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WO1990000619A1 - Analyse de substances de modification cellulaire - Google Patents

Analyse de substances de modification cellulaire Download PDF

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Publication number
WO1990000619A1
WO1990000619A1 PCT/GB1989/000775 GB8900775W WO9000619A1 WO 1990000619 A1 WO1990000619 A1 WO 1990000619A1 GB 8900775 W GB8900775 W GB 8900775W WO 9000619 A1 WO9000619 A1 WO 9000619A1
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WO
WIPO (PCT)
Prior art keywords
cell
reagent
hormone
cellular component
modifier
Prior art date
Application number
PCT/GB1989/000775
Other languages
English (en)
Inventor
Nicholas J. MARSHALL
Patricia A. EALEY
Stanley J. Holt
Original Assignee
University College London
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 GB888816302A external-priority patent/GB8816302D0/en
Priority claimed from GB888818508A external-priority patent/GB8818508D0/en
Application filed by University College London filed Critical University College London
Publication of WO1990000619A1 publication Critical patent/WO1990000619A1/fr
Priority to FI910081A priority Critical patent/FI910081A0/fi

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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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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

  • This invention relates to the bioas ⁇ ay of cell-modifying reagents, e.g. hormones.
  • a cell-modifying reagent such as a hormone in a sample, e.g. of a body fluid
  • im unoa ⁇ say measures the immuno-reactivity of the cell-modifier which may not, in some circumstances, accurately correlate with its actual biological activity (bioactivity) . It is therefore desirable to be able to measure the bioactivity of cell-modifying reagents such as hormones more directly.
  • the present invention provides a novel method for the assay of cell-modifying reagents such as hormones (and autoantibodies that mimic their effect) which is capable of relatively rapid large scale automated operation and thus avoids the above-mentioned disadvantages of known me the s .
  • the method of the present invention for the assay of an extra-cellular cell-modifying reagent comprises:
  • colorimetric reagent means a reagent which absorbs or generates, in a quantifiable way, electromagnetic radiation in the ultraviolet (10-400 nm), visible (400-750 nm) or infra-red (750-10 5 nm) part of the spectrum.
  • chromogenic reagent means a reagent which is capable of being converted under the conditions of the assay into a colorimetric reagent (as defined above) under the influence of the cellular component.
  • Cell cultures have previously been used in other types of assay. For example, Mo ⁇ mann (J. Immunological Methods, 6_5 pp 55-63 1983) described a method for the colorimetric assay of cellular growth and survival.
  • the method relied on the conversion of a tetrazolium salt (MTT) into dark blue formazan by the dehydrogenase present in living but not dead cells.
  • MTT tetrazolium salt
  • the method was operated in a multiwell microtitre plate and the results were read on a microtitre plate reader.
  • the method was used to measure the effect of extra cellular cell-modifying reagents such as proliferative lymphokine ⁇ , e.g. interleukin 2, mitogen stimulators, e.g. Concanavallin A, and complement mediated lysis.
  • a similar method has been used in cytotoxicity assays for bacterial enterotoxins, e.g. cholera toxin, see Barer e_t al, Histochemical Journal 33 pp.
  • the present invention is based on the discovery that it is possible to monitor, in cultures of cells, changes in cellular components rather than changes in cell numbers. Such changes can occur much more rapidly, e.g. in hours rather than days, so that the new method can be much quicker to operate than the known methods.
  • the method of the invention may be operated on a cell culture in which some cellular division has taken place, especially in those cases where it is the quantity rather than the activity of the cellular component which is altered, it is preferred to operate the method using a cellular component which is altered before there is any substantial alteration in the number of cells in the culture as this makes for a substantially more rapid method.
  • the new method may be operated in the wells of a microtitre plate, and provides a rapid and convenient method for the assay of extra-cellular cell modifiers, especially when the assay is performed by the eluted stain assay method described herein.
