WO2009029039A1 - Procédé de mesure de la viabilité d'une cellule - Google Patents

Procédé de mesure de la viabilité d'une cellule Download PDF

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Publication number
WO2009029039A1
WO2009029039A1 PCT/SE2008/050974 SE2008050974W WO2009029039A1 WO 2009029039 A1 WO2009029039 A1 WO 2009029039A1 SE 2008050974 W SE2008050974 W SE 2008050974W WO 2009029039 A1 WO2009029039 A1 WO 2009029039A1
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WO
WIPO (PCT)
Prior art keywords
cells
measurement
status
marker
viability
Prior art date
Application number
PCT/SE2008/050974
Other languages
English (en)
Inventor
Karl Andersson
Hans Lundqvist
Lina VENNSTRÖM
Original Assignee
Ridgeview Instruments Ab
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 Ridgeview Instruments Ab filed Critical Ridgeview Instruments Ab
Publication of WO2009029039A1 publication Critical patent/WO2009029039A1/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
    • G01N33/5014—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 toxicity

Definitions

  • the present invention relates to the field of biological research. More in particular, it relates to biological research utilizing living cells. Even more in particular, it relates to the measurement of the status of cells, such as the viability of cells.
  • Biological research of today is to a significant fraction relying on cells as model organisms.
  • the measurement of how cell status is dependant of a certain treatment is of great interest.
  • Cell status may include qualities as cell growth, changes in cell morphology, triggering of repair mechanisms after damage, viability, and the like.
  • One common example of cell status measurement is the viability measurement, i.e. the measurement of the ability of a cell to survive a certain treatment is of great interest.
  • the reverse is also common, i.e. the measurement of how efficient a certain treatment is in killing cells.
  • Yet another viability measurement of interest is to follow natural cell death (apoptosis) either through triggering of apoptotic molecular pathways (i.e. not poisoning the cell, but rather trick it to commit suicide) or as it occurs without use of molecular effectors.
  • PI propidium iodide
  • toxicity measurements using the antonym toxicity There are other methods available for the measurement of viability (such methods are sometimes referred to as toxicity measurements using the antonym toxicity).
  • One common method relies on radioactive chromium (51Cr), which is absorbed by living cells and released when the cells die. Such measurements typically comprise an incubation period, during which the cells are loaded with 51Cr, a toxin treatment period (and optionally a waiting period) prior to harvesting the cell culture medium. Since dead cells have release their chromium into the cell culture medium, the radioactivity of the medium is proportional to the number of killed cells. Measurements relying on 51Cr are terminal and can therefore only provide information on the cell viability status at the time of harvesting.
  • the object of the present invention is to provide an improved method for measuring cell status in general.
  • the invention is particularly useful for time-resolved measurements of cell viability.
  • the method according to the invention is defined in claim 1.
  • the method for measuring cell viability uses of
  • the invention in a preferred embodiment comprises three characteristic components.
  • Suitable markers include (but are not limited to) radioactive markers, fluorescent markers and chemoluminiscent markers.
  • Suitable cells include (but are not limited to) prokaryotic or eukaryotic cells that can be cultured in a laboratory.
  • suitable cells are human cancer cell- lines, for example the cell-line A431 (ATCC, CLR 1555, Rocksville MD USA). The time resolved measurement is typically made in a device described in WO2005080967, which is incorporated by reference herein.
  • Another application is the study of apoptosis and potential apoptotic inducers and apoptotic inhibitors.
  • Yet another application is the development of cancer treatments, i.e. finding suitable means for killing tumor cells.
  • Still another application is the study of cell viability when put under influence of external treatments, such as cell resistance to electromagnetic radiation.
  • Yet another application is to study the function or effect of the immune system when killing intruding cells.
