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WO2007040117A1 - Procédé permettant de mesurer simultanément un nombre pluriel de réponses cellulaires - Google Patents

Procédé permettant de mesurer simultanément un nombre pluriel de réponses cellulaires Download PDF

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Publication number
WO2007040117A1
WO2007040117A1 PCT/JP2006/319156 JP2006319156W WO2007040117A1 WO 2007040117 A1 WO2007040117 A1 WO 2007040117A1 JP 2006319156 W JP2006319156 W JP 2006319156W WO 2007040117 A1 WO2007040117 A1 WO 2007040117A1
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WIPO (PCT)
Prior art keywords
cell
cells
fluorescence
fluorescence intensity
detected
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Ceased
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PCT/JP2006/319156
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English (en)
Japanese (ja)
Inventor
Ritsu Honda
Hiroyuki Kishi
Atsushi Muraguchi
Eiichi Kanaumi
Kihachiro Tohbo
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TOYAMA NEW INDUSTRY ORGANIZATION
University of Toyama NUC
NS Materials Inc
Original Assignee
TOYAMA NEW INDUSTRY ORGANIZATION
University of Toyama NUC
NS Materials Inc
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Publication of WO2007040117A1 publication Critical patent/WO2007040117A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56966—Animal cells
    • G01N33/56972—White blood cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6452—Individual samples arranged in a regular 2D-array, e.g. multiwell plates
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/645—Specially adapted constructive features of fluorimeters
    • G01N21/6456—Spatial resolved fluorescence measurements; Imaging
    • G01N21/6458—Fluorescence microscopy
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
    • G01N33/5047—Cells of the immune system
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label