  • any extracellular cell modifier e.g. an interleukin, growth factor (GF-1 GF-2, EGF or FGF) gamma-interferon, mitogen such as Concanavallin A, or any drug that mimics their action.
  • Autoantibodies that mimic the effect of a particular hormone can also be assayed.
  • the coloured or chromogenic reagent which interacts in a measurable way with a cellular component altered by the hormone may be one which simply interacts in a quantitative way with that cellular component and thus provides a quantitative value for the total amount of that component present in the cells.
  • the reagent may be one which binds to a cellular component, e.g. a protein, and thus provides a measure of the total amount of protein present in the cell culture.
  • the reagent may be a compound (or combination of compounds) which are altered by a cellular component, e.g. an enzyme, the amount or activity of which is altered by the action of the hormone on the cells.
  • a cellular component e.g. an enzyme
  • the reagent may be a vital dye, e.g. neutral red, that is to say a dye which is taken up by unfixed living cells, especially if the cellular uptake proces ⁇ it ⁇ elf i ⁇ stimulated by the hormone.
  • the cellular component may be a cell constituent, e.g. fat, which is substantially only present in cells which have been exposed to the hormone being a ⁇ ayed.
  • the cellular component i ⁇ activated by the hormone without being altered in total amount. In all these cases, the interaction between the reagent and the cellular component must be measurable so as to provide a measure of the quantity of hormone in the sample.
  • cell numbers preferably substantially do not change.
  • the main advantages of this over assays which rely upon increases (or reductions) in cell number are: (i) Since only a short exposure time (hours as opposed to days) i ⁇ required, the assay need not be operated under sterile conditions. This greatly enhances the convenience and technical ease of the system and increases its commercial potential. (ii) A fa ⁇ ter as ⁇ ay, e.g. same-day, a ⁇ say is achieved. This i ⁇ important in the context of routine Clinical Chemistry, (iii) Enzymic amplification of the response i ⁇ often obtained. This system i ⁇ thereby capable of a much larger magnitude of response than that derived solely from a change in cell number. This may be demonstrated if longer exposure times are selected and a comparison i ⁇ made between increases in cell number and the increased response of the new assay system. This feature increases the precision of the bioassay.
  • the interaction between the colorimetric reagent and the cellular component gives rise to a colour which can be measured spectrophotometrically.
  • a colour which can be measured spectrophotometrically.
  • Such colour may either be originally present in the reagent, e.g. in the form of a dye, the amount of which bound by the cellular component depends on the amount of the cellular component which itself depends upon the amount of the hormone being assayed.
  • the colour may itself be the result of the interaction between a chromogenic reagent and the cellular component, e.g. the result of the interaction between a dehydrogenase system and a tetrazolium salt. In either case, the colour is eluted from the cells of the cell culture and then measured spectrophotometrically in the elution medium.
  • a suitable chromogenic reagent capable of fluorescence or luminescence.
  • a suitable chromogenic reagent capable of fluorescence or luminescence.
  • Such a reagent can be used in a similar manner to the colorimetric reagents, and the result of the interaction between the reagent and the cell i ⁇ read with an appropriate photometer.
  • Suitable cell lines for use in the method of the invention are generally available; see for example the catalogues of the various International Collection ⁇ of Cell Culture ⁇ , e.g. the European Collection of Animal Cell Cultures, published by the Public Health Laboratory Service Centre for Applied Microbiology and Research, Porton Down, U.K.
  • the 3rd Edition (1988) of the catalogue of this collection includes the stable T-cell line MOLT-4 which i ⁇ respon ⁇ ibe to gamma-interferon (Dadmarz e_t al , Leukemia Re ⁇ earch, JL0_, 1279-1285, 1986).
  • the mou ⁇ e T-cell ly phobla ⁇ t CTLL-M line which i ⁇ dependent upon Interleukin-2 (Sander ⁇ on et al, Nature 268, 154-156, 1977) or the mouse embryo fibroblast 3T3 cell line (Swis ⁇ 3T3 cells) which responds to Epidermal Growth Factor (Rozengurt e_t aJL, Proc. Natl. Acad. Sci.) U.S.A., 7J3, 4392-4396, 1981).
  • many cell lines are respon ⁇ ive to hormones.
