EP1590652A1 - Procede de triage et d'identification de cellules a plusieurs parametres et dispositif correspondant - Google Patents

Procede de triage et d'identification de cellules a plusieurs parametres et dispositif correspondant

Info

Publication number
EP1590652A1
EP1590652A1 EP04707905A EP04707905A EP1590652A1 EP 1590652 A1 EP1590652 A1 EP 1590652A1 EP 04707905 A EP04707905 A EP 04707905A EP 04707905 A EP04707905 A EP 04707905A EP 1590652 A1 EP1590652 A1 EP 1590652A1
Authority
EP
European Patent Office
Prior art keywords
examination
particles
carrier
cells
cell
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.)
Withdrawn
Application number
EP04707905A
Other languages
German (de)
English (en)
Inventor
Torsten Müller
Stefan Hummel
Annette Pfennig
Gabriele Gradl
Axel Bonsen
Thomas Schnelle
Rüdiger Meyer
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.)
PerkinElmer Cellular Technologies Germany GmbH
Original Assignee
Evotec Technologies GmbH
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
Priority claimed from DE10320956A external-priority patent/DE10320956B4/de
Application filed by Evotec Technologies GmbH filed Critical Evotec Technologies GmbH
Publication of EP1590652A1 publication Critical patent/EP1590652A1/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1456—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals
    • G01N15/1459—Optical investigation techniques, e.g. flow cytometry without spatial resolution of the texture or inner structure of the particle, e.g. processing of pulse signals the analysis being performed on a sample stream
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M47/00—Means for after-treatment of the produced biomass or of the fermentation or metabolic products, e.g. storage of biomass
    • C12M47/04—Cell isolation or sorting
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1031—Investigating individual particles by measuring electrical or magnetic effects
    • G01N15/12—Investigating individual particles by measuring electrical or magnetic effects by observing changes in resistance or impedance across apertures when traversed by individual particles, e.g. by using the Coulter principle
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1468—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle
    • G01N15/147—Optical investigation techniques, e.g. flow cytometry with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/1484—Optical investigation techniques, e.g. flow cytometry microstructural devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/1023—Microstructural devices for non-optical measurement
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N15/149—Optical investigation techniques, e.g. flow cytometry specially adapted for sorting particles, e.g. by their size or optical properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1006—Investigating individual particles for cytology
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1019—Associating Coulter-counter and optical flow cytometer [OFC]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N2015/1028—Sorting particles
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1477—Multiparameters
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume or surface-area of porous materials
    • G01N15/10—Investigating individual particles
    • G01N15/14—Optical investigation techniques, e.g. flow cytometry
    • G01N2015/1497—Particle shape
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/00029—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor provided with flat sample substrates, e.g. slides
    • G01N2035/00099—Characterised by type of test elements
    • G01N2035/00158—Elements containing microarrays, i.e. "biochip"

Definitions

  • the invention relates to an examination method for preferably biological particles, in particular for the examination of biological cells in a cell sorter, according to claim 1 and a corresponding examination device according to claim 14.
  • a sorting device which consists of a dielectrophoretic electrode arrangement arranged downstream in the carrier stream behind the dielectrophoretic cage.
  • a disadvantage of the known examination method described above is that the cells to be examined are in a of a sample are often very different.
  • the target cells In the case of very heterogeneous samples, from which, for example, certain target cells are identified by a method and these target cells are then to be isolated, the target cells often make up only a small proportion of the total sample.
  • the other cells do not have the desired properties or are no longer vital, ie already dead.
  • it often happens that the cells are not completely isolated, but that some cells pass through the system as an aggregate of two or more cells. This is an undesirable result.
  • the detailed examination of individual cells or aggregates in a field cage is a time-consuming process, so that the examination of the entire cell sample in the field cage would take a very long time.
  • the invention is therefore based on the object of improving the known examination method described above in such a way that an examination of biological cells of no interest (e.g. dead cells) or cell clumps in the dielectrophoretic cage can be avoided.
  • biological cells of no interest e.g. dead cells
  • cell clumps in the dielectrophoretic cage can be avoided.
  • the invention encompasses the general technical teaching that, prior to the examination of the particles suspended in the carrier stream in the dielectrophoretic cage, a preliminary examination of the particles moving with the carrier stream is carried out in order to subsequently catch the particles of interest for further investigation in the dielectrophoretic cage and to be able to examine.
  • the preliminary examination can concern, for example, the intensity of a fluorescence, the vitality of a cell and / or the question of whether it is a single cell or an aggregate.
