WO2009157385A1 - 細胞の識別およびソーティング方法およびその装置 - Google Patents
細胞の識別およびソーティング方法およびその装置 Download PDFInfo
- Publication number
- WO2009157385A1 WO2009157385A1 PCT/JP2009/061219 JP2009061219W WO2009157385A1 WO 2009157385 A1 WO2009157385 A1 WO 2009157385A1 JP 2009061219 W JP2009061219 W JP 2009061219W WO 2009157385 A1 WO2009157385 A1 WO 2009157385A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cell
- cells
- sorting
- transmitted light
- light information
- Prior art date
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1468—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle
- G01N15/147—Electro-optical investigation, e.g. flow cytometers with spatial resolution of the texture or inner structure of the particle the analysis being performed on a sample stream
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N15/1434—Electro-optical investigation, e.g. flow cytometers using an analyser being characterised by its optical arrangement
- G01N2015/1438—Using two lasers in succession
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1493—Particle size
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N15/00—Investigating characteristics of particles; Investigating permeability, pore-volume, or surface-area of porous materials
- G01N15/10—Investigating individual particles
- G01N15/14—Electro-optical investigation, e.g. flow cytometers
- G01N2015/1497—Particle shape
Definitions
- the present invention relates to a method for identifying and sorting cells, and in particular, transmitted light that reflects at least one of features on the morphology of the cell mainly including the size and shape of the cell and features on the internal structure of the cell including the cell nucleus and cytoplasm.
- the present invention relates to a cell identification and sorting method and apparatus using only the above information, or transmitted light information and information of side scattered light reflecting characteristics of the internal structure of the cell.
- a technique called flow cytometry in which each cell is analyzed by flowing a cell suspension at a high speed and irradiating it with a laser beam, is used.
- the relative size and shape of each cell, the difference in internal structure, and fluorescence labeling are performed to measure the fluorescence intensity and the type of fluorescence, thereby identifying various cells and cell groups. It is known that the abundance ratio of cells can be analyzed in a short time.
- the cell suspension is flowed to the center of a high flow rate sheath liquid (sheath flow) and is narrowed down by passing through a conical flow path so that each cell flows on the same line. .
- sheath flow sheath liquid
- SSC side scattered light
- the forward scattered light (FSC) reflects the size of the cell, and the side scattered light (SSC) reflects the complexity of the internal structure of the cell.
- Fluorescence (FL) detects each wavelength component by spectrally extracting and extracting light emitted in the same 90-degree direction as side scattered light (SSC) with an optical filter.
- the cells are irradiated with light and the fluorescent dye is detected.
- a fluorescent dye absorbs a certain wavelength and converts its high energy light (short wavelength) into lower energy light (long wavelength).
- Each fluorescent dye has a unique excitation wavelength distribution and emission wavelength distribution. That is, it absorbs a certain range of light wavelengths and emits radiated light (fluorescence) having a certain range of wavelengths.
- the intensity of the obtained fluorescence (fluorescence intensity: fluorescence intensity) is measured by a detector, and the obtained value is digitally converted and displayed as a cytogram or a histogram together with the fluorescence intensity of other cells.
- a cytogram displays scattered light and fluorescence information obtained from a single cell on two-dimensional coordinates, and a histogram is represented, for example, with the horizontal axis representing light intensity and the vertical axis representing the number of cells.
- the scattered light measurement value is obtained and analyzed as shown in FIG. 8 to recognize the difference in cell size and internal structure. . Scattered light is acquired at the front and side, respectively.
- the cell cycle refers to a process in which a daughter cell generated by cell division becomes a mother cell again to undergo cell division and become a new daughter cell.
- the cell cycle can be divided into G1 phase, S phase, G2 phase, and M phase. Particularly, in G2 phase and M phase, the number of chromosomes increases, so that the cell nucleus or the size of the cell is increased.
- some special cells including stem cells are known to have a higher proportion of cell nuclei in the cells than in general cells.
- Japanese Patent No. 2973387 Japanese Patent Laid-Open No. 6-323987 Published by Shujunsha Co., Ltd. Hiromitsu Nakauchi "Cell Engineering Separate Volume Experiment Protocol Series New Edition Flow Cytometry Freedom-From Multi-Color Analysis to Clone Sorting" P113
- stem cells and cancer stem cells which is required for regenerative medicine and drug development, utilizes the feature of stem cells and cancer stem cells that discharges fluorescent chemical substances out of the cells. Sorting cells.
- stem cells and cancer stem cells are damaged by fluorescent chemical substances, which adversely affects the differentiation and induction of stem cells and cancer stem cells.
- the object of the present invention is to recognize and sort special cells including the cell cycle of living cells and stem cells without staining the cell nucleus, without killing the cells, and without damaging the cells. It is an object of the present invention to provide a method and apparatus for identifying and sorting cells that can be performed.
- SM single mode
- the signal that has passed through the cell containing the irradiated light is received in the arbitrary region, or the light that has passed through the cell containing the irradiated light is received in the arbitrary region.