  • the cell lines known as FRTL-5 Ambesi-Impiombato et al , Proc. Ntl . Acad.
  • the FRTL-5 cell line has been used to assay auto-antibodies from patient ⁇ with Grave ⁇ ' di ⁇ ea ⁇ e (an auto-immune thyroid disease) using conventional assays requiring the use of radioactive materials (Ambesi-Impiombato, USP 4608341).
  • the cell lines Ro ⁇ 17/2 (Majeska and Rodan, Calcif. Tissue. Int. 1 59-66, 1982) and UMR 104, 105, 106 or 108 (Partridge et al , Cancer Res.
  • 3T3-F442A cells are sensitive to growth hormone (Morikawa, M. et al . , Mol. and Cellular Biology, 4_, 228-231, 1984) while rat Nb2 lympho a cells are sensitive to both growth hormone and prolactin (Tanaka et al, J. Clin. Endocrinol. Metabl. 5_6, 18-20, 1983).
  • MA10 cells are responsive to luteinizing hormone and hCG (Ascoli, Endocrinology 108, 88-93, 1981) and Yl cells are responsive to adrenocorticotrophin (ACTH) (Kowal, Rec. Prog. Horm. Res. 2_6, 623-633 1970).
  • the sensitivity of the cell-line is shown by redirected cell differentiation.
  • the UMR104-108 cell ⁇ and ROS17/2 show a marked reduction in alkaline phosphatase activity following exposure to parathyroid hormone, while the 3T3-F442A cells are transformed into fat cell ⁇ under the influence of growth hormone. This is accompanied by a marked increase in alpha glycerophosphate dehydrogenase activity.
  • the reagent added to the cell culture is designed to. interact with the cell constituent which has been modified by exposure to the hormone.
  • the enzyme content of the cell culture may be activated by exposure to the hormone even though the total amount of the enzyme has not changed.
  • the adenylate cycla ⁇ e activity is activated almost in ⁇ tantly by expo ⁇ ure of the cell ⁇ to thyroid ⁇ timulating hormone.
  • the reagent added to the cell culture would be cho ⁇ en to re ⁇ pond to the increa ⁇ ed activity of the enzyme pre ⁇ ent in the cell culture.
  • the cell line used to assay the hormone i ⁇ cho ⁇ en for its ability to respond to that hormone it i ⁇ po ⁇ ible to enhance the response using certain cell stimulators.
  • Thi ⁇ can be used to potentiate the response of FRTL-5 cells to TSH ( see Fig.4) .
  • the new method for screening novel cell cultures for their sensitivity to a hormone.
  • the assay method is carried out in exactly the same way except that the new cell culture is used together with the appropriate known hormone.
  • the addition of the reagent and the measurement of the interaction between the reagent and the cellular component altered by the hormone i ⁇ carried out in exactly the same way as when the hormone itself is assayed.
  • the reagent which interacts with the cellular component altered by the hormone is of course cho ⁇ en with regard to the nature of the component which i ⁇ altered, e.g. a protein (which may be an enzyme) a lipid or a nucleic acid.
  • Suitable dyes (stains) which interact with cellular proteins in a reproducible way include Naphthol Yellow S, Coomassie blue, dinitrofluorobenzene , light green and orange II.
  • Staining methods for DNA include the use of the Feulgen reaction and methyl green pyronin for both DNA and RNA.
  • Other stains may be used, including more particularly those already used in the microscopic examination of cells. Fluore ⁇ cent probes for many cell components (e.g. as di ⁇ closed in the catalogue of Molecular Probes Inc., Eugene, Oregon, U.S.A.) exist and can be used in the same way.
  • the reagent is chosen for its ability to interact with that enzyme system.
  • the ability of MTT to interact with the cellular dehydrogena ⁇ e system to produce dark blue formazan has already been mentioned.
  • Other enzymes which may be present in cell cultures include peroxida ⁇ es, acid, neutral and alkaline pho ⁇ phatase ⁇ , specific and non-specific estera ⁇ e ⁇ , adenylate cycla ⁇ e and ATPase. Methods for the detection of some of these enzymes and therefore reagents which interact with them, are already well known.
  • the ' " assay referred to above may be performed on cell ⁇ , and, after the formazan produced has been eluted and measured, a second colorimetric assay, for example, to measure the protein or DNA content of the cell ⁇ , can be performed.
  • the sequential ⁇ taining technique may be useful to measure the effects of more than one hormone on the same target cell, if different cellular components (which interact with different stain ⁇ ) which respond to different hormones can be identified.