  • it can be determined whether it is cells or material whose shape and size is not the primary objective of the detailed examination, for example impurities or other cells, provided that they differ from the target cells.
  • a preliminary examination of the particles suspended in the carrier stream and a selection of certain particles depending on the result of the preliminary examination are therefore carried out, while the actual main examination is carried out only for the previously selected particles which are slowed down by one to enable meaningful main inspection, which would be made difficult by a movement of the particles.
  • the particles selected as a function of the preliminary examination are brought to a complete standstill before the main examination, for example by capturing them in a dielectrophoretic cage. Rather, it is also possible within the scope of the invention that the particles selected as a function of the preliminary examination are braked in the carrier stream only to such an extent that a meaningful examination of the particles is possible.
  • particle used in the context of the invention is to be understood generally and is not restricted to individual biological cells. Rather, this term also includes synthetic or biological particles, with particular advantages if the particles are biological materials, for example biological cells, cell groups, cell components or biologically relevant macromolecules, each possibly in combination with other biological particles or synthetic carrier particles. Synthetic particles can be solid particles, liquid particles or particles separated from the suspension medium
  • the particle selected as a function of the preliminary examination and subsequently examined in more detail as part of the main examination is sorted and / or treated depending on the result of the main examination. For example, different types of cells can be distinguished during the main inspection and then sorted accordingly.
  • dielectrophoretic elements The above-mentioned publication by Müller, T. et al. described dielectrophoretic elements can be used.
  • a transmitted light measurement for example, a transmitted light measurement, a fluorescence measurement and / or an impedance spectroscopy can be carried out.
  • a transmitted light measurement is carried out first and then a fluorescence measurement, the transmitted light measurement and the fluorescence measurement preferably taking place in spatially separate examination windows (“region of interest”).
  • the transmitted light measurement can make it possible, for example, to distinguish between living and dead biological cells, while the fluorescence measurement can be used to investigate whether the particles suspended in the carrier stream carry a fluorescence marker.
  • both a transmitted light measurement and a fluorescence measurement are carried out in spatially separate examination windows as part of the preliminary examination, then it is advantageous if the examination window for the transmitted light measurement in the carrier stream is upstream of the examination window for the fluorescence measurement.
  • the examination window for the transmitted light measurement it is alternatively also possible for the examination window for the transmitted light measurement to be arranged downstream in the carrier stream behind the examination window for the fluorescence measurement.
  • optical image is preferably recorded in the course of the preliminary examination of the particles moving with the carrier stream, which enables digital image evaluation to classify the particles.
  • the particles are preferably examined morphologically in order, for example, to be able to distinguish a single biological cell from a cell clump.
  • optical image used in the context of the present description is to be understood generally and is not restricted to two-dimensional images in the conventional sense of the word. Rather, the term optical image in the sense of the invention also includes a point-like or line-shaped optical scanning of the carrier stream or of the particles suspended in the carrier stream.
  • the brightness can be integrated along a line transverse to the carrier current channel in order to detect and classify individual particles.
  • living and dead cells in the context of the preliminary examination can be made in a transmitted light measurement by evaluating the intensity distribution in the recorded optical image.
  • a special principle of this transmitted light measurement with the properties mentioned is, for example, phase contrast illumination.
  • living biological cells have a ring structure with a light measurement relatively bright edge and a darker center, whereas dead biological cells have an approximately uniform brightness when transmitted light measurement and appear dark against the background.
  • certain molecules can be localized within a cell.
  • molecules can be located within a cell that are marked with a fluorescent dye.
  • the fluorescent dye can be, for example, molecularly produced “tags” of “green fluorescent protein” and its derivatives, other autofluorescent proteins.
  • fluorescent dyes which bind covalently or non-covalently to a cellular molecule are also suitable as fluorescent dyes.
  • fluorogenic substances which are converted by cellular enzymes into fluorescent products or so-called FRET pairs (fluorescence resonance energy transfer) can also be used as fluorescent dyes.
  • the state of the fluorescent dyes used can be distinguished, for example, on the basis of their spectral properties or by means of bioluminescence.
  • the structure and function of the molecules can also be determined on the basis of the localization of molecules within a cell.
  • a distinction can be made here, for example, according to the occurrence in the plasma membrane, in the cytosol, in the mitochondria, in the Golgi apparatus, in endosomes, in lysosomes, in the cell nucleus, in the spindle apparatus, in the cytoskeleton, colocalization with actin, tubulin.
