WO1996030760A1 - Procede d'identification de substances biologiquement actives de par leur effet sur des cellules vivantes - Google Patents

Procede d'identification de substances biologiquement actives de par leur effet sur des cellules vivantes Download PDF

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Publication number
WO1996030760A1
WO1996030760A1 PCT/DK1996/000133 DK9600133W WO9630760A1 WO 1996030760 A1 WO1996030760 A1 WO 1996030760A1 DK 9600133 W DK9600133 W DK 9600133W WO 9630760 A1 WO9630760 A1 WO 9630760A1
Authority
WO
WIPO (PCT)
Prior art keywords
flow chamber
matrix
cells
chamber
wall
Prior art date
Application number
PCT/DK1996/000133
Other languages
English (en)
Inventor
Ole Thastrup
Kurt Scudder
Jaromir Ruzicka
Original Assignee
Novo Nordisk A/S
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Novo Nordisk A/S filed Critical Novo Nordisk A/S
Priority to AU51430/96A priority Critical patent/AU5143096A/en
Priority to JP8528816A priority patent/JPH11504507A/ja
Priority to EP96908028A priority patent/EP0817965A1/fr
Publication of WO1996030760A1 publication Critical patent/WO1996030760A1/fr

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—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
    • 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
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/16—Particles; Beads; Granular material; Encapsulation
    • 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
    • C12M41/00—Means for regulation, monitoring, measurement or control, e.g. flow regulation
    • C12M41/46—Means for regulation, monitoring, measurement or control, e.g. flow regulation of cellular or enzymatic activity or functionality, e.g. cell viability
    • 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