  • Still another application is the measurement of total DNA in the target cells as an indirect measure of growth.
  • Figure 1 shows a flow chart of the method
  • Figure 2 shows a suitable instrument, known in prior art, for performing the measurement in the viability method
  • Figure 3 shows results from one 51Cr incubation experiment
  • Figure 4 shows results from two 51Cr incubations on untreated cells and from one 51Cr incubation on treated cells
  • Figure 5 shows results from two 51Cr incubations on untreated cells, wherein the 51Cr concentration was different; and Figure 6 shows results from a series of combined 51Cr incubations and treatments.
  • the term "cell status” refers to the general condition of a cell population with respect to one of more aspects including (but not limited to) DNA content, intracellular metal ions concentrations, intensity of metabolism, fraction necrotic cells, and the like.
  • viability refers to the viability status of the cells subject to measurement. Viable cells are alive and growing, and poor viability is recognized as slow or absent growth, poor cell morphology, or necrosis.
  • the cells attached to the solid support are denoted "target cells”.
  • Target cells include (but are not limited to) pro kary otic cells, eukaryotic cells, tissue slices (thinner than 2 mm), and small organisms (less than 2mm in diameter) that can be cultured in a laboratory.
  • target cells include (but are not limited to) human cancer cell lines, human embryonic stem cell lines (na ⁇ ve or differentiated), cancer cell lines from other mammals, embryonic cell lines from other mammals, insect cells and xenopus cells.
  • the method requires a compound to be used for monitoring the status of the target cells, said compound being denoted "status marker”.
  • status markers include, but are not limited to, radioactive markers (e.g. 51Cr, 18-F-deoxyglucose), fluorescent markers (e.g.
  • the present invention aims at providing a method of measuring cell status by enabling a time-resolved detection of status markers in target cells. Due to the time-resolved detection, the effect of different chemical or environmental conditions can be detected as it appears.
  • target cells are loaded with viability markers (110), followed by an optional wash (120).
  • the cells are treated with the agent or exposed to a condition for which the status should be determined (130), followed by an optional wash (140).
  • the last step in the method is the time-resolved detection of status marker associated with target cells (150), which is a direct or indirect measure of the status of the cells.
  • cytotoxic drugs e.g. Doxorubicin, Daunorubicin, Fluorouracil, Tiotepa, Hydroxykarbamid, Karboplatin, Metotrexat, Etoposid, Paklitaxel, Vinkristin, Dakarbazin, Epirubicin, Oxaliplatin, Lomustin, Cytarabin, Vinorelbin, Docetaxel, Pemetrexed, Topotekan, Amsakrin, Merkaptopurin, Busulfan, Melfalan, Metotrexat, Idarubicin, Mitoxantron, Mitomycin, or Capecitabin) .
  • cytotoxic drugs e.g. Doxorubicin, Daunorubicin, Fluorouracil, Tiotepa, Hydroxykarbamid, Karboplatin, Metotrexat, Etoposid, Paklitaxel, Vinkristin, Dakarbazin, Epirubicin, Oxaliplatin,
  • cytotoxic proteins e.g. monoclonal antibodies like Rituximab, Trastuzumab, and Cetuximab
  • growth factors e.g. EGF, NGF, TGF-alpha, TGF-beta
  • other proteins e.g. insulin
  • detergents e.g. tween 40, tween 20, Triton XlOO, octylglycoside or NP40
  • inorganic compounds or metals e.g. Copper, Lead, Cadmium, or Mercury
  • radioactive compounds e.g.
  • Possible conditions include (but are not limited to) starvation, hyperthermia, hypothermia, increased or decreased ionic strength compared to physiological conditions, increased or decreased pH compared to physiological conditions, non-ionizing radiation, ionizing radiation, static electromagnetic fields, fluctuating electromagnetic fields (e.g. microwave treatment), and mechanical treatment (e.g.
  • apoptosis- inducing effector compounds can be used, including (but not limited to) bisindolylmaleimide compounds (as defined in US6284783, which is incorporated by reference herein), butylated hydroxyanisole, allyl sulfide, benzyl isothiocyanate, and dimethyl fumarate.
  • the method can be conducted in slightly different ways.
  • One other example is as described in figure Ib, wherein the treatment of the target cells could be the first step (111), followed by an optional wash (121), after which the treated target cells are loaded with status marker (131). After an optional wash (141), status marker is detected using a time-resolved detection technology.