  • the morphology of a cell can be determined in the course of the main and / or preliminary examination, it also being possible to use dyes.
  • two or more states of a cell population can be distinguished in the course of the main and / or preliminary examination.
  • a cellular signal based on the translocation of a fluorescence-labeled molecule, e.g. Receptor activation followed by receptor internalization, receptor activation followed by binding of arrestin, receptor aggregation, transition of a molecule from the plasma membrane into the cytosol, from the cytosol into the plasma membrane, from the cytosol into the cell nucleus or from the cell nucleus into the cytosol.
  • a fluorescence-labeled molecule e.g. Receptor activation followed by receptor internalization, receptor activation followed by binding of arrestin, receptor aggregation, transition of a molecule from the plasma membrane into the cytosol, from the cytosol into the plasma membrane, from the cytosol into the cell nucleus or from the cell nucleus into the cytosol.
  • the interaction of two molecules can be determined in the course of the main and / or preliminary examination, preferably at least one of the interacting molecules carrying a fluorescent marker and the interaction e.g. is shown by colocalization of two fluorescent colors, a FRET or a change in the fluorescence lifetime.
  • the status of a cell within a cell cycle can also be determined in the course of the main and / or preliminary examination, the morphology of the cell or the staining of the cellular chromatin preferably being evaluated.
  • Another possibility for the main and / or preliminary examination is to determine the membrane potential of a cell, preferably using membrane-sensitive dyes. Dyes are preferably used here which are sensitive to the plasma membrane potential and / or the mitochondrial membrane potential.
  • the vitality of a cell can also be determined in the course of the main and / or preliminary examination, the cell morphology preferably being evaluated and / or fluorine substances being used which can distinguish between living and dead cells.
  • cytotoxic effects can also be examined and / or the intracellular pH values determined during the main and / or preliminary examination.
  • An enzymatic activity within a cell can also be determined during the main and / or preliminary examination, wherein fluorine or chromogenic substances, in particular kinases, phosphatases or proteases, can preferably be used.
  • the production output of cells which produce biological products such as proteins, peptides, antibodies, carbohydrates or fats, can be determined, it being possible to use one of the methods described.
  • cell stress paths can also be determined as part of the main investigation.
  • the invention comprises a corresponding examination device for carrying out the examination method described above.
  • the examination device according to the invention preferably has optics to record an image of the particles.
  • the optics of the examination device according to the invention can preferably be adjusted in order to adjust the magnification, the focus and / or the field of view or to select a specific optical filter, wherein the optics can be adjusted by an actuator (e.g. an electric motor).
  • an actuator e.g. an electric motor
  • the braking of the particles is preferably carried out by a dielectrophoretic cage, which is known per se.
  • the dielectrophoretic cage not only serves to brake the suspended particles for a detailed examination, but also fulfills the function of a switch or switch, in that the suspended particles are dependent on the detailed examination by the Cage can be fed to one of several output lines.
  • the individual electrodes of the dielectrophoretic cage can preferably be controlled independently of one another.
  • the dielectrophoretic cage is preferably arranged at the branching point of the output lines for this purpose.
  • a funnel-shaped electrode arrangement (“funnel”) can be arranged in one or more of the output lines in order to prevent the suspended particles in the output lines from sinking.
  • the carrier stream in the outlet lines has a speed profile that shows only a low flow velocity near the wall, so that a sinking of the particles in the outlet lines could lead to deposits near the wall.
  • the suspended particles are supplied via two separate carrier flow lines which open into a common carrier flow line.
  • a partition can be arranged in the common carrier flow line, which also separates two separate partial flows from one another in the common carrier flow line, the two partial flows being able to be subjected to an examination.
  • the particles suspended in the two partial streams can then be combined.
  • the combined particles can then be fixed in a dielectrophoretic cage in the manner described above and subjected to a detailed examination.
  • the cells released from the dielectrophoretic cage can then be fed to one of several output lines.
  • a particular advantage of the invention is the fact that cells can be examined under aseptic, low-germ conditions and isolated accordingly.
  • FIG. 1 shows a fluidic diagram of a cell sorter according to the invention with a sorting chip
  • FIG. 2 shows the carrier current channel of the sorting chip with several dielectrophoretic elements
  • FIG. 3 shows a schematic illustration of the examination optics of the cell sorter from FIG. 1,
  • Figure 4 is a graph to illustrate the distinction between dead and living biological
  • Figure 5a-5e an example of the examination method according to the invention in the form of a flow chart
  • FIGS. 6-9 alternative embodiments of the carrier current channel of the sorting chip with a plurality of dielectrophoretic elements.