  • FRTL-5 cell ⁇ were maintained in stock cultures in Coon's modified F-12 medium supplemented with 5% newborn calf serum, 100 units penicillin/ml, 100 ⁇ q streptomycin per ml, 10 "3 units of thyroid stimulating hormone (TSH) per 1, 10 ⁇ g insulin per ml, 10 nM cortisol, 5 ⁇ g tran ⁇ ferrin per ml, lOng glycyl-L-hi ⁇ tidyl-L-lysine acetate per ml and 10 ng somatostatin per ml.
  • TSH thyroid stimulating hormone
  • the cell ⁇ were plated out in well ⁇ of a microtitre plate at a density of 5 X 10 4 cells/ml (150 / 1/well) in the above medium.
  • a repeater Eppendorf pipette was u ⁇ ed to dispense the cell ⁇ , which were mixed at frequent interval ⁇ to prevent the cells settling to the bottom of the container from which they were being withdrawn.
  • After 3 days in this medium it was removed and replaced by a medium containing all the above constituents apart from the TSH in order to allow the cells to come to a resting state. At least 7 days were allowed for this to happen.
  • the medium was changed every 3-4 days.
  • Thi ⁇ cell culture and manipulation technique may be used with other strain ⁇ of cell ⁇ which adhere to well ⁇ .
  • Rat lymphoma cell ⁇ Nb2 which grow in suspension culture were grown in RPMI medium containing 10 ⁇ 4 M 2-mercaptoethanol , 50 units penicillin/ml, 50 ⁇ g streptomycin/ml, 2x 10 ⁇ 3 M glutamine, 10% foetal calf serum and 10% horse serum. Exposure to Hormone
  • the medium in the well ⁇ wa ⁇ replaced with medium containing the sample to be assayed and appropriate controls for 2 to 48 hours.
  • the as ⁇ ay of hormone ⁇ i ⁇ undertaken on a routine basis it i ⁇ po ⁇ ible to maintain a con ⁇ tant supply in the incubator of microtitre plates with cell ⁇ attached thereto in order to minimise delays between receiving and assaying samples of hormones.
  • the hormone was added to a suspension of cells in the well ⁇ of the microtitre plates.
  • a ⁇ tock ⁇ upply of cell ⁇ in suspension can be maintained in the incubator for routine use, by culture in tis ⁇ ue culture fla ⁇ k ⁇ from which they can be dispensed into the microtitre plates.
  • the reagent which reveals the effect of the hormone may then be added in accordance with one of the following procedures: 1. COLORIMETRIC DYE STAINING PROCEDURE
  • Dulbecco's pho ⁇ phate buffered saline pH7.3.
  • Dulbecco' ⁇ phosphate buffered saline consists of 8g NaCl/1; 200mg KCl/1; 1.15g Na 2 HP0 4 /l , 132mg CaCl 2 .2H 2 0/1; 200mg KH 2 P0 4 /I and lOO g MgCl 2 .6H 2 0/1. 1ml 25% glutaraldehyde . was added to 11.5ml PBS to make the fixer solution. This may be stored at 4° C for up to 2 weeks.
  • fixer solution 50 ⁇ l was added to each microtitre well which already contained 150 ⁇ l of culture medium, and left to fix for lh at room temperature.
  • the medium was tipped off with a ⁇ harp flick of the wri ⁇ t and the plate was blotted on a paper towel to remove as much liquid as po ⁇ ible from the well ⁇ .
  • the plate was inverted and allowed to drain to dryne ⁇ . It wa ⁇ stored in the dark until required.
  • the cells were plated out at the required density in lOO ⁇ l of medium per well of a microtitre plate. After a suitable period of growth of the cells, their dehydrogenase activity was assayed by adding 10 ⁇ l of MTT solution at 5 mg/ml. After 40 minutes 150 l of 10% Triton-X in 0.5 M HCl wa ⁇ addded to each well. The plate wa ⁇ shaken gently on a plate shaker for 1 hour to release the formazan into the medium, which wa ⁇ mea ⁇ ured using a microtitre plate reader at a test wavelength of 570 nm and a reference wavelength of 630 nm. RESULTS The results show:
  • cell stimulators such a ⁇ the anterior pituitary polypeptide hormones, e.g. thyroid stimulating hormone (TSH) induce early enzymic activation, which may be detected by the eluted stain system;
  • TSH thyroid stimulating hormone
  • Fig. 1 of the accompaning drawings the responses of the two eluted stain systems are compared at selected time intervals subsequent to the addition of TSH to resting FRTL-5 cell ⁇ .
  • dehydrogenase activity a ⁇ measured by cleavage of MTT, i ⁇ enhanced by the earliest reading taken (6 hours) at which time there has been no detectable increase in the NYS staining which reflects overall cell protein.
  • TSH trophic hormone
  • the metaphase index i the number of cells exhibiting metapha ⁇ e figure ⁇ expre ⁇ ed a ⁇ a % of the total cells in a given culture (Ealey et al, J. Immunol. Methods 3 1, 117-123, 1988). This demonstrates that a wave of mito ⁇ i ⁇ pa ⁇ sed through the cultures after 20 hours exposure to the hormone. Thi ⁇ i ⁇ con ⁇ i ⁇ tent with a doubling time of 30 hour ⁇ for FRTL-5 cell ⁇ , and supports the observation that the bioas ⁇ ay ⁇ yste can measure hormonal activation of the enzymic system.