- the SM light that has passed through the flow path, the light that has passed through the cell, and the light reflected, scattered, and diffracted by the cell are all referred to as transmitted light.
- transmitted light information In an arbitrary region for receiving transmitted light, SM light is always received, but the SM light receiving power varies during cell measurement.
- the first aspect of the cell identification and sorting method according to the present invention is characterized by the morphological characteristics of the cell including the size and shape of the cell obtained by irradiating the cell with light, and the internal structure of the cell including the cell nucleus and cytoplasm Identification and sorting of cells characterized by identifying and sorting a particular cell population, or a portion of living cells of that particular cell population, using transmitted light information reflecting at least one of the above features Is the method.
- the living cells are identified and sorted using the transmitted light information and the side scattered light information reflecting the characteristics of the internal structure of the cell.
- a method for identifying and sorting cells are provided.
- a third aspect of the cell identification and sorting method of the present invention is characterized in that some living cells of the specific cell population are part of cells in the G1 phase of the cell cycle (cell cycle). Cell sorting and sorting method.
- a part of living cells in the G1 phase of the cell cycle is a cell histogram formed by transmitted light information displayed on the horizontal axis, or a horizontal
- the cell smaller than the smallest cell in the S phase in the analysis scatter diagram of the cell formed by the transmitted light information displayed on the axis and the side scattered light information displayed on the ordinate, that is, the curve in the analysis scatter diagram This is a cell identification and sorting method, characterized in that it is a so-called left foot cell.
- a cell identification and sorting method of the present invention there is provided a cell identification characterized in that a part of living cells of the specific cell population is a part of cells in the M phase of the cell cycle. And a sorting method.
- a cell histogram formed by transmitted light information displayed on the horizontal axis or a horizontal histogram of a part of the M cells in the cell cycle is displayed.
- the cell larger than the cell with the largest S phase in the analysis scatter diagram of the cell formed by the transmitted light information displayed on the axis and the side scattered light information displayed on the ordinate, that is, the curve in the analysis scatter diagram It is a cell identification and sorting method characterized by being a cell in a so-called right foot portion.
- the cell identification and sorting method determines whether a part of living cells of the specific cell population is subjected to fluorescence treatment including cell surface antibody / antigen reaction and intracellular fluorescent protein expression.
- the cell identification and sorting method is characterized in that the specific cell population or a part of living cells of the cell population is sorted without performing the fluorescence labeling treatment of nuclear staining.
- the eighth aspect of the cell identification and sorting method of the present invention is formed by transmitted light information displayed on the horizontal axis by identifying some special cells including stem cells using at least transmitted light information.
- This is a cell identification and sorting method that sorts special cells including stem cells larger than the largest cells, that is, cells at the so-called right foot of the curve in the analysis scatter diagram.
- the cell identification is characterized in that one cell is sorted into one well from a part of living cells of the specific cell population. And a sorting method.
- cells in an area within 1% from the maximum value are selected as adiploid nuclear cells or polyploid nuclear cells. Identifying and sorting as a cell identification and sorting method.
- a value obtained by subtracting 4 ⁇ from an average value (mean) of the 1% region is an average value of transmitted light information of the specific cell population ( A cell identification and sorting method that identifies and sorts cells as adiploid or polyploid nuclei cells when greater than mean).
- a value obtained by subtracting 4 ⁇ from an average value (mean) of the 1% region is an average value of transmitted light information of the specific cell population ( greater than mean), and when the value (CV) obtained by dividing the standard deviation ( ⁇ ) of the cells in the 1% region by the mean value (mean) is 8% or more, the aneuploid nucleus cell or the polyploidy
- CV value obtained by dividing the standard deviation ( ⁇ ) of the cells in the 1% region by the mean value (mean)
- the specific cell population is characterized in that a part of living cells is subjected to fluorescence treatment including cell surface antibody / antigen reaction and intracellular fluorescent protein expression.
- the method of identifying and sorting cells which are blood cells sorted without performing fluorescence labeling treatment for nuclear staining.
- cancer diagnosis For example, cancer cells are often polyploid nuclei having many cell nuclei or aneuploid nuclei. It can also be applied as a method for predicting the prognosis and therapeutic effect of cancer by measuring the number of polyploid nuclei and adiploid nuclei cells in blood.
- the first aspect of the cell sorting apparatus includes a light irradiating unit for irradiating light to a cell, a morphological feature of the cell including the size and shape of the cell, and an internal structure of the cell including the cell nucleus and cytoplasm Transmitted light information that reflects at least one of the above features, side scattered light information that reflects the characteristics of the cell's internal structure, and cell transmitted information, measured transmitted light and side scattered light information
- a cell sorting apparatus comprising: an analysis unit that analyzes a cell; and a cell sorting unit that sorts a specific population of cells obtained by the analysis unit, or a part of living cells of the specific cell population.