Definitions

  • the present invention relates to a multi-cell response simultaneous measurement method using an image sensor such as a CCD image scanner.
  • the present invention is a method that allows the fluorescence emitted by a large number of cells to be tracked independently for each cell over time and in a massively parallel manner.
  • Patent Document 1 Japanese Unexamined Patent Application Publication No. 2004-173681
  • Patent Document 2 Japanese Patent Application Laid-Open No. 2004-187676
  • Patent Documents 1 and 2 describe that it has been confirmed that cells are identified on a cell-by-cell basis, and describe that the identified cells can also be collected. However, it was not easy to detect many lymphocyte forces from lymphocytes that actually reacted specifically to the antigen.
  • a detection method for example, an increase in calcium ion concentration in lymphocytes in response to an antigen is used, and a change in the calcium ion concentration is detected as fluorescence to identify an antigen-specific lymphocyte.
  • the manner in which fluorescence is generated and the intensity, etc. are not always constant.
  • the calcium ion concentration increases after a certain period of time, resulting in an increase in fluorescence intensity.
  • the fluorescence microscope 'imaging device has an imaging device in addition to a normal fluorescence microscope.
  • Fluorescence microscope ⁇ The imaging device has no problem in terms of speed, but with an ordinary microscope, the imaging range is narrow, and up to 1000 cells cannot be detected at a time, and tens of thousands to hundreds of thousands of cells cannot be detected. It was impossible to detect strong fluorescence at once.
  • a cell chip detection device based on a DNA microarray scanner has been developed to detect cell fluorescence arrayed on a cell chip, and can detect intracellular or extracellular fluorescence.
  • this system uses a laser to excite the fluorescent dye and detect the excitation light, a detection system that tracks changes over time cannot simultaneously detect a large number of cell regions with a slow line scan speed.
  • the object of the present invention is to simultaneously measure the state of a large number of cells retained on the chip, more than 10,000, preferably more than 100,000, for example, lymphocyte reactivity to antigen stimulation,
  • the object is to provide a method for individually grasping the state of each cell.
  • Fluorescence having at least a part of the position of the plurality of positions on a cell chip that holds a plurality of cells independently at a plurality of positions is detected by an image sensor
  • lymphocyte is a B lymphocyte and the receptor is a B lymphocyte receptor.
  • the image sensor is a CCD image scanner or a CMOS image scanner
  • the cell chip that holds a plurality of cells independently at a plurality of positions has a spot or a hole at a position for holding the cell on one surface of the substrate, and at least the spot or the hole.
  • a stimulus response that an antigen exerts on lymphocytes can be rapidly detected on a large scale and quantitatively for individual cells. As a result, it becomes easier to produce an antibody having a high affinity for a specific antigen.
  • lymphocytes of patients with B cell leukemia have a reduced calcium response to stimulation by anti-IgM antibodies.
  • B cell leukemia leukemia
  • the power of being able to diagnose Beel 1 Leukemia (leukemia) can also be applied in this way, thereby enabling rapid diagnosis of leukemia, life expectancy tests, and the like. .
  • the necessary treatment can be performed accurately, and it can be used effectively in clinical examinations.
  • the method of the present invention comprises:
  • Fluorescence of at least a part of the position force of the plurality of positions on the cell chip holding a plurality of cells independently at a plurality of positions is detected by an image sensor (fluorescence detection step),
  • a method of measuring a cell state including displaying (recording) fluorescence intensity or a converted value from fluorescence intensity (display step). Then, at least the detection and recording are repeated over time.
  • the measurement target in the method of the present invention is a cell chip that holds a plurality of cells independently at a plurality of positions. If the cell chip is to hold a plurality of cells independently at a plurality of positions, the method of holding the cell, the number of held cells, the density of the held cells on the chip surface, etc. are particularly limited. There is no.
  • the cell chip for example, a plurality of microchips for storing cells on one surface of the substrate described in Patent Documents 1 and 2 above.
  • a microwell array chip having a clowell may contain one cell (independently) in each microwell.
  • a plurality of microwells (holes) correspond to the “plural positions”.
  • the culture solution can be stored together with the cells.
  • the cell chip may be one in which cells are individually fixed on the chip surface using a thermosensitive polymer described in JP-A-2005-102628.
  • the fixed positions of a plurality of cells on the chip surface correspond to the “plural positions”.
  • the cell chip can be held in a plurality of 1S clusters that can hold cells uniformly (at equal intervals) throughout. For example, if one cluster is a group of 10 to 1000 cells X 10 to 1000 cells (positions), a plurality of clusters, for example, n cells x m (n and m are independently 2 to 50, for example) ) On the substrate.
  • the number of cells held on the cell chip is not particularly limited. However, since the object of the present invention is to simultaneously measure the state of a large number of cells, for example, at least 10,000 cells. Yes, preferably 50,000 or more, more preferably 100,000 or more, and even more preferably 200,000 or more. In particular, from the viewpoint of searching for lymphocytes that are antigen-specific and low in frequency, the number of cells retained on the cell chip is preferably at least 200,000.
  • the condition for the number of cells held on the cell chip can be determined appropriately in consideration of the purpose of measurement and the sensitivity of the image sensor, which is a measuring instrument, and the resolution required for fluorescence detection. For example, about 1 million cells And preferably 500,000. However, the upper limit of the number of cells retained on the cell chip can be further increased if the capabilities of the image sensor and the equipment required for recording processing of the detected fluorescence intensity improve as the technology advances. It is a spear.
  • lymphocytes are used as cells in the method of the present invention
  • the state before and after stimulation with an antigen for lymphocytes can be measured. More specifically, measurement of the state before and after stimulation with antigen is based on intracellular calcium generated by a signal through a receptor possessed by lymphocytes. Can be measured by a change in fluorescence intensity using a Ca ion-dependent fluorescent dye. More specifically, for example, an increase in intracellular calcium caused by a signal through the B cell receptor can be measured by a change in fluorescence intensity. The same applies to T cell receptors.
  • Fluorescence from at least some of the plurality of positions is detected by an image sensor. Fluorescence from the position where the cell is fixed is generated when the cell reacts with the antigen by adding a Ca ion-dependent fluorescent dye to the fixed cell in advance.
  • an antigen binds to the antigen receptor (immunoglobulin) of B lymphocytes, intracellular signal transduction occurs first, and as a result, the concentration of intracellular Ca ions changes, and when Ca ion-dependent fluorescent dyes coexist, Fluorescence is generated.
  • the fluorescent dye for example, Fura-2, Fluo_3 or Fluo-4 can be used.
  • the fluorescent dye is not particularly limited as long as it is a Ca ion-dependent fluorescent dye.
  • the fluorescence may be a deviation of intracellular fluorescence, fluorescence on the cell membrane surface, or fluorescence emitted from a cell! ,.
  • a cell chip containing 200,000 cells is placed in the measurement range.
  • an image scanner that has sufficient sensitivity to fully examine cell responses.
  • This device 1) is a cell derived from a biological host regularly arranged on a microwell array chip !, or puts all the intracellular or extracellular fluorescence of a subcultured cell line into the field of view, 2) By allowing time-lapse photography, the reaction of cells after drug loading can be performed for each of several hundreds of thousands of cells. This can be analyzed.
  • the resolving power of the image sensor does not necessarily have to be remarkably high, and may be appropriate.