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  • Health & Medical Sciences (AREA)
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  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Molecular Biology (AREA)
  • Hematology (AREA)
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  • Urology & Nephrology (AREA)
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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
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Abstract

On titre des agents de modification cellulaire extracellulaires tels que des hormones (et des autoanticorps mimant leurs effets) en les ajoutant à une culture de cellules contenant un composant cellulaire dont l'activité ou la quantité est sensible à cet agent de modification cellulaire, puis on mesure le changement dans le composant cellulaire par l'emploi d'un réactif colorimétrique ou chromogénique approprié. On met adéquatement en ÷uvre le procédé dans les puits d'une plaque de microtitrage utilisant un réactif colorimétrique ou chromogénique pouvant être mesuré spectrophotométriquement. On élue le produit coloré à partir des cellules jusque dans les puits et on lit les mesures spectrophotométriques directement dans les puits avec un lecteur de plaque de microtitrage.
PCT/GB1989/000775 1988-07-08 1989-07-07 Analyse de substances de modification cellulaire WO1990000619A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
FI910081A FI910081A0 (fi) 1988-07-08 1991-01-07 Analys av cellmodifierande substanser.

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB888816302A GB8816302D0 (en) 1988-07-08 1988-07-08 Novel bioassay
GB8816302.7 1988-07-08
GB888818508A GB8818508D0 (en) 1988-08-04 1988-08-04 Improvements in bioassay of hormones
GB8818508.7 1988-08-04

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WO1990000619A1 true WO1990000619A1 (fr) 1990-01-25

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JP (1) JPH03505669A (fr)
WO (1) WO1990000619A1 (fr)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1993005172A1 (fr) * 1991-08-30 1993-03-18 Creative Biomolecules, Inc. Procede de triage de proteines morphogeniques
ES2071567A1 (es) * 1993-05-21 1995-06-16 Inia Metodo de valoracion toxicologica de un medio acuatico.
US5650276A (en) * 1991-03-11 1997-07-22 Creative Biomolecules, Inc. Morphogenic protein screening method
US5707810A (en) * 1991-03-11 1998-01-13 Creative Biomolecules, Inc. Method of diagnosing renal tissue damage or disease
AU696364B2 (en) * 1991-08-30 1998-09-10 Creative Biomolecules, Inc. Morphogenic protein screening method II
US6407060B1 (en) 1996-03-22 2002-06-18 Curis, Inc. Method for enhancing functional recovery following central nervous system ischemia or trauma
US6524797B1 (en) 1999-05-10 2003-02-25 Bernhard O. Palsson Methods of identifying therapeutic compounds in a genetically defined setting