  • FIG. 1c Yet another example is as described in figure Ic, wherein the treatment of the target cells and the loading of status marker is performed simultaneously (112), followed by an optional wash (122). Next, status marker is detected using a time-resolved detection technology (132).
  • Still another example is as described in figure Id, wherein the first step is the treatment of the target cells (113), followed by an optional wash (123). Next, status marker is loaded and simultaneously detected using a time- resolved detection technology (133).
  • FIG. 1e Yet another example is as described in figure Ie, wherein the first step is the loading of status marker during time- resolved detection (114), followed by an optional wash (124). Next, the treatment of the cells is started and the effect of it being simultaneously detected using a time-resolved detection technology (134).
  • the preferred method for completing steps 114, 150, 151, 132, 133, and 134 in figure 1, i.e. the detection, has been previously disclosed [WO2005080967, which is incorporated by reference herein] and is schematically described in figure 2.
  • the method relies on target cells (202) being attached to a defined area on a solid support (201).
  • a reference area in this case opposite to the target area.
  • a liquid is in contact with the solid support to enable a suitable environment for the target cells.
  • the liquid may possibly contain status marker, so that the loading of target cells takes place during detection.
  • the solid support is inclined and slowly rotated using a motor (203).
  • a detector capable of detecting the label attached to the species used is mounted (204) over the elevated portion of the solid support.
  • an elevated signal will be registered in case the status marker has bound to or is present in the target cells.
  • the rate of change of status marker density can be followed by depicting the difference between the detected signal from target area and reference area over time.
  • target cells are attached to a selected portion of a solid support
  • the present invention makes it possible to follow cell status in real time, thereby monitoring the progress of processes toxic to the cell.
  • step 113 and 123 were ignored and the baseline measurement started at step 133.
  • a time-resolved measurement was performed using the preferred device as described in figure 2, using a detector sensitive to gamma radiation in the energy range 100- 1000 keV.
  • the resulting graph of amount of 51Cr versus time is shown in figure 3. As seen, the cells absorb 51Cr during approximately 16 hours after which the 51Cr is declining.
  • the baseline measurement was repeated in order to verify the stability of the method.
  • target cells were treated with Agent during one hour (113), followed by a wash (123) and approximately 15 hours after Agent treatment, the target cells were loaded with 51Cr during measurement (133).
  • the resulting curve of amount of 51Cr versus time is shown in figure 4, together with the two baseline curves.
  • Figure 4 shows that the decline of 51Cr is delayed in the treated cells compared to the baseline measurement. Furthermore, the results from the two baseline measurements are in agreement, indicating that the viability measurement is robust.
  • a probable cause for the general shape of the curves in figure 3 and figure 4 is that the target cells might be sensitive to 51Cr itself.
  • the decline of 51Cr appeared earlier at higher concentration of 51Cr as shown in figure 4.
  • the fact that the cytotoxic Agent is shifting the decline towards later points in time indicates that Agent is forcing target cells into cell-cycle arrest, because dividing cells are likely more sensitive to metal poisoning than static cells.
  • Example 2 The method described above (figure 1, steps 114-124- 134) was tested with target cells of type A431 (a human cancer cell-line) grown on approximately one quarter of a 10cm circular cell-dish.
  • the cellular status detected in this example was viability and the status marker was 51 -chromium.
  • the cells While detecting the 51Cr level in the cells, the cells were incubated with 51Cr during approximately 2h. After incubation, a toxic agent (Triton XlOO) was added to the cell dish. The release of 51Cr was then followed approximately 7 hours. The procedure was repeated several times with varying concentrations of the toxic agent Triton XlOO.
  • Triton XlOO Triton XlOO