  • CMOS type image scanner that is slightly inferior in resolution if it has the ability to detect fluorescence of cells that use CCD type image scanners.
  • the design is such that a stimulus response in the detection range can be detected in real time for each individual cell.
  • the image scanner is, for example, It is appropriate that the imaging area has a resolution of 3 million pixels or more and has a function of capturing fluorescence per cell (position) by 4 pixels or more of the imaging element. The number of pixels required for the image scanner is determined appropriately by the product of the number of pixels that capture fluorescence per cell (position) and the number of cells (positions) that include the imaging area. Is done.
  • a fluorescence measuring device including the image sensor can be used.
  • An explanatory diagram of this fluorescence measuring apparatus is shown in FIG. It consists of an image sensor 10, a split filter 20, a telecentric optical system 30, a light source 40, a shutter 50, and a computer 60 that controls them.
  • the image sensor 10 receives fluorescence emitted from the cell chip 70 and converts it into digital data.
  • the image sensor 10 has a sufficient number of pixels necessary for data acquisition, and it is appropriate that the image sensor 10 has a function of photographing fluorescence per sphere with four or more pixels of the image sensor. 9 pixels or more is desirable to stabilize From this, the number of lymphocytes that can be handled by the device is determined by the total number of image sensors.
  • the split filter 20 reflects and transmits the excitation light wavelength selected from the light emitted from the light source. In addition, it has a function to select and pass the fluorescence wavelength emitted from the sample.
  • the telecentric optical system 30 has a function of efficiently irradiating the sample with excitation light and efficiently condensing the fluorescent light emitted from the sample onto the image sensor 10.
  • a telecentric optical system (lens system) as the optical system in order to minimize the distortion of the peripheral area.
  • the light source 40 has a function of generating light necessary for excitation with a sufficient output.
  • the shutter 50 has a function of irradiating the cell chip 70 with excitation light only when necessary in order to minimize damage to the cell chip 70, and is controlled by the computer 60.
  • the computer 60 has a function of controlling the image sensor 10 and the shutter 50, and has a function of acquiring, analyzing, and storing fluorescence data output from the image sensor 10. It also monitors and controls the entire device.
  • the cycle of fluorescence detection by the image sensor can be appropriately determined according to the state of the cell to be measured. For example, if the cell is a lymphocyte and the cell condition is the lymphocyte's reactivity to antigen stimulation, fluorescence detection by the image sensor should be performed at least once every 60 seconds, preferably every 30 seconds. Is appropriate. The fluorescence detection cycle is limited by the data processing capability of the device used for fluorescence detection and recording. With the fluorescence measurement device shown in Fig. 1, fluorescence detection and recording is performed once every 10 seconds.
  • One fluorescence detection and recording (1 cycle) includes a series of processes including all fluorescence detection, including fluorescence detection, transfer of image data to a computer, and image correction on a personal computer. .
  • the fluorescence detected by the image sensor 10 is digitized by the image sensor 10 and recorded in the computer 60.
  • Data digitized by the image sensor 10 is digitized in proportion to the amount of fluorescence emitted.
  • the data that can be stopped here is the relative fluorescence.
  • the image sensor 10 includes a plurality of light receiving units, and each light receiving unit has a respective light receiving characteristic.
  • a correction map for the light intensity of each light receiving unit is created to correct the input light quantity and obtain an accurate fluorescence amount.
  • Fluorescence data digitized by the image sensor is captured by a plurality of pixels for one lymphocyte.
  • the converted value of the fluorescence intensity can be a calcium concentration.
  • the fluorescence emission amount can be converted into the calcium concentration and the calcium concentration can be quantitatively measured.
  • Display of recorded fluorescence intensity or converted value of fluorescence intensity force may be performed at the appropriate time after being stored over time in parallel with the above detection and recording, or after being temporarily stored in the computer. You can also Each position where the cell is held is assigned an address, and the fluorescence intensity detected in association with the address is recorded. Therefore, the acquired fluorescence intensity or calcium concentration can be displayed on a computer monitor in real time for each location (cell).
  • the addresses can be numbered in the direction of the entire raster, the number of the cluster, and the lymphocytes in the cluster.
  • the fluorescence intensity and the calcium concentration can be displayed in time series. It is also possible to display data of only lymphocytes selected arbitrarily. In addition, fluorescence intensity and calcium concentration can be compared individually for the time acquired. The change in overall brightness can be confirmed at a glance. In addition, any range of data can be selected and displayed on the graph.
  • the computer display in Fig. 1 shows the changes over time in the amount of fluorescence reflecting the intracellular calcium concentration in individual cells.
  • lymphocyte fractions are collected from peripheral blood B lymphocytes using a known method (density gradient centrifugation).
  • the B lymphocyte fraction is preferably collected using a commercially available kit.
  • the collected B lymphocytes should be preliminarily introduced with a calcium indicator that increases or decreases the amount of fluorescence when excited by binding to calcium.
  • the amount of fluorescent dye introduced is controlled by the amount of fluorescent dye in the liquid in which the cells are reacted, and is preferably between 0.1 micromole / liter and a concentration force of 5 micromole Z liter.
  • the action time is preferably 15 minutes or 60 minutes to maintain cell activity. For temperature, room temperature Or it is desirable to do it at the temperature of body temperature.
  • lymphocytes are isolated from the spleen after splenectomy, and isotonic solution After washing well, suspend in a culture solution such as RPMI1640 containing 10% FCS or a buffer solution that retains cell function.
  • Samples shall be arrayed on cell chips.
  • Cell chips are mainly 10x5 clusters (45000 pieces: 900 quenore per cluster) as experimental chips! / ⁇ i3 ⁇ 415xl 5 (203 ⁇ 4 "2500) ⁇ @
  • the substrate of the cell chip it is necessary to use a silicon chip that has been surface-treated, or polyethylene glycolanol 2000 (PEG2000) and polyethyleneglycolole 6000 (PEG6000) in a ratio of about 2 to 1.
  • PEG2000 polyethylene glycolanol 2000
  • PEG6000 polyethyleneglycolole 6000
  • lipid membrane coating agent called lipidid
  • lipidid in the case of ethanol solvent, use a commercially available 10X or 50X solution and apply it to the array area on the cell chip. If this happens, the subsequent operation may be affected, so the ethanol may be washed twice or three times with more ethanol than the amount applied before it dries. This removes excess coating agent and prevents it from peeling off during measurement or adversely affecting cells.
  • aqueous type lipidids in a solution diluted 10 to 50 times, vibrate evenly inside the well on the cell chip in an ultrasonic cleaner. After leaving it for a while, clean it with water again in water to remove the excess coating agent.
  • the cell suspension obtained by suspending the cells in a culture solution containing 10% FCS or a serum-free medium having a function of maintaining the cell function was replaced with ethanol. Fills a buffer with the same composition as the cell suspension with the vacuum expelling the air in the well on the cell chip, and gently places a sufficient amount of the cell suspension on it.
  • a rubber spacer is placed on the cell chip after the array work, and a thin glass (such as a cover glass) cut to an appropriate size is placed on the spacer.
  • the cover glass and the surface of the cell chip are filled with the same culture solution as the cell suspension or a buffer with a composition having an appropriate function of maintaining cell responsiveness.
  • a rubber or other glass, plastic or metal spacer is placed directly on a glass, plastic or metal base to which the cell chip is fixed, and a bridge is formed on the spacer. Install the cover glass so that Hereinafter, the cell arrayed on the cell chip is referred to as a prep.