Citations (2)

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EP0005032A2 (fr) * 1978-04-20 1979-10-31 Howard Maurice Shapiro Méthode d'essai biologique
EP0159653A1 (fr) * 1984-04-16 1985-10-30 Innogenetics N.V. Utilisation analytique de lignées cellulaires de phagocytes

Patent Citations (2)

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EP0005032A2 (fr) * 1978-04-20 1979-10-31 Howard Maurice Shapiro Méthode d'essai biologique
EP0159653A1 (fr) * 1984-04-16 1985-10-30 Innogenetics N.V. Utilisation analytique de lignées cellulaires de phagocytes

Non-Patent Citations (6)

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Title
BIOCHEMISTRY, Vol. 13, No. 16, 1974, PETER JAY SIMS et al., "Studies on the Mechanism by Which Cyanine Dyes Measure Membrane Potential in Red Blood Cells and Phosphatidylcholine Vesicles", pages 3315-3330. *
CHEM. PHARM. BULL., Vol. 34, No. 11, 1986, SHUU MATSUMOTO and HIDEYO IMANISHI, "Effect of Interferon on Activated Oxygen Production in Human Monocytes in Vitro: Direct Measurement of Phagosomal Activated Oxygens by a New Method Using Luminol-Binding Microspheres", pages 4774-4781. *
DEVELOP. BIOL. STANDARD., Vol. 64, 1986, (Basel), M.R. BARER et al., "A Semi-Automated System for the Assessment of Toxicity to Cultured Mammalian Cells Based on Detection of Changes in Staining Properties", pages 251-259. *
EUR. J. IMMUNOL., Vol. 17, 1987, JANICE TAVERNE et al., "Cytotoxicity of Tumor Necrosis Factor for Thyroid Epithelial Cells and Its Regulation by Interferon-gamma", pages 1855-1858. *
HISTOCHEMICAL JOURNAL, Vol. 18, 1986, M.R. BARER et al., "Quantitation of Dye Binding by Cell Monolayers in a Microtiter System", pages 122-128. *
JOURNAL OF IMMUNOLOGICAL METHODS, Vol. 65, 1983, TIM MOSMANN, "Rapid Colorimetric Assay for Cellular Growth and Survival: Application to Proliferation and Cytotoxicity Assays", pages 55-63. *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5650276A (en) * 1991-03-11 1997-07-22 Creative Biomolecules, Inc. Morphogenic protein screening method
US5707810A (en) * 1991-03-11 1998-01-13 Creative Biomolecules, Inc. Method of diagnosing renal tissue damage or disease
US5741641A (en) * 1991-03-11 1998-04-21 Creative Biomolecules, Inc. Morphogenic protein screening method
US5994131A (en) * 1991-03-11 1999-11-30 Creative Biomolecules, Inc. Morphogenic protein screening method
WO1993005172A1 (fr) * 1991-08-30 1993-03-18 Creative Biomolecules, Inc. Procede de triage de proteines morphogeniques
AU678345B2 (en) * 1991-08-30 1997-05-29 Creative Biomolecules, Inc. Morphogenic protein screening method
AU696364B2 (en) * 1991-08-30 1998-09-10 Creative Biomolecules, Inc. Morphogenic protein screening method II
ES2071567A1 (es) * 1993-05-21 1995-06-16 Inia Metodo de valoracion toxicologica de un medio acuatico.
US6407060B1 (en) 1996-03-22 2002-06-18 Curis, Inc. Method for enhancing functional recovery following central nervous system ischemia or trauma
US6524797B1 (en) 1999-05-10 2003-02-25 Bernhard O. Palsson Methods of identifying therapeutic compounds in a genetically defined setting
US7374880B2 (en) 1999-05-10 2008-05-20 Bernhard O Palsson Methods of identifying therapeutic compounds in a genetically defined setting

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EP0423206A1 (fr) 1991-04-24

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