Abstract

L'invention concerne un procédé pour la mesure de l'état cellulaire, à savoir des qualités telles que la croissance cellulaire, des changements de la morphologie de la cellule, le déclenchement de mécanismes de réparation après endommagement, la viabilité et similaires. Le procédé comprend la fixation de cellules cibles sur un support solide, le marquage de la cellule cible par un marqueur d'état (par exemple, une molécule fluorescente ou chimioluminescente ou un émetteur de rayonnement radioactif), et la détection de la quantité de marqueur d'état en résolution temporelle. L'invention concerne en outre un tel procédé de mesure, consistant à mettre le support solide étant en contact avec un liquide, puis à effectuer une mesure permettant détecter la présence d'un marqueur d'état, ainsi qu'une mesure de référence. Le liquide est temporairement retiré pendant la mesure.
PCT/SE2008/050974 2007-08-28 2008-08-28 Procédé de mesure de la viabilité d'une cellule WO2009029039A1 (fr)

Applications Claiming Priority (2)

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SE0701925 2007-08-28
SE0701925-0 2007-08-28

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WO2009029039A1 true WO2009029039A1 (fr) 2009-03-05

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8993259B2 (en) 2012-05-02 2015-03-31 Charles River Laboratories, Inc. Method of viability staining with membrane permeable fluorescent dye and membrane impermeable fluorescence quencher
EP3077814A4 (fr) * 2013-11-04 2017-06-07 Charles River Laboratories, Inc. Procédé de détection de cellules viables dans un échantillon de cellules
US9709500B2 (en) 2012-05-02 2017-07-18 Charles River Laboratories, Inc. Optical method for detecting viable microorganisms in a cell sample
US10324036B2 (en) 2012-05-02 2019-06-18 Charles River Laboratories, Inc. Porous planar cell capture system

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005080967A1 (fr) * 2004-02-20 2005-09-01 Ridgeview Instruments Ab Procede et dispositif pour la caracterisation des interactions entre differentes especes

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005080967A1 (fr) * 2004-02-20 2005-09-01 Ridgeview Instruments Ab Procede et dispositif pour la caracterisation des interactions entre differentes especes

Non-Patent Citations (3)

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Title
BRUNNER K.T. ET AL: "Quantitative assay of the lytic action of immune lymphoid cells on 51-Cr-labelled allogeneic target cells in vitro; inhibiton by isoantibody and by drugs", IMMUNOLOGY, vol. 14, no. 2, 1968, pages 181 - 196, XP003025141 *
MUELLER H. ET AL: "Comparison of the usefulness of the MTT,ATP and calcein assays to predict the potency of cytotoxic agents in various human cancer cell lines", JOURNAL OF BIOMOLECULAR SCREENING, vol. 9, no. 6, 2004, pages 506 - 515, XP009081506 *
PROVINCIALI M. ET AL: "Optimization of cytotoxic assay by target cell retention of the fluoroscent dye carboxyfluoroscein diacetate (CFDA) and comparison with conventional chromium-51 release assay", JOURNAL OF IMMUNOLOGICAL METHODS, vol. 155, 1992, pages 19 - 24, XP023657846 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8993259B2 (en) 2012-05-02 2015-03-31 Charles River Laboratories, Inc. Method of viability staining with membrane permeable fluorescent dye and membrane impermeable fluorescence quencher
US8993260B2 (en) 2012-05-02 2015-03-31 Charles River Laboratories, Inc. Fluorescence-based viability staining method using a membrane permeable flourescent dye and membrane impermeable fluorescence quencher
US9709500B2 (en) 2012-05-02 2017-07-18 Charles River Laboratories, Inc. Optical method for detecting viable microorganisms in a cell sample
US10324036B2 (en) 2012-05-02 2019-06-18 Charles River Laboratories, Inc. Porous planar cell capture system
US10976258B2 (en) 2012-05-02 2021-04-13 Charles River Laboratories, Inc. Porous planar cell capture system and method of use
EP3077814A4 (fr) * 2013-11-04 2017-06-07 Charles River Laboratories, Inc. Procédé de détection de cellules viables dans un échantillon de cellules

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