Definitions

  • the invention relates to the technique of identifying biologically active substances and to be more specific to identification by the effect of the substance on living cells.
  • biologically active compounds include, but are not limited to, drugs, insecticides, pesticides, and herbicides.
  • the practice of screening large libraries of samples of unknown composition for the few which may contain a compound of specific biological activity is one of the more common methods of new drug discovery.
  • the samples of unknown composition are in most cases biological material, such as plant extracts or microbial fermentation broths. Screening these for biological activity is normally accomplished by performing binding assays or, more recently, functional assays.
  • binding assay is an attempt to find compounds of interest by identifying those which adhere with a minimum degree of affinity to cells or cell products. This can be done using fluorescent, luminescent, or radioactive detection methods. These assays are based not on a biological response, but a passive process which results from biological activity, namely the generation of specific molecules by biological systems. They cannot be construed as functional assays or as real-time assays.
  • Gene Expression Assays Another way to determine biological activity is to measure up-regulation or down-regulation of expression of a known gene. This is done by inserting DNA which codes for something which can be readily measured into a cell's genome such that the expression of interest is coupled to expression of the inserted DNA. While this is a true functional assay, it also is not a real time assay. In addition, it is only capable of finding compounds which affect gene expression. In many cases this is not the response of interest. Other Functional Assays.
  • the CytoSensor described in US 4 915 812 and US 5 395 503 is a commercial instrument which has been billed as a screening instrument. It is based on the detection of increased cellular proton flux by means of a semiconducting electrode. The instrument is applicable to high through-put screening, but can only detect cellular events that result in changes in extracellular pH. Again, many responses of interest are not associated with changes in extracellular pH.
  • luminescent probes Imaging of cellular functions using luminescent probes. Visualization of intracellular function using luminescent (fluorescent or bioluminescent) probes has become one of the mainstay techniques in modern cell biology. Using traditional optical microscopes with quantitative detectors in place of the human eye, both the concentration and distribution in the cell of a variety of intracellular molecules of interest can be measured. While luminescent probes can be measured in large populations of cells using other techniques, imaging is the only way to learn what is going on in single cells or small populations of cells. It also has the advantage of much higher optical efficiency when compared with traditional spectrophotomethc techniques for measuring optical signals.
  • These cells are ones which have been specifically chosen to exhibit the desired biological behaviour. They may be primary cells cultured from fresh tissue, or they may be genetically engineered cells transfected with one or more genes of interest.
  • the method according to the invention allows monitoring of dynamic cellular responses in living cells, while the cells are attached to a matrix (e.g. microcarrier beads) that can be fluidically controlled. Under continuous flow operation it is possible to obtain very short sample exposure times which make the invention suited to high through-put screening for novel biologically active substances.
  • the method will also find application in the search for rare biological events associated with expression of specific proteins in transfected cell lines.
  • the matrix may be provided as microcarrier beads or as microdroplets of agarose.
  • the method employs detection of cellular signals from cells cultured on the surface of microcarrier beads or encapsulated in microdroplets of agarose gel, the beads/droplets not only providing the support for the cells but also the vehicle for transportation of said cells from tissue culture facilities (incubators) to detection system.
  • the flow chamber is capable of positioning the carriers in a fixed position without the aid of a human operator, and simultaneously allowing visualization through a microscope equipped with a high numerical aperture objective lens.
  • the flushing of the flow chamber may advantageously be a forward flushing.
  • a forward flushing is meant that the flush liquid passes the chamber in the same direction as do the injected matrix carrying flow fluid.
  • a more effective flushing is obtained as the flushing fluid simply displaces the matrix out of the chamber instead of sucking it out.
  • a further object of the invention is to provide an apparatus for use by the method according to the invention.
  • Such an apparatus is characterized in that it comprises a flow chamber having a wall through which the content of said chamber may be monitored, a fluidic handling system for controlled sequential injection of buffer, cell carrying matrix and sample or samples into the flow chamber and for final removing of waste during a flushing of this chamber.
  • the flow chamber of the apparatus may comprise a plane optical surface as a wall through which the content of the chamber may be monitored, a ring shaped wall having an edge adjacent to the plane optical surface leaving a gap between said edge and said optical surface which gap allows liquid but not the matrix to pass.
  • a volume of buffer carrying the matrix beads or droplets When injected into the flow chamber the beads or droplets will be retained on the optical surface whereas the buffer may escape through the gap between this surface and the edge of the ring shaped wall. Injected sample will trickle between the beads or the droplets in close contact with these beads and droplets and escape through the gap.
  • the chamber may be flushed by sucking flushing buffer in through the gap and removing it via the inlet of the chamber until all beads and droplets are flushed away.
  • a quicker flushing may be obtained when the distance between the edge of the ring shaped wall and the plane optical surface may be controlled so that it momentarily allows the matrix beads or microdroplets to pass.
  • This way the flow through the chamber may be made unidirectional and all supplies may be made to the inlet of the chamber and all draining of waste may take place from an area surrounding the inlet.
  • the width of the gap may be changed by electromagnetic means which are controlled to momentarily pull the ring shaped wall and the plane surface away from each other. This makes it easy to control the width of the gap so that it is enlarged during the flushing step of the identification cycle.
  • Figure 1-6 show schematically a diagram of the fluidic system of an apparatus in six successive step positions during the sequence run through when carrying out the method according to the invention
  • Figure 7 shows schematically a sectional view of the flow chamber during the fixation of the matrix at an optical surface of an end wall of the chamber
  • Figure 8 shows the chamber according to figure 7 seen through the end wall of the chamber
  • Figure 9 shows schematically a sectional view of the flow chamber during flushing
  • Figure 10 shows the chamber according to figure 9 seen through the end wall of the chamber.
  • An apparatus for carrying out the identification cycle comprises as shown in figure 1 -6 a flow chamber 1 into which a charge of matrix beads carrying the living cells chosen for the identification is transported and exposed to the sample the influence of which shall be identified.
  • the content of the flow chamber may be observed through a wall 2 of this chamber.
  • the optical portion used for the observation is illustrated by the arrow 3.
  • This optical portion may be a standard microscope equipped for epi-illumination.
  • the light source may be a rapid scanning monochromator which allows switching among selected wavelengths of light for excitation of the fluorescent molecules in the cells in a time scale appropriate for the response being measured.
  • the fluorescence detector may be a photomultiplier, camera, image detector or other with sufficiently fast response to allow the signal to be detected without undersampling of the process. Both the light source and the detector are computer controlled for synchronizing the acquisition of the fluorescence signals with the appropriate excitation wavelength.
  • the fluidic part of the apparatus further comprises a container 4 for a buffer fluid, waste containers 5 and 6 and a container 7 for matrix beads or droplets carrying the living cells.
  • the fluidic part of the apparatus comprises three syringes 8, 9, and 10 which under computer control are sequentially operated to act as pumps providing the driving force which sends liquid through the apparatus in a way which is further controlled by two-way valves 11 , 12, and 13 at the outlet ends of the respective syringes and a multi-way selection valve 14. All valves are controlled by the computer to establish the appropriate connection during the different steps of the process.
  • the selection valve has a main lead 15 which by the different positions of this valve may be connected to one of a number of other leads 16, 17, 18, 19 and 20. The choice of connection is controlled by the computer.
  • Figure 1 shows the first step during which the syringe 8 is loaded with buffer from the container 4 whereas the syringe 9 is empty, i.e. with its piston in its innermost position.
  • the selection valve 14 in its position connecting the main lead 15 to the lead 17 which is connected to the container 7 containing the matrix beads carrying the living cells.
  • the piston of the syringe 8 is pressed inward, as illustrated by the arrow 23 in figure 3, and the valve 11 at the outlet of this syringe 8 is changed to the position connecting this syringe 8 to the main lead 15 of the selection valve 14 which valve itself connects its main lead 15 to the lead 19 leading to the flow chamber 1.
  • the piston is pressed into the syringe 8 its content of buffer liquid is pressed into the coil 21 where it displaces the liquid with suspended matrix beads.
  • This matrix bead carrying liquid is led through the lead 19 to the flow chamber 1 as illustrated by the arrows 25 and 26.
  • the matrix beads are trapped as it will be described below and the buffer liquid carrying the beads flows past and into the waste container 5.
  • the syringe 8 is refilled with buffer from the container 4 before proceeding to the fourth step.
  • the piston of the syringe 8 is drawn out and the selection valve provides a connection to the lead 16 which is inserted into a sample 27 which shall be tested. Therefore, when the piston of the syringe 9 is drawn outward, the sample is sucked into the coil 21 as illustrated by the arrows 29 and 30, and by the fifth step shown in figure 5 the sample is propelled to the flow chamber 1 by pressing in the piston of syringe 8 as shown by an arrow 31.
  • the selection valve connects the main lead 15 with the lead 19 leading to the flow chamber 1.
  • the syringe 8 is again filled with buffer liquid from the container 4.
  • Figure 6 illustrates the sixth and last step of the process during which step the selection valve is moved to make connection to the lead 20 which may be connected to a waste container not shown.
  • the piston of syringe 10 is moved rapidly outward, aspirating liquid from the waste container 5 through the flow chamber in the reverse direction and into the syringe 10 as illustrated by the arrows 33 and 34.
  • the valve 13 is in the opposite position of the position shown in figure 6.
  • the valve 13 is turned to the position shown in figure 6 and the piston of the syringe 10 is moved inward to dispense the content of the syringe 10 into the waste container 6 as shown by the arrow 35.
  • the syringe 9 is emptied through the selector valve 14 into the not shown waste container connected to the lead 20.
  • the row of samples is conveyed one step forward so that not the sample 27 but the sample 28 is conducted into the flow chamber and so on until a sufficient number of cycles have been performed to test all the samples in the row. As it is seen the whole detecting process may be carried out automatically.
  • valves and syringes may be varied and modified widely without deviating from the scope of the invention. Also, the construction of the flow chamber may be varied from that which is described below.
  • Figure 7 shows schematically a sectional view of a flow chamber during the filling with matrix carrying beads as described in step three.
  • the flow chamber comprises a ring shaped wall 36 adjacent to a plane optical wall 37.
  • the position of the ring shaped wall 36 relative to the optical wall 37 leaves a gap between an edge 38 of the ring shaped wall and the optical WEIII which gap is just small enough to keep the matrix beads 40 or droplets trapped in the chamber whereas the liquid may pass through the gap to be led away to a waste container when a liquid with suspended beads or droplets are discharged into the chamber.
  • the escaping liquid is symbolized by the arrows 39.
  • Figure 8 shows the chamber according to figure 7 seen through the optical wall 37.
  • Figure 9 shows schematically a flow chamber as the one shown in figure 7 and 8 only during the emptying of the chamber as described in the sixth step of the process.
  • the liquid is sucked up through the flow chamber as symbolized by the arrows 41 and 42.
  • Figure 10 shows the chamber of figure 9 seen through the wall 37.
  • the components of the apparatus may be modified widely.
  • the ring shaped wall 36 is movable in relation to the optical wall 37. This allows unidirectional flow of liquid through the chamber.
  • the edge 38 is maintained so close to the optical wall 37 that the matrix beads or droplets are trapped and only the liquid may escape through the gap.
  • the wall 36 and its edge 38 is moved away from the optical wall 37 so far that the width of the gap is larger than the diameter of the beads. Thereby the beads may be flushed out through the gap to a waste container. In this way a more effective and quicker flushing of the chamber may be obtained.
  • the changing of the distance between the edge 38 and the optical wall 37 may be controlled by the computer controlling the course and sequence of the steps.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Health & Medical Sciences (AREA)
  • Hematology (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Toxicology (AREA)
  • Genetics & Genomics (AREA)
  • General Engineering & Computer Science (AREA)
  • Sustainable Development (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