  • FIG. 1 shows a cell sorter according to the invention, which uses a microfluidic sorting chip 1 to sort biological cells dielectrophoretically.
  • the sorting chip 1 has a plurality of connections 2-6 for fluidic contacting, the fluidic contacting of the connections 2-6 being described in DE 102 13 272, the content of which is attributable to the present description.
  • the connection 2 of the sorting chip 1 serves to receive a carrier current with the biological cells to be sorted, while the connection 3 of the sorting chip 1 serves to discharge the selected biological cells, which are not further examined on the sorting chip 1.
  • the selected biological cells can be collected by a suction syringe 7, which can be connected to the connection 3 of the sorting chip 1.
  • the output 5 of the sorting chip 1 serves to remove the biological cells of interest, which can then be further processed or examined.
  • connections 4 and 6 of the sorting chip 1 serve to supply a so-called envelope current, which has the task of connecting the selected biological cells to the connection 5 of the
  • connections 4 and 6 of the sorting chip are connected via two sheath flow lines 8, 9, a Y-piece 10 and a four-way valve 11 to a pressure vessel 12 in which a cultivation medium for the sheath flow is located.
  • the pressure vessel 12 is pressurized via a compressed air line 13 so that the buffer solution (for example a cultivation medium) in the pressure vessel 12 with a corresponding position of the four-way valve 11 via the Y-piece 10 and the sheath flow lines 8, 9 flows to the connections 4, 6 of the sorting chip 1.
  • the sheath flow can alternatively also be realized using principles other than the pressure container 12 with the buffer solution, such as, for example, a syringe pump or a peristaltic pump.
  • connection 2 of the sorting chip 1 is connected to a particle injector 15 via a carrier current line 14.
  • the particle injector 15 is connected via a T-piece 16 to a carrier flow syringe 17, which is mechanically driven and injects a predetermined liquid flow of a carrier flow.
  • the T-piece 16 is connected upstream via a further four-way valve 18 and a sheath flow line 19 to a three-way valve 20.
  • the three-way valve 20 enables the sheath flow lines 8, 9 and the carrier flow line 14 to be flushed before the actual operation.
  • the three-way valve 20 is connected upstream via a peristaltic pump 21 to three three-way valves 22.1-22.3, to each of which a syringe reservoir 23.1-23.3 is connected.
  • the syringe reservoirs 23.1-23.3 are used to supply a filling stream for flushing the entire fluidic system before the actual operation, the
  • Syringe reservoir 23.1 e.g. Contains 70% ethanol, while the syringe reservoir 23.2 preferably contains aqua destillata as filler substance.
  • the syringe reservoir 23.3 contains e.g. a buffer solution as a filling flow substance.
  • the cell sorter has a collecting container 27 for excess envelope flow and a collection container 28 for excess filling flow.
  • the rinsing process which is carried out before the cell sorter is actually operated is first described below in order to free the sheath flow lines 8, 9, the carrier flow line 14 and the remaining fluid system of the cell sorter from air bubbles and contaminants.
  • the three-way valve 22.1 is first opened and ethanol is injected from the syringe reservoir 23.1 as a filling stream, the ethanol being initially conveyed to the three-way valve 20 by the peristaltic pump 21.
  • the ethanol is used both to reduce the number of bacteria in the system (for setting up an aseptic analysis and selection process) and to completely displace the air from the fluidic system.
  • the three-way valve 20 is set such that a part of the filling flow conveyed by the peristaltic pump 21 is passed on via the filling flow line 19, while the remaining part of the filling flow conveyed by the peristaltic pump 21 is passed on to the four-way -Valve 11 arrives.
  • the two four-way valves 11, 18 are in turn set so that the filling flow is passed through the sheath flow lines 8, 9 and the carrier power line 14. Cultivation medium also flows from the pressure container 12 into the collecting container 27 in order to briefly flood the lines.
  • the three-way valves 22.1-22.3 are closed and the peristaltic pump 21 is switched off.
  • the four-way valve 11 is set in such a way that the pressure vessel 12 is connected to the Y-piece 10, so that the culture medium located in the pressure vessel 12, due to the excess pressure prevailing in the pressure vessel 12, into the sheath flow lines 8, 9 is pressed.
  • the four-way valve 18 is set during the sorting operation so that there is no flow connection between the T-piece 16 and the four-way valve 18.