- the cell sorting unit includes a cell histogram formed by transmitted light information displayed on the horizontal axis, or transmitted light information displayed on the horizontal axis. Characterized in that it has a function of sorting one or a plurality of living cells in a predetermined region in a cell analysis scatter diagram formed by side scattered light information displayed on the vertical axis into a predetermined well, Cell sorting device.
- SM single mode
- SM single mode
- FIG. 1 is a histogram created by detecting transmitted light information according to the present invention and displaying a peak in the transmitted light information on the horizontal axis.
- FIG. 2 shows a histogram in which transmitted light information including all phases of the cell cycle of HeLa cells is detected and a peak in the transmitted light information is displayed on the horizontal axis.
- FIG. 3 is an image diagram showing a state in which transmitted light information and side scattered light information are dot-plotted.
- FIG. 4 is a measurement principle diagram of the cell sorting apparatus according to the present invention.
- FIG. 5 is a diagram illustrating a configuration of the sorting unit.
- FIG. 6 shows a histogram of conventional forward scattered light information. A peak in the forward scattered light information is displayed on the horizontal axis.
- FIG. 7 is a diagram showing the results of cell cycle analysis by nuclear staining (PI) of cells.
- FIG. 8 is a diagram showing measurement / analysis of scattered light by a conventional flow cytometer.
- FIG. 9 is a conceptual diagram showing peak values, width values, and area values as parameters of transmitted light information.
- FIG. 10 is a conceptual diagram of a method for identifying a special cell including a stem cell identified by the peak value of transmitted light information and the width value of transmitted light information in a certain flow velocity range.
- FIG. 11 shows the result of analysis after extracting almost the same flow rate data.
- FIG. 12 is a dot plot graph in which the horizontal axis represents the transmitted light peak in the transmitted light information, and the vertical axis represents the transmitted light peak in the side scattered light information.
- FIG. 13 is a histogram showing the fluorescence intensity of the cell nucleus on the horizontal axis in the region R1 shown in FIG.
- FIG. 14 shows a histogram showing the transmitted light peak in the transmitted light information on the horizontal axis in the data in the region R1 shown in FIG.
- FIG. 15 shows a histogram showing the fluorescence intensity of the cell nucleus on the horizontal axis in the region gated in the region R3.
- FIG. 16 shows a histogram obtained by measuring a population in which plural types of cells are mixed. A peak in transmitted light information is displayed on the horizontal axis.
- One aspect of the cell identification and sorting method according to the present invention is the transmission light information reflecting the morphological characteristics of the cell including the size and shape of the cell obtained by irradiating the cell with light, or the light on the cell.
- the information of transmitted light that reflects the morphological characteristics of the cell including the size and shape of the cells obtained by irradiating and the information of side scattered light that reflects the characteristics of the internal structure of the cell A cell sorting method characterized by identifying and sorting a specific cell population or a part of living cells of the specific cell population.
- Some living cells of the specific cell population described above are, for example, cells in the G1 phase or M phase of the cell cycle (cell cycle).
- the period of mitosis is called the M phase, and it ends in about 1 hour in most cells and is only a part of the whole cycle.
- the period between the M phase and the next M phase is called the interphase, and the cells synthesize and grow the necessary substances during the interphase, and most proteins and other substances remain throughout the interphase. It continues to be synthesized.
- the interphase is further divided into G1, S and G2.
- S phase is the time when nuclear DNA replication occurs.
- the period from the end of the M period to the start of the S period is the G1 period, that is, the first gap period. In cells that are actively dividing, enzymes necessary for DNA synthesis are activated during the G1 phase. This activation usually allows the cell to enter S phase.
- the M period is further divided into the first, middle, latter and final periods.
- chromosome condensation occurs, and at this time, chromosomes are observed under a microscope.
- the nuclear membrane disappears and the chromosomes line up on the equator plane.
- the spindle is also completed at this time.
- sister chromosomes that have joined together in the vicinity of the centromere are separated in such a way that they are pulled by the spindle and begin to move in the polar direction.
- centromere centromere
- the aggregated chromosomes are unwound and the nuclear membrane is reformed.
- cytokinesis begins and cell division ends.
- the S phase is the gene amplification / repair phase and is unstable, and therefore, the recognition and sorting of the G1 phase and M phase where the chromosome is stable. I do.
- the transmitted light reflects the morphological characteristics of the cell including its size and shape as its information.
- Side scattered light reflects the characteristics of the internal structure of the cell as its information.
- a specific period of the cell cycle (cell cycle) is recognized by combining transmitted light or such two pieces of information.
- the cell cycle has been analyzed by nuclear staining (PI) of cells.
- PI nuclear staining
- the G1, S, and M periods were clearly recognized.
- the cells were damaged by the nuclear staining of the cells (some types of nuclear staining could be damaged without killing the cells), were killed and could not be applied to live cells.
- FIG. 4 is a measurement principle diagram of the cell identification and sorting apparatus of the present invention.