Procédé d'identification de substances biologiquement actives de par leur effet sur des cellules vivantes, constituant à: cultiver des cellules sur ou dans une matrice qu'il est possible de contrôler fluidiquement; b) charger les cellules de molécules chimiquement luminescentes ou transfecter préalablement ces cellules avec des molécules de recombinaison présentant des propriétés de luminescence; c) injecter un volume bien défini de la suspension de la matrice hébergeant les cellules dans un flux d'écoulement transportant la suspension dans une chambre d'écoulement présentant une paroi au travers de laquelle il est possible de surveiller le contenu de la chambre d'écoulement considérée; d) perfuser les cellules avec un échantillon contenant une substance potentiellement biologiquement active; e) mesurer la réponse en luminescence du contenu de la chambre d'écoulement; f) rincer la chambre d'écoulement afin qu'elle soit prête pour un nouveau cycle d'essais.
PCT/DK1996/000133 1995-03-31 1996-03-29 Procede d'identification de substances biologiquement actives de par leur effet sur des cellules vivantes WO1996030760A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
AU51430/96A AU5143096A (en) 1995-03-31 1996-03-29 Method for identifying biologically active substances by the ir effect on living cells
JP8528816A JPH11504507A (ja) 1995-03-31 1996-03-29 生物学的に活性な基質を、生きている細胞に関するこれらの影響によって同定するための方法
EP96908028A EP0817965A1 (fr) 1995-03-31 1996-03-29 Procede d'identification de substances biologiquement actives de par leur effet sur des cellules vivantes