  • the carrier flow injected by the carrier flow syringe 17 then flows via the T-piece 16 into the particle injector 15, wherein 29 biological cells are injected into the carrier flow by a further injection syringe.
  • the carrier stream with the injected biological cells then flows from the particle injector 15 via the carrier stream line 14 to the connection 2 of the sorting chip.
  • a temperature sensor 30 is attached to the particle injector 15 in order to measure the temperature T of the particle injector 15.
  • a temperature control element 31 in the form of a Peltier element around which the To be able to heat or cool the particle injector 15 and the sorting chip 1.
  • the heating or cooling energy Q is predefined here by a temperature controller 32, which is connected on the input side to the temperature sensor 30 and regulates the temperature T of the particle injector 15 to a predefined setpoint.
  • a carrier current channel 33 is now described below with reference to FIG. 2, which is arranged in the sorting chip 1 of the cell sorter and branches into two output lines 34, 35, the output line 34 being connected to the connection 5 of the sorting chip 1 and for forwarding the positively selected particle is used, while the output line 35 is connected to the connection 3 of the sorting chip 1 and is used to discharge the selected particles.
  • a funnel-shaped electrophoretic electrode arrangement 36 is arranged, which has the task of arranging the particles suspended in the carrier flow one behind the other in the carrier flow channel 33.
  • the exact technical structure and the mode of operation of the electrode arrangement 36 is described in the publication by Müller, T. et al. described, the content of which is attributable to the present description, so that a detailed description of the electrode arrangement 36 can be dispensed with below.
  • a dielectrophoretic cage 37 is arranged in the carrier flow channel 33, which enables the particles suspended in the carrier current 33 to be captured and used for an in-depth examination. fixation in an examination window UF.
  • the dielectrophoretic cage 37 reference is also made to the publication by Müller, T. et al. referenced, so that a detailed description can be dispensed with in this regard.
  • a sorting device which consists of a dielectrophoretic electrode arrangement 38, with regard to the structure and the functioning of the electrode arrangement 38 also referring to the publication by Müller, T. et al , is referred.
  • the electrode arrangement 38 sorts the particles suspended in the carrier stream either into the outlet line 34 or into the outlet line 35, the selection taking place as a function of a main inspection carried out on the particles fixed in the cage 37, as will be described in detail below.
  • a flow guide device is arranged, which likewise consists of a dielectrophoretic electrode arrangement 39 and has the task of preventing particles from flowing back from the outlet line 35 into the outlet line 34.
  • the electrode arrangement 39 is V-shaped and has two legs, one leg of the electrode arrangement 39 projecting into the output line 34, while the other leg of the electrode arrangement 39 projects into the output line 35.
  • FIGS. 2 and 3 describe how the particles suspended in the carrier stream are examined in the sorting chip 1.
  • a transmitted light measurement is first carried out in an examination window ROH (region of interest 1) and a fluorescence measurement in another examination window ROI2 (region of interest 2), the examination window ROH in the carrier flow channel 33 upstream of the examination window ROI2 for the fluorescence measurement is arranged.
  • Both the transmitted light measurement and the fluorescence measurement are carried out here by the detection unit D shown schematically in FIG. 3, which has a CCD camera 40 for image acquisition, which is arranged below the sorting chip 1 and is aligned with a deflection mirror 41.
  • the sorting chip 1 is a light source for the
  • a light-emitting diode 42 is arranged, a condenser 43 being located between the light-emitting diode 42 and the sorting chip 1, which can have, for example, a phase contrast diaphragm.
  • a lens 44 is arranged below the sorting chip 1 in the beam path of the condenser 43.
  • the CCD camera 40 therefore takes an image of the examination window ROH via the deflection mirror 41 and the objective 44.
  • the detection unit D has a plurality of electromotive actuators 45.1-45.3, which enable the objective 44, the filter block 47 or the deflection mirror 41 to be adjusted.
  • the adjustment of the objective 44 enables the magnification and the focus to be changed.
  • the filter block 47 can be adjusted to select different filters.
  • the adjustment of the deflecting In contrast, gel 41 has the purpose of shifting the field of view along the carrier flow channel 33 in order to be able to recognize any deposits in the carrier flow channel 33.
  • the detection unit D has a light source 46 (e.g. a laser), which enables a fluorescence excitation of the biological cells suspended in the carrier current line 33 via a filter block 47, the CCD camera 40 taking a corresponding fluorescence image.