- a laminar flow of a sample (cell) floating flow 2 and a sheath (sheath) liquid flow 3 is formed. Both the sheath liquid and the sample liquid are pushed out by the pressure of a compressor or the like.
- the sample liquid is hydrodynamically constricted at the stage of forming a laminar flow with the sheath liquid, and is wrapped in the sheath flow.
- the flow diameter of the sample flow becomes very narrow and the cells are arranged in a row.
- each cell is arranged in a row, and a state is formed in which the cells flow in the flow cell one by one.
- a pair of predetermined optical connectors (for example, multi-core connectors) 4 are provided so as to sandwich the flow cell 1 from both sides, and the light from the laser 5 is irradiated to the cells 6 passing through the flow cell 1 and transmitted through the cells 6.
- the laser 5 and the optical fiber 8 are arranged and fixed so that the transmitted light is received by the transmitted light receiving device 7. That is, the SM light transmitted through the SM (single mode) optical fiber 8 from the laser 5 is irradiated to the flow cell 1.
- the cells 6 flow in order one by one as described above across the SM light.
- One end of the optical fiber 8 is connected to the laser 5, and the transmitted light receiving device 7 is disposed on the other end side.
- parameters of transmitted light information a peak value of transmitted light information, a width value of transmitted light information, and an area value of transmitted light information are taken.
- a side scattered light receiving device 9 that detects a laser beam and side scattered light (SSC) scattered in a 90-degree direction among the scattered light from the cells 6 is provided. Further, a fluorescent device 10 is provided that performs spectral analysis using an optical filter in the same 90-degree direction as side scattered light (SSC) and detects each wavelength component.
- the sample suspension and sheath liquid that have passed through the flow cell form droplets and are sorted into a predetermined well.
- the sheath liquid and the sample liquid flow from the bottom to the top, but may flow from the top to the bottom. In normal sorting, it flows from top to bottom.
- a plurality of excitation wavelengths are required or a special wavelength is required, a plurality of lasers can be mounted and switched according to the purpose.
- a measuring unit for measuring the flow rate of the sample liquid is provided.
- the light receiving device When the light receiving device receives light, it generates a current, which is converted into a voltage pulse for pulse processing.
- the voltage pulse is A / D converted (the voltage pulse is an analog value, and this is usually measured on a scale of 1024 divisions and replaced with any integer value from 0 to 1023. (Referred to as “A / D conversion”), various histograms are created by a computer (analysis software) using numerical data of each parameter.
- FIG. 1 is a histogram created by detecting transmitted light information according to the present invention.
- FIG. 9 shows a conceptual diagram of the above-described transmitted light information.
- FIG. 9 shows a peak value, a width value, and an area value as parameters of transmitted light information.
- the transmitted light receiving region always receives SM (single mode) light and has a certain light receiving level (power). When the cell passes, its light reception level (power) fluctuates as shown in the quadratic curve shown.
- SM single mode
- power light receiving level
- the width value is the same, a cell having a large peak value has a low transmittance of transmitted light, for example, means that the cell nucleus is large (the cell nucleus has a smaller transmittance than the cytoplasm).
- the horizontal axis indicates the peak in the transmitted light information
- the vertical axis indicates the frequency.
- the sample (cell) size increases as it goes to the right of the horizontal axis.
- the sample (cell) size is getting smaller as it goes to the left of the horizontal axis.
- FIG. 1 clearly shows the change in cell size described above.
- the magnitude relationship of each period is as follows, which is consistent with the substance.
- the living cells are sorted, the sorted cells continue to repeat cell division, and the G1, S, and M phases having the characteristics shown in FIG. Show. Further, sorting can be performed in all regions in each recognized period (for example, sorting can be performed in all regions, not just the left skirt portion of the G1 period).
- FIG. 6 shows a histogram of conventional forward scattered light information.
- the horizontal axis indicates the forward scattered light peak in the forward scattered light information.
- the correlation between the magnitudes of the G1, S, and M periods is unknown. Therefore, it is impossible to sort each stage with live cells.
- FIG. 2 shows a histogram of transmitted light information including all phases of the cell cycle of HeLa cells.
- a peak in transmitted light information is displayed on the horizontal axis.
- the boundary line shown by the dotted line in FIG. 2 is the same as the boundary line shown in FIG.
- the left skirt portion of the curve indicates the G1 phase
- the right skirt portion of the curve indicates the M phase. Therefore, sorting the left skirt that protrudes to the left in G1 phase (about 1% of the total) can ensure that only G1 live cells are sorted separately from the others, and protrudes to the right in M phase.
- the right skirt portion about 7% of the whole
- FIG. 3 is an image diagram showing a state where transmitted light information and side scattered light information are dot-plotted.
- the vertical axis represents side scattered light information
- the horizontal axis represents transmitted light information. The shape increases as it goes to the right of the horizontal axis.
- FIG. 3 (a) shows the G1 period
- FIG. 3 (b) shows the S period
- FIG. 3 (c) shows the M period information.
- the G1, S, and M periods are gradually increasing.