  • the light source of the scanner is activated and the warm-up operation is performed until the temperature inside the casing reaches equilibrium.
  • the warm-up operation it is possible to perform highly accurate measurement while suppressing fluctuations in the amount of excitation light.
  • the prep is placed on the moving stage of the array scanner, and set so that the optical axis and the cell chip surface are vertical. By making the optical axis and the cell chip surface perpendicular to each other and preventing tilting, accurate measurement is possible.
  • the dedicated data capture software is started up on the computer for analysis that has been started up. Under the red LED ring illumination light, data is collected for the part to be measured on the prep. Adjust the XY stage under the scanner receiver so that it is within the field of view of the insertion software, and focus the optical system on the chip surface. At this time, the ⁇ stage does not drift easily, and precise position adjustment is possible.
  • the position detection image is digitally processed, noise is removed, enhancement processing and rotation correction are performed, and the positions of the wells are arranged so as to form individual grids.
  • the pixel of each image sensor constituting each cell is 2x2 or more, preferably 4x4 pixels or more. This cell is large enough to contain a single cell, and is used for detection as if the cell is in this position.
  • a desirable example that fits within the field of view of an image sensor in the case of a 15x15 cluster prep would be a 4x4 pixel comprising a single cell area.
  • fluorescence detection matrixes These images showing cell regions created from the position detection images are called fluorescence detection matrixes.
  • the creation of the fluorescence detection matrix is performed fully automatically by the automatic image processing process on the computer as shown in FIG. 2 after the position detection image is collected.
  • the fluorescence detection matrix In order to detect the fluorescence of cells, we automatically create a matrix for fluorescence detection that corresponds one-to-one with the number of cells as many as the number of cells using our digital image processing technology.
  • the fluorescence value data recorded on the memory is recorded on a hard disk of a personal computer as a text file or binary data.
  • the recorded data is a general-purpose format and can be read using a commercially available program.
  • the fluorescence value data stored in the memory can be used for calculation without any special operation, and analysis can be performed at high speed.
  • the program starts shooting immediately, and is released from a calcium indicator combined with intracellular free calcium excited under the excitation light of a mercury lamp for a limited time before and after each shooting cycle. Take the captured fluorescence with an image sensor.
  • the fluorescence imaging is performed every 10 seconds, and the image acquisition and analysis process is completed by the time the next imaging sequence starts. Even if image processing is prolonged, shooting, image data transfer by high-speed serial bus, and image analysis processing are processed in separate processes, and after the data transfer, the next shooting process is not affected.
  • the fluorescence image including the fluorescence of the photographed cell is compared with the fluorescence detection matrix as electronic information, and the information of the pixels in the target cell region is integrated, or the cell region (16 pieces) is integrated. It is recorded as the maximum value or average value or median value of the pixels. Images taken over time are processed before the next shooting, and the output numerical values are recorded on a memory or hard disk as electronic information in time series.
  • Cell fluorescence analysis mainly uses at least one, preferably three of the following three filters (see Fig. 4).
  • Figure 4-a shows the distribution of cell fluorescence using a histogram filter and selects a uniform cell population.
  • Fig. 4-b is a scatter plot filter, showing the distribution of the fluorescence values of cells at two time points before and after, and the cells with changed fluorescence values can be detected at positions away from the population.
  • Figure 4- c is a time-series filter, which shows a change with time for each cell.
  • the histogram filter shown in Fig. 4a is mainly used to separate cell fluorescence from background fluorescence.
  • the fluorescence of cells before stimulation also varies depending on intracellular calcium.
  • a very well-prepared prep is divided into a group of empty wells that contain nothing and a group of cells that contain fluorescence. Cells that appear scattered at positions with higher fluorescence values than this population are generally activated cells from the beginning, and are inappropriate as the initial cell state.
  • the histogram 'filter is very useful for extracting a group of cells to be analyzed and facilitating the subsequent sorting operation.
  • the scatter diagram filter shown in Fig. 4b displays on the XY axis the fluorescence value at one time point before stimulation and the fluorescence value at an arbitrary time point after stimulation.
  • the time point displayed on the Y-axis is changed along the time series, the change in intracellular calcium concentration of cells activated in response to antigens or stimulating substances in the cell group selected on the histogram In response, it can be recognized as a separated cell population compared to the cell population.
  • This cell group is set as an area connecting arbitrary points on the screen of the personal computer, and only the information on the cells contained in the enclosed area is selected. The screen can be enlarged or reduced to select a more accurate group.
  • the time-series filter shown in Fig. 4c shows a time-series fluorescence change on the horizontal axis for each individual cell, and among them, a cell having a desired intracellular calcium mobilization pattern can be selected. It is a filter that can. By adding analysis to the cells selected with other filters, it is possible to operate more efficiently.
  • filters By using these filters properly, they can be used for various purposes. Furthermore, by using a plurality of filters, a more detailed evaluation can be performed on the cells selected by the previous filter.
  • RATIO indicating the degree of response of the cell, which is not just the absolute value data of fluorescence, or a primary differential value for sensitively detecting fluorescence changes, or the like is used. This makes it possible to examine qualitative changes in intracellular calcium, and to distinguish calcium mobilization caused by a specific antigen from calcium fluctuations as part of normal cellular activity.
  • the fluorescence information selected by the narrowing operation of the fluorescence information includes the fluorescence information.
  • the position information on the obtained image is attached. Therefore, based on this information, by referring to the cluster address and the address of the well in the prep cell area, cells can be collected by a micromanipulator, or the address can be passed automatically. This makes it possible to collect cells with an automatic cell collection device.
  • FIG. 3 shows an example of actual cell fluorescence acquisition.
  • the calcium response of each cell can be analyzed with parallelism of 200,000 or more simultaneous processes.
  • Stimulation is an anti-IgM antibody that stimulates the B cell receptor itself. (Displayed! /, The data for 250 samples randomly selected to improve visibility: Create images in Excel) 1% of mouse splenocytes that actually react with antigen were mixed When antigen stimulation was performed on a prep of a cell group (0.3-0.7% as reactive B cells), a clear cellular response that was considered to be antigen-specific was observed (bottom).
  • This method is not limited to antibody production techniques, and it is possible to examine more detailed cell profiles by measuring cell calcium mobilization on a prep and then staining the cell membrane antigen on the chip. . Using this technology, it can be expected to be used in fields such as clinical diagnosis.
  • the present invention is very useful in the case where it is necessary to grasp the state of a large number of cells, particularly lymphocytes at once, and is useful for preparing antigen-specific antibodies and diagnosing diseases involving lymphocytes, etc. Useful.
  • FIG. 1 is an explanatory diagram of a fluorescence detection apparatus.
  • FIG. 2 is an explanatory diagram of the operation of the automatic recognition function of the CCD scanner shown in FIG.
  • FIG. 4 is an explanatory diagram of three filters used for cell fluorescence analysis.