  • a light source 46 e.g. a laser
  • FIG. 4 shows a living cell 48 and a dead cell 49 in the upper region and the associated intensity profiles 50, 51 in the fluoroscopic image in the lower region. It can be seen from this that the living cell 48 has a relatively dark nucleus, whereas the dead cell 49 is uniformly illuminated on the inside, which makes it possible to distinguish the living cell 48 from the dead cell 49, as will be described in detail.
  • the carrier flow line 14 and the sheath flow lines 8, 9 are first rinsed with a 70% ethanol solution, then with aqua destillata and finally with a buffer solution in order to clean the fluid sorter of the cell sorter and, in particular, to remove air bubbles and dirt.
  • the carrier stream is then injected from the carrier stream syringe 17 into the carrier stream line 14, the biological cells to be examined being injected from the injection syringe 29 at the particle injector 15 into the carrier stream after the supply of the sheath stream as described below.
  • the culture medium in the pressure vessel 12 for the enveloping stream is pressed out of the pressure vessel 12 into the enveloping stream lines 8, 9 by the compressed air supplied via the compressed air line 13, which leads into the connections 4 and 6 of the sorting chip 1 and the forwarding support the particles selected in the sorting chip 1 via the connection 5 of the sorting chip 1.
  • the suspended particles are first lined up in succession in the flow direction by the electrode arrangement 36, as is indicated schematically by a dashed arrow.
  • phase contrast images Bi, ..., B n are recorded one after the other in order to determine the speed of movement of the suspended particles and to distinguish living from dead cells, as will be described in detail below.
  • an intensity signal Ii, ..., I n is determined for each of the phase contrast images Bi, ..., B n by the image intensity in the phase contrast images B ⁇ , ..., B n in columns, ie at right angles to the direction of flow, is integrated.
  • the individual intensity signals Ii, ..., I n therefore each have a signal peak at the location of a biological cell, with a signal peak between the in- shifts intensity signals I ⁇ , ..., I n according to the speed of movement of the cells and the time interval between the intensity signals I ⁇ , ..., I n .
  • a cross correlation function ⁇ i is calculated for successive intensity signals Ii, Ii + i.
  • the calculation of the cross-correlation function ⁇ i serves to determine the speed of movement of the cells in the carrier flow channel 33 of the sorting chip 1 so that the electrophoretic cage 37 can be actuated at the right time to catch a specific cell.
  • the maxima are then calculated for the individual cross-correlation functions ⁇ i (x) as a function of the displacement x in the longitudinal direction of the carrier flow channel 33.
  • the movement speed v of the cells in the carrier current channel 33 results from this as the quotient of the mean value of the maxima of the cross-correlation functions and the time interval between the successive phase contrast images Bi, ..., B n .
  • the speed of movement v of the cells can be used in a feedback for pump control, i.e. to check whether the calculated and actual pumping rate match and how it may need to be adjusted.
  • the speed of movement v can be used to identify whether there are faults in the system which cause the cells to flow too slowly (constipation), stop or even flow backwards. All of these faults can be identified and eliminated in this way, e.g. by flushing the system.
  • the above-described determination of the speed of movement v of the cells can alternatively also be carried out outside the examination window ROH, ROI2 take place. Basically, it is possible to track the cell movement within the entire carrier flow channel 33 or any areas of the carrier flow channel 33.
  • the signal form of the intensity signals I ⁇ , ..., I n provides information about the size of the particles and any aggregate formation.
  • the evaluation of the intensity signals is important for the control and automation of the entire system, namely the pumps, the dielectrophoretic electrode elements (for example, when is “switching” and when “switching"), the detailed image recording in the cage 37 and the sample storage.
  • the capture time t F is then calculated at which the cage 37 has to be activated in order to capture the examined particles for the subsequent main inspection in the examination window UF.
  • the capture time t F is simply the result of the speed of movement v of the particle and the distance to the cage 37.
  • the particle spacing d P between adjacent particles is also determined. This is important for the distinction between a single cell and a cell aggregate, as will be described in detail.
  • cell edge points i, x r are determined at which the intensity in the phase contrast image exceeds a predetermined limit value I TH . It is then checked whether there is an intensity minimum between the cell edge points xi, x r . If this is the case and there is a minimum intensity, then the cell is a living cell, as can be seen from FIG. 4. Otherwise, the cell is classified as dead, in order to subsequently make a corresponding selection, as will be described in detail below.