- FIG. 3D shows information obtained by superimposing the G1, S, and M periods.
- a dotted boundary line shown in FIG. 3D corresponds to the size of the S period.
- the left skirt portion indicates the G1 period
- the right skirt portion indicates the M period.
- some special cells including stem cells are identified by the transmitted light information without staining the cells containing the antigen and antibody reaction of the cells with a fluorescent dye. Since most of some special cells including stem cells are nuclei, the transmittance is lower than that of normal cells, so the transmitted light signal is strong (large) (because of the attenuated signal).
- a histogram is created based on transmitted light information, a region of some special cells including stem cells corresponds to a region of the right skirt portion of the curve.
- an analysis scatter diagram may be created by combining other light information such as transmitted light and scattered light. The stem cells identified in the above-mentioned right skirt region are sorted.
- the special cells are cells such as stem cells and cancer stem cells having self-replicating ability or multipotency, and cancer cells that become polyploid nuclei. Cancer cells are often polyploid nuclei with many cell nuclei, and as a result, the ratio of nuclei to cells is large and the light transmittance is low. is there.
- the present invention can be applied to identification and sorting of polyploid nuclear cells and adiploid nuclear cells. That is, identification and sorting of polyploid nuclei cells and adiploid nuclei cells are performed based on the transmitted light information.
- identification and sorting of polyploid nuclei cells and adiploid nuclei cells are performed based on the transmitted light information.
- cytokinesis performed following chromosome division cells that failed to undergo cytokinesis will have twice the normal number of nuclei (or one nucleus with twice the number of chromosomes). Among them, cells that have escaped cell death but have various abnormalities as a result of the abnormal number of chromosomes appear. These are called polyploid nuclear cells and adiploid nuclear cells.
- the transmitted light information when used to sort the cells in the upper 1% area shown in the peak histogram of the transmitted light information, the polyploid nucleus cell and the adiploid nucleus cell can be obtained with high accuracy. And can be sorted separately.
- the value obtained by subtracting 4 ⁇ from the above average value (mean) of the 1% region shown in the peak histogram in the transmitted light information is larger than the average value (mean) of the peak in the transmitted light information of the HeLa cell.
- 1% of the cells described above are polyploid or adiploid nuclei. This is because the average value (mean) of the whole histogram is 4 ⁇ away from the average value (mean) of the top 1% region.
- the value obtained by subtracting 4 ⁇ from the average value (mean) of the above-mentioned 1% region shown in the peak histogram in the transmitted light information is larger than the overall average value (mean) in the above-described histogram, and is 1%.
- the accuracy is further increased by setting a value (CV) obtained by dividing the standard deviation ( ⁇ ) of 1% of cells by the mean value (mean) to a predetermined value or more.
- cancer diagnosis by identifying polyploid nuclei cells and adiploid nuclei cells that should not originally exist in blood.
- cancer cells are often polyploid nuclei having many cell nuclei or aneuploid nuclei. It can also be applied as a method for predicting the prognosis and therapeutic effect of cancer by measuring the number of polyploid nuclei and adiploid nuclei cells in blood.
- FIG. 5 is a diagram showing the configuration of the sorting unit. Identification, disposal, and sorting are performed in the sorting unit. Each state will be described. That is, the operation differs between the state until the target cell is detected and the state where the target cell is detected.
- the sample liquid continuously flows in the direction of the arrow, and the cells flowing in the flow cell are irradiated with laser light at the identification unit, or transmitted light information, or Based on the information on the transmitted light and the information on the side scattered light, the target cell is identified. Until the target cell is detected, the dispensing nozzle is inserted into the waste liquid tank, and the non-target cell is discarded into the waste liquid tank.
- attains the nozzle tip is as follows.
- a plurality of optical signal detectors optical fibers
- the flow velocity is calculated from the interval between the detection units and the time difference passing through each measurement unit. Since the distance from the detection unit to the nozzle tip is known, the arrival time of the target cell to the nozzle tip is calculated.
- Example 1 Using a cell sorting device that can sort cells according to the measurement principle shown in FIG. 4, HeLa cells that pass through the flow cell are irradiated with laser light, and light that has passed through the cells and light that has passed through the cells. And the light scattered to the side by the cells was detected, and a histogram with peaks in the transmitted light information was created.
- FIG. 1 shows a histogram based on transmitted light information. As shown in FIG. 1, the G1 phase of HeLa cells is measured and analyzed in FIG. 1a, the S phase in FIG. 1b, and the M phase in FIG. 1c.
- the peak in the transmitted light information displayed on the horizontal axis of FIG. 1 reflects the information on the cell morphology.
- the horizontal axis increases in size toward the right, and decreases in size toward the left.
- a boundary line indicated by a dotted line vertically corresponds to the size of cells in the S phase.
- the G1 phase of FIG. 1a is definitely the G1 phase of the HeLa cell (3% of the total) in the left foot of FIG. 1a, and the G1 phase of the cell cycle is specified. Is done.