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  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Toxicology (AREA)
  • Optics & Photonics (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Physiology (AREA)
  • Zoology (AREA)
  • Virology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L’invention porte sur un procédé grâce auquel l’état d’un grand nombre de cellules contenues sur une puce (par exemple, les réactivités de lymphocytes à une stimulation antigène) peut être mesuré simultanément et l’état des cellules individuelles peut être compris. L’invention concerne donc un procédé de mesure d’états cellulaires consistant à maintenir indépendamment un nombre pluriel de cellules en un nombre pluriel de positions sur une puce de cellules, à détecter la fluorescence provenant d’au moins une partie du nombre pluriel de positions en utilisant un capteur d’image, à enregistrer les intensités de la fluorescence ainsi détectée pour chacune des positions, et à indiquer les intensités fluorescentes ou les données converties à partir des intensités fluorescentes ainsi enregistrées. On répète par la suite au moins la détection et l’enregistrement tels que décrits ci-dessus.
PCT/JP2006/319156 2005-09-30 2006-09-27 Procédé permettant de mesurer simultanément un nombre pluriel de réponses cellulaires Ceased WO2007040117A1 (fr)

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JP2005-287567 2005-09-30
JP2005287567A JP4089916B2 (ja) 2005-09-30 2005-09-30 多細胞応答同時測定法