  • the luminance L of the individual cells is determined in the method section in FIG. 5d by integrating the intensity I of a cell between the cell edge groups xi and x r .
  • the luminance L so determined is compared to the cell with a minimum value L m i n and a maximum value L max.
  • the transmitted light illumination is switched off and the fluorescence excitation by the light source 46 is switched on.
  • a fluorescence image is then recorded in the examination window ROI2 and the fluorescence I of the cell is measured.
  • the fluorescence excitation it is also possible for the fluorescence excitation to be switched on permanently, with only the transmitted light illumination being switched off if the determined luminance of the cell is within the aforementioned window.
  • the predetermined threshold may I m i n below, so assumed that the cell in question does not carry a fluorescent amplifier.
  • certain cells are then selected, taking into account the distinction between living and dead cells and the check for a fluorescent marker. For example, those cells can be selected which are alive and which carry a fluorescent marker, whereas other cells are selected.
  • the cells selected in this way are then captured in the dielectrophoretic cage 37 at the predetermined capture time t F and thereby fixed, so that a main inspection of the captured cell with a higher resolution and a longer exposure time is subsequently possible.
  • the selected cells ie generally the living cells provided with a fluorescent marker, are then passed from the electrode arrangement 38 into the output line 34. left, whereas the selected cells (eg dead cells) are conveyed into the output line 35.
  • the main examination in the examination window UF can be images with fluorescence excitation, one or more excitation wavelengths being used simultaneously or at different times.
  • suitable dichroic mirrors are used in the filter block 47.
  • the fluorescent light from one or more wavelengths is simultaneously directed to one or more cameras.
  • Suitable emission filter inserts in filter block 47 or suitable emission splitters are used for this purpose. It is thus possible to generate images of the selected cell with several fluorescent colors simultaneously or in succession. It is also possible to generate an image of the selected cell with white-light phase contrast illumination. This is necessary to determine whether one or more non-fluorescence-labeled cells are still attached to a fluorescence-labeled cell, which leads to a — generally undesirable — contamination of this one fluorescence-labeled cell.
  • FIG. 6 largely corresponds to the exemplary embodiment shown in FIG. 2, so that in order to avoid repetition reference is made to the above description and the same reference numerals are used below for corresponding components, which are identified only by an apostrophe for differentiation.
  • a special feature of this exemplary embodiment is the simpler structural design of the dielectrophoretic electrode arrangement 36 'arranged in the carrier flow channel 33' on the input side, oscillated particles in the carrier flow channel 33 'one behind the other.
  • a further peculiarity of this exemplary embodiment is that a hook-shaped electrode arrangement 52 'is arranged downstream of the electrode arrangement 36' in the carrier flow channel 33 ', which is also referred to as a "hook” in accordance with its shape and has the task of holding particles and virtually parking them ,
  • the exact structure and mode of operation of the electrode arrangement 52 ' is described, for example, in Müller, T. et al. : “Life Cells in Cellprocessors" in Bioworld 2-2002, so that a detailed description of the electrode arrangement 52 'can be dispensed with here and the content of the above publication can be fully attributed to this description.
  • Another examination window 54 ' is located in the dielectrophoretic cage 37', so that the decelerated particles can be examined in the di-electrophoretic cage 37 '.
  • a further special feature of this exemplary embodiment is that a funnel-shaped electrode arrangement 55 'is arranged in the output line 34' for the positively selected particles, the function of which corresponds to the electrode arrangement 36 'and the function of which is to supply the particles in the output line 34' center.
  • This is advantageous because the particles in the outlet line 34 'have a tendency to have sunk and can therefore deposit close to the wall where the flow velocity is low.
  • the electrode arrangements 36 ', 52' and the dielectrophoretic cage 37 ' are arranged off-center in the carrier current line 33'.
  • the particles contained in the carrier stream after being released by the dielectrophoretic cage 37 ', automatically enter the output line 35' for negatively selected particles if the electrode arrangement 38 'is not activated.
  • FIG. 7 largely corresponds to the exemplary embodiment described above and shown in FIG. 6, so that in order to avoid repetition reference is made to the above description and in the following the same reference numerals are used for corresponding components which are used to distinguish them two apostrophes are marked.
  • a special feature of this exemplary embodiment is that the dielectrophoretic cage 37 "is arranged at the point at which the carrier current channel 33" branches into the two output lines 34 ", 35".
  • the individual electrodes of the dielectrophoretic cage 37 " can be controlled separately, so that the dielectrophoretic cage 37" can perform two functions, namely the function of a cage ("cage") and others the function of a switch.