- FIG. 2 shows a histogram of peaks in transmitted light information of HeLa cells (including all phases of the cell cycle).
- the left skirt portion of the horizontal axis shown in FIGS. 1 and 2 (3% of the whole, 1%) was gated, and one or a plurality of cells were sorted into a predetermined well.
- Example 2 Similarly, by using a cell sorting apparatus capable of sorting cells according to the measurement principle shown in FIG. 4, HeLa cells passing through the flow cell are irradiated with laser light, and the light transmitted through the cells and the cells are used. Light scattered sideways was detected, and an analysis scatter plot based on transmitted light and side scattered light information was created.
- FIG. 3 shows an image diagram thereof. As shown in FIG. 3, the G1 phase of HeLa cells is measured and analyzed in FIG. 3a, the S phase in FIG. 3b, and the M phase in FIG. 3c.
- the transmitted light information displayed on the horizontal axis in FIG. 3 reflects information on the cell morphology
- the side scattered light information displayed on the vertical axis reflects information on the internal structure of the cell.
- the shape of the horizontal axis increases as it goes to the right, and the shape decreases as it goes to the left.
- a boundary line indicated by a vertical dotted line in FIG. 3D generally corresponds to the size of cells in the S phase.
- the M period protrudes from the right end of the S period to the right (corresponding to the right skirt portion of the histogram). Therefore, in the M phase, the right protruding portion protruding from the S phase is surely the M phase of the HeLa cell, and the M phase of the cell cycle is specified.
- FIG. 4 shows a configuration of a measurement unit that can measure transmitted light information and side scattered light information.
- the flow velocity information of the cell was able to be calculated from the transmitted light information at the measurement point.
- FIG. 5 shows a configuration of a sorting unit that can sort one or a plurality of cells from a predetermined cell population into a predetermined well.
- the cells to be sorted can know the time from the flow rate information to the dispensing nozzle, so the dispensing nozzle is set according to the time.
- the whole term tergate cells could be sorted by transferring from the waste tank to a predetermined well.
- Example 4 The G1 and M phase cells sorted in Example 1 and Example 2 are subjected to the same analysis as in Example 1 and Example 2 after culturing under predetermined conditions, and the cells are sorted. It was. As a result, almost the same results as in Example 1 and Example 2 were obtained.
- FIG. 10 is a conceptual diagram of a method for identifying a special cell including a stem cell identified by the peak value of transmitted light information and the width value of transmitted light information in a certain flow velocity range. If the width value of the transmitted light information is within a certain flow velocity range, the cell size means almost the same size, and the cell in the region where the peak value of the transmitted light information is large has a lower transmittance than the cytoplasm. Reflects the cell nucleus. That is, at the same cell size, the larger the cell nucleus, the larger the peak value of transmitted light information, and the higher the possibility of special cells including stem cells. Since the flow rate varies depending on the position of the cross section of the light receiving surface through which cells flow as shown in FIG. 9, it means that the same level of flow velocity passes through the same position of the cross section of the light receiving surface.
- Stem cells or cancer stem cells have the characteristics that the size of the nucleus is about twice as large as normal cells, so the conventional fluorescence chemical substance (Hoechst33342) was discharged out of the cell. There are problems when considering the effects and certainty on cells.
- the right side (large value) of the histogram of transmitted light information displayed on the horizontal axis, or the transmitted light displayed on the horizontal axis Use analysis scatter plots that reflect information on the cell shape and internal structure of the transmitted light information width value or side scattered light information displayed on the right side (large value) of the peak value of information and the vertical axis.
- sorting was performed by identifying a group with a large amount of transmitted light information, and candidate stem cells or cancer stem cells were extracted.
- Example 6 Approximately half of the cells in the region of 1% from the maximum value of the transmitted light information (peak of the transmitted light waveform) (that is, the right end of the peak histogram in the transmitted light information shown in FIG. 2) is an aneuploid / polyploid nucleus. It was confirmed as follows by measuring HeLa cells stained for nuclei. That is, based on the correlation between the result of measuring HeLa cells stained with nuclei and the identification based on transmitted light information, it is confirmed that identification sorting can be performed with high accuracy based on the transmitted information as follows. did.
- FIG. 12 shows a dot plot graph in which the horizontal axis shows the peak of transmitted light and the vertical axis shows the side scattered light peak.
- the transmitted light reflects the size of the cell, and a larger value is obtained as the cell size increases.
- Side scattered light reflects the internal structure of the cell, and the more complicated the internal structure (large nuclei, multiple nuclei, etc.), the larger the value obtained.
- the lower left part is removed as noise information (cell debris etc.). The remaining area from which this noise information has been removed is defined as R1.
- This technology is premised on the measurement of a single cell population, but if there is a concern about cell aggregation or contamination of microscopic cell debris from the cell being measured, the signal width of the transmitted / scattered light signal is confirmed. You may put the process to do. If these signal width values fall outside the range of 2 ⁇ with respect to the average value, it may be an aggregation of multiple cells or cell debris, and the signal from a single cell may not be analyzed. is there. Therefore, the analysis accuracy is further improved by excluding data in which the signal width value of the transmitted light / scattered light signal is out of the range of 2 ⁇ with respect to the average value.
