising the step of determining the inflection point of each photocurrent voltage curve.
7. Verfahren nach Anspruch 5 oder 6, wobei die Lichtquellen der Reihe nach oder in einer vorbestimmten Abfolge angesteuert werden.7. The method according to claim 5 or 6, wherein the light sources are driven in sequence or in a predetermined sequence.
8. Computerprogrammprodukt mit Programmcodemitteln, die auf einem computerlesbaren Datenträger gespeichert sind, um das Verfahren nach Anspruch 5, 6 oder 7 auszuführen, wenn das Programmprodukt auf einem Computer ausgeführt wird.8. Computer program product with program code means, which are stored on a computer-readable data carrier, in order to achieve the method according to claim 5, 6 or 7 when the program product is executed on a computer.
9. Verwendung der Vorrichtung nach einem der Ansprüche 1 bis 4 oder des Verfahrens nach Anspruch 5, 6 oder 7 oder des Computerprogrammprodukts nach9. Use of the device according to one of claims 1 to 4 or of the method according to claim 5, 6 or 7 or of the computer program product
Anspruch 8 in der Diagnostik oder für die Wirkstoffuntersuchung. Claim 8 in diagnostics or for drug testing.
PCT/EP2001/009752 2000-08-24 2001-08-23 Device and method for a local resolution study of cross-linked cells and/or cell systems and a use for the study of active ingredients using a microphysiometer WO2002016936A1 (en)

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