  • the dielectrophoretic cage 37 can thus fix the particles in the carrier stream on the one hand for the examination in the examination window 54" and on the other hand feed the particles to one of the two output lines 34 ", 35".
  • branch point used in the context of the present description is to be understood generally and is not restricted to the geometric intersection of the output lines. Rather, it is also possible for the cage 37 "or the switch to be arranged upstream of the intersection of the output lines.
  • branching point also includes the so-called" sepa-matrix ". This is the dividing line of the laminar flow in the carrier flow channel.
  • the electrode arrangements 36 ′′, 52 ′′, the cage 37 ′′ and the measuring stations 53 ′′, 54 ′′ are arranged centrally in the carrier current channel 33 ′′ here and in the following exemplary embodiments.
  • FIG. 8 largely corresponds to the exemplary embodiment described above and shown in FIG. 7, so that in order to avoid repetition, reference is made to the above description and the same reference numerals are used below for corresponding components, which, however, are used to distinguish them three apostrophes are marked.
  • a special feature of this exemplary embodiment is the constructive design of the dielectrophoretic cage 37 '", which here only consists of six spatially arranged electrodes, the individual electrodes being controllable separately are so that the cage 37 '"can either act as a switch or switch or as a cage.
  • FIG. 9 finally shows a further exemplary embodiment of a possible arrangement in a sorting chip.
  • two carrier flow lines 56, 57 open into a common carrier flow line 58, suspended particles being supplied in each case via the two carrier flow lines 56, 57.
  • a funnel-shaped electrode arrangement 59, 60 is arranged in each of the two carrier flow lines 56, 57 in order to center the particles contained in the carrier flows of the two carrier flow lines 56, 57.
  • the common carrier flow channel 58 there is a partition 61 upstream at the junction of the two carrier flow lines 56, 57, so that the particles suspended in the carrier flows of the two carrier flow lines 56, 57 are first carried in parallel next to one another and separately from one another in the carrier flow line 58.
  • a further electrode arrangement 67 Downstream of the dielectrophoretic cage 65 there is a further electrode arrangement 67 which, after being released by the cage 65, supplies the particles suspended in the carrier stream to one of three output lines 68, 69, 70 in the examination window 66 depending on the result of the examination ,
  • the output lines 68, 70 serve to discharge the negatively selected particles, while the outlet line 69 serves to continue the positively selected particles.
  • the electrode arrangement 67 must therefore be actively activated if particles are to be conveyed into the output lines 68, 70 for the negatively selected particles, whereas there is no activation for the positively selected particles. This arrangement is therefore particularly suitable for investigations in which only a few particles are selected negatively.

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Abstract

L'invention concerne un procédé d'étude de particules biologiques, notamment destiné à être mis en oeuvre dans un dispositif de triage cellulaire. Ledit procédé consiste à introduire les particules à étudier dans un courant porteur ; à mettre en oeuvre une première étude des particules se déplaçant avec le courant porteur ; à sélectionner au moins une particule en fonction du résultat de la première étude ; à ralentir la particule sélectionnée ; et, à mettre en oeuvre une deuxième étude de la particule sélectionnée à l'état ralenti. L'invention concerne également un dispositif d'étude correspondant.
EP04707905A 2003-02-05 2004-02-04 Procede de triage et d'identification de cellules a plusieurs parametres et dispositif correspondant Withdrawn EP1590652A1 (fr)

Applications Claiming Priority (5)

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DE10304653A DE10304653B4 (de) 2003-02-05 2003-02-05 Mehrparametrige Detektion in einem fluidischen Mikrosystem
DE10304653 2003-02-05
DE10320956A DE10320956B4 (de) 2003-02-05 2003-05-09 Untersuchungsverfahren für biologische Zellen und zugehörige Untersuchungseinrichtung
DE10320956 2003-05-09
PCT/EP2004/001034 WO2004070362A1 (fr) 2003-02-05 2004-02-04 Procede de triage et d'identification de cellules a plusieurs parametres et dispositif correspondant

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EP04707910A Withdrawn EP1590653A1 (fr) 2003-02-05 2004-02-04 Detection multiparametre dans un microsysteme fluidique

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EP1590653A1 (fr) 2005-11-02
JP2006517292A (ja) 2006-07-20
US20060139638A1 (en) 2006-06-29
US20060152708A1 (en) 2006-07-13
DE10304653B4 (de) 2005-01-27

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