- FIG. 13 shows a histogram showing the fluorescence intensity of cell nuclei on the horizontal axis in the region R1 described above.
- the large peak indicates the fluorescence intensity of the cell nucleus in the G1 phase
- the small peak indicates the fluorescence intensity of the cell nucleus in the G2 / M phase.
- a region larger than the peak of G2 / M phase is considered to be an adiploid / polyploid nucleus cell region.
- the region larger than the peak of the G2 / M phase was defined as being 3 ⁇ or more away from the center of the G2 / M region. Assuming that this area is R2, and the number of data in the R2 area was examined, it was 205 data (that is, 1.34% of the total).
- FIG. 14 a histogram showing the transmitted light peak in the transmitted light information on the horizontal axis is shown in FIG.
- the area of 1% from the larger value (the right end in the figure) is R3
- the number of data in the R3 area is 154 data.
- the average value (mean) of this R3 region was 673.9, and the standard deviation ( ⁇ ) was 69.9.
- the ratio (CV) of the standard deviation ( ⁇ ) to the mean value (mean) was 10.4%.
- the overall average value (mean) in FIG. 14 is 371.5, and the overall average value (mean) is located 4 ⁇ away from the average value (mean) of the R3 region.
- FIG. 15 shows a histogram showing the fluorescence intensity of the cell nucleus on the horizontal axis in the region gated in the region R3.
- FIG. 15 displays the same area as the area R2 determined in FIG. 13, and this area is designated as R4.
- the number of data in the region R4 was 74, which was 48.1% of the data gated in the region R3.
- FIG. 16 shows a histogram obtained by measuring a population in which plural types of cells are mixed.
- the horizontal axis indicates the peak in the transmitted light information
- the vertical axis indicates the frequency.
- R may be arbitrarily determined from a population in which a plurality of types of cells are mixed, and the analysis performed in the present embodiment may be applied to the population 2 existing in the region R.
- peaks in transmitted light information and scattered light information are used. However, peaks, widths and areas in transmitted light information, and peaks, widths and areas in scattered light information may be used. .
- the present invention it is possible to analyze a specific live cell population regardless of the presence or absence of fluorescence treatment including cell surface antibody / antigen reaction, intracellular fluorescent protein expression, and nuclear staining. Furthermore, as shown in the first to sixth embodiments, the specific cell population or a part of the living cells of the cell population is identified without performing the fluorescence labeling treatment for nuclear staining regardless of the presence or absence of the fluorescence treatment. And can be sorted.
- the laser beam that is normally blocked by a beam mask or the like before entering the detector and that has traveled straight through the flow cell without being scattered is used as transmitted light information. It can be used to identify and sort live cells in a specific phase of the cell cycle of live cells, and aneuploid / polyploid nuclear cells.