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DK35595 1995-03-31
DK0355/95 1995-03-31

Publications (1)

Publication Number Publication Date
WO1996030760A1 true WO1996030760A1 (fr) 1996-10-03

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PCT/DK1996/000133 WO1996030760A1 (fr) 1995-03-31 1996-03-29 Procede d'identification de substances biologiquement actives de par leur effet sur des cellules vivantes

Country Status (4)

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EP (1) EP0817965A1 (fr)
JP (1) JPH11504507A (fr)
AU (1) AU5143096A (fr)
WO (1) WO1996030760A1 (fr)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998005959A1 (fr) * 1996-08-02 1998-02-12 Axiom Biotechnologies, Inc. Appareil et procede de mesure en temps reel de la reponse cellulaire
WO1998049337A1 (fr) * 1997-05-01 1998-11-05 Eastman Chemical Company Bacterie de marquage bioluminescente et procedes de surveillance de toxicite dans des systemes de traitement d'eaux usees biologiques
US6242209B1 (en) 1996-08-02 2001-06-05 Axiom Biotechnologies, Inc. Cell flow apparatus and method for real-time measurements of cellular responses
WO2001096597A2 (fr) * 2000-06-13 2001-12-20 Glaxo Group Limited Procede de criblage a haut rendement de composes potentiels pour une activite biologique
EP1248107A2 (fr) * 2001-03-22 2002-10-09 IPF Pharmaceuticals GmbH Procédé et appareil de criblage à résolution temporelle
US6558916B2 (en) 1996-08-02 2003-05-06 Axiom Biotechnologies, Inc. Cell flow apparatus and method for real-time measurements of patient cellular responses
US6632619B1 (en) 1997-05-16 2003-10-14 The Governors Of The University Of Alberta Microfluidic system and methods of use
US6900021B1 (en) 1997-05-16 2005-05-31 The University Of Alberta Microfluidic system and methods of use