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Cited By (1)

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CN113155791A (zh) * 2015-10-23 2021-07-23 阿贝里奥仪器有限责任公司 用于对试样的以荧光记号标记的结构高分辨率成像的方法和设备

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JPWO2009110614A1 (ja) * 2008-03-07 2011-07-14 泰信 小林 エフェクター細胞の機能測定法及び測定用キット並びに測定システム
CN102272561B (zh) * 2009-01-06 2014-05-14 古河电气工业株式会社 光测量装置及检测体识别分注装置
JP6787561B2 (ja) * 2016-04-18 2020-11-18 学校法人 工学院大学 情報処理装置、方法、及びプログラム
JP2020054234A (ja) 2017-01-31 2020-04-09 富士フイルム株式会社 細胞培養装置、撮像ユニット及び培養監視方法
JP7113435B2 (ja) * 2017-06-23 2022-08-05 国立大学法人 東京大学 解析装置、解析プログラム及び解析方法

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JP2003329681A (ja) * 2002-05-14 2003-11-19 Hitachi Ltd 生体試料検査装置
JP2004173681A (ja) * 2002-11-14 2004-06-24 Atsushi Muraguchi 抗原特異的リンパ球検出用マイクロウェルアレイチップ、抗原特異的リンパ球の検出法及び製造方法
JP2005148048A (ja) * 2003-04-25 2005-06-09 Jsr Corp バイオチップおよびバイオチップキットならびにその製造方法および使用方法

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JP2003329681A (ja) * 2002-05-14 2003-11-19 Hitachi Ltd 生体試料検査装置
JP2004173681A (ja) * 2002-11-14 2004-06-24 Atsushi Muraguchi 抗原特異的リンパ球検出用マイクロウェルアレイチップ、抗原特異的リンパ球の検出法及び製造方法
JP2005148048A (ja) * 2003-04-25 2005-06-09 Jsr Corp バイオチップおよびバイオチップキットならびにその製造方法および使用方法

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113155791A (zh) * 2015-10-23 2021-07-23 阿贝里奥仪器有限责任公司 用于对试样的以荧光记号标记的结构高分辨率成像的方法和设备

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