Citations (2)

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Publication number Priority date Publication date Assignee Title
US4559299A (en) * 1983-02-04 1985-12-17 Brown University Research Foundation Inc. Cytotoxicity assays in cell culturing devices
US5278048A (en) * 1988-10-21 1994-01-11 Molecular Devices Corporation Methods for detecting the effect of cell affecting agents on living cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US4559299A (en) * 1983-02-04 1985-12-17 Brown University Research Foundation Inc. Cytotoxicity assays in cell culturing devices
US5278048A (en) * 1988-10-21 1994-01-11 Molecular Devices Corporation Methods for detecting the effect of cell affecting agents on living cells

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 351, DERWENT WPI, Dialog Accession No. 010246555, WPI Accession No. 95-147810/20, DARLING G.D. et al., "Aminostyryl-Pyridinium Salt Fluorescent Probes for Viscosity Measurement - Partic. in Individual Biological Cells e.g. for Drug Screening"; & CA,A,2 129 933, 13-02-1995, 9520 (Basic). *

Cited By (15)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6280967B1 (en) 1996-08-02 2001-08-28 Axiom Biotechnologies, Inc. Cell flow apparatus and method for real-time of cellular responses
WO1998005959A1 (fr) * 1996-08-02 1998-02-12 Axiom Biotechnologies, Inc. Appareil et procede de mesure en temps reel de la reponse cellulaire
US6958221B2 (en) 1996-08-02 2005-10-25 Caliper Life Sciences, Inc. Cell flow apparatus and method for real-time measurements of patient cellular responses
US5919646A (en) * 1996-08-02 1999-07-06 Axiom Biotechnologies, Inc. Apparatus and method for real-time measurement of cellular response
US6558916B2 (en) 1996-08-02 2003-05-06 Axiom Biotechnologies, Inc. Cell flow apparatus and method for real-time measurements of patient cellular responses
US6242209B1 (en) 1996-08-02 2001-06-05 Axiom Biotechnologies, Inc. Cell flow apparatus and method for real-time measurements of cellular responses
US5804436A (en) * 1996-08-02 1998-09-08 Axiom Biotechnologies, Inc. Apparatus and method for real-time measurement of cellular response
US6110661A (en) * 1997-05-01 2000-08-29 Eastman Chemical Company Bioluminescent reporter bacterium
WO1998049337A1 (fr) * 1997-05-01 1998-11-05 Eastman Chemical Company Bacterie de marquage bioluminescente et procedes de surveillance de toxicite dans des systemes de traitement d'eaux usees biologiques
US6632619B1 (en) 1997-05-16 2003-10-14 The Governors Of The University Of Alberta Microfluidic system and methods of use
US6900021B1 (en) 1997-05-16 2005-05-31 The University Of Alberta Microfluidic system and methods of use
WO2001096597A2 (fr) * 2000-06-13 2001-12-20 Glaxo Group Limited Procede de criblage a haut rendement de composes potentiels pour une activite biologique
WO2001096597A3 (fr) * 2000-06-13 2003-09-12 Glaxo Group Ltd Procede de criblage a haut rendement de composes potentiels pour une activite biologique
EP1248107A2 (fr) * 2001-03-22 2002-10-09 IPF Pharmaceuticals GmbH Procédé et appareil de criblage à résolution temporelle
EP1248107A3 (fr) * 2001-03-22 2002-10-16 IPF Pharmaceuticals GmbH Procédé et appareil de criblage à résolution temporelle

Also Published As

Publication number Publication date
EP0817965A1 (fr) 1998-01-14
JPH11504507A (ja) 1999-04-27
AU5143096A (en) 1996-10-16

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