Abstract
Description
また、幹細胞を含む一部の特殊な細胞では、細胞に占める細胞核の大きさの割合が、一般の細胞より高いこととして知られている。
特に、細胞周期における細胞の染色体が安定しているG1期と、染色体の数が増えて細胞核または細胞のサイズが大きくなるM期を認識し、ソーティングすることが可能であることが判明した。
例えば癌細胞は多くの細胞核を有する多倍体核、もしくは異倍体核のケースが多い。血液中の多倍体核、異倍体核細胞の数を測定することにより癌の予後や治療効果を予測する方法としても応用することができる。
2 サンプル流
3 シース流
4 光コネクタ
5 レーザー
6 細胞
7 透過光受光デバイス
8 光ファイバ
9 側方散乱光受光デバイス
10 蛍光デバイス
この発明の細胞の識別およびソーティング方法の1つの態様は、細胞に光を照射して得られる細胞の大きさや形状等を含む細胞の形態上の特徴を反映する透過光の情報、もしくは細胞に光を照射して得られる細胞の大きさや形状等を含む細胞の形態上の特徴を反映する透過光の情報と、細胞の内部構造の特徴を反映する側方散乱光の情報を合わせて用いて、ある特定の細胞集団、またはその特定の細胞集団の一部の生細胞を識別およびソーティングすることを特徴とする細胞のソーティング方法である。上述した特定の細胞集団の一部の生細胞は、例えば、セルサイクル(細胞周期)のG1期、もしくはM期の細胞の一部である。
G1<S<G2/M
例えば癌細胞は多くの細胞核を有する多倍体核、もしくは異倍体核のケースが多い。血液中の多倍体核、異倍体核細胞の数を測定することにより癌の予後や治療効果を予測する方法としても応用することができる。
図5に示すように、サンプル液が矢印の方向に連続して流れ、フローセル中を流れる細胞に、識別部においてレーザー光を照射し、透過光の情報、または、透過光の情報および側方散乱光の情報に基づいて、目的細胞であるか否かの識別を行う。目的細胞が検出されるまでは、分注ノズルは廃液槽に挿入されており、非目的細胞は廃液槽に廃棄される。
透過光の情報、または、透過光の情報および側方散乱光の情報に基づいて、目的細胞が検出されると、目的細胞が分注ノズル先端に到達するまでの時間が算出される。算出された到達時間に細胞がノズル先端に到達するタイミングで廃液槽が退避し、その次にステージ(培養プレート)が上昇する。このようにして、分注ノズルの先端が各ウエル(well)に挿入されて、目的細胞が分注される。
光信号の検出部(光ファイバ)はサブミクロンの精度でサンプル進行方向に複数設置されている(測定部の間隔は既知)。各検出部の間隔と各測定部を通過する時間差より流速が算出される。検出部からノズル先端までの距離は既知であるため、目的細胞のノズル先端までの到達時間が計算される。
実施例1
図4に示した計測原理に従って細胞のソーティングを行うことができる細胞のソーティング装置を使用して、フローセルを通過するHeLa細胞にレーザー光を照射して、細胞を透過した光、細胞を透過した光および細胞によって側方に散乱した光を検出し、透過光情報におけるピークによるヒストグラムを作成した。図1に透過光情報によるヒストグラムを示す。図1に示すように、HeLa細胞のG1期は図1aに、S期は図1bに、M期は、図1cにそれぞれ計測され、解析されている。
同様に、図4に示した計測原理に従って細胞のソーティングを行うことができる細胞のソーティング装置を使用して、フローセルを通過するHeLa細胞にレーザー光を照射して、細胞を透過した光および細胞によって側方に散乱した光を検出し、透過光および側方散乱光情報による解析散布図を作成した。図3にそのイメージ図を示す。図3に示すように、HeLa細胞のG1期は図3aに、S期は図3bに、M期は、図3cにそれぞれ計測され、解析されている。
図4に、透過光情報と側方散乱光情報を計測できる計測部の構成を示す。この計測部を備えた細胞のソーティング装置を使用することによって、細胞の流れ方向に設けた測定点によって、細胞の蛍光情報、透過光情報及び側方散乱光情報が計測できることに加え、複数間の計測点の透過光情報によって、細胞の流速情報を計算することができた。
実施例1と実施例2においてソーティングされたG1期とM期の細胞が、所定の条件下の培養後に、再度、実施例1と実施例2のような解析をして、細胞のソーティングを行った。その結果、実施例1および実施例2と概ね同様な結果が得られた。
図10は、透過光情報のピーク値と、一定の流速範囲にある透過光情報の幅値によって、特定された幹細胞を含む特殊細胞の特定方法の概念図である。一定の流速範囲以内の透過光情報の幅値であれば、細胞のサイズがほぼ同じ大きさを意味し、その中の透過光情報のピーク値の大きい領域の細胞では、細胞質より透過率の小さい細胞核を反映している。つまり同じ細胞の大きさでは、細胞核が大きければ大きいほど透過光情報のピーク値が大きく、幹細胞を含む特殊細胞の可能性が高い。流速については、図9に示すように細胞が流れる受光面断面の位置によって異なる為、同じ程度の流速では、ほぼ同じ受光面断面の位置を通過することを意味する。
この場合、細胞の流れる位置によって幅値のばらつき(=流速ばらつき)が考えられるが、図11のようにほぼ同じ流速データを抽出した後に解析することで精度を向上させることができる。
透過光情報(透過光波形のピーク)の最大値(即ち、図2に示す透過光情報におけるピークのヒストグラムに示す右端)から1%の領域における細胞のおよそ半数が異倍体・多倍体核細胞であったことを、核を染色したHeLa細胞を測定することにより次の通り確認した。即ち、核を染色したHeLa細胞を測定した結果と、透過光情報に基づく識別との間の相関関係に基づき、透過情報に基づいて、高い精度で識別ソーティングが可能であることを次の通り確認した。
Claims (15)
- 細胞に光を照射して得られる細胞の大きさや形状等を含む細胞の形態上の特徴と細胞核や細胞質等を含む細胞の内部構造上の特徴の少なくとも一方を反映する透過光の情報を用いて、ある特定の細胞集団、またはその特定の細胞集団の一部の生細胞を識別およびソーティングすることを特徴とする細胞の識別およびソーティング方法。
- 前記透過光の情報と、細胞の内部構造の特徴を反映する側方散乱光の情報とを用いて、前記生細胞を識別およびソーティングすることを特徴とする、請求項1に記載の細胞の識別およびソーティング方法。
- 前記特定の細胞集団の一部の生細胞は、セルサイクル(細胞周期)のG1期の細胞の一部であることを特徴とする、請求項1または2に記載の細胞の識別およびソーティング方法。
- 前記セルサイクルのG1期の一部の生細胞が、横軸に表示される透過光情報によって形成される細胞のヒストグラム、または、横軸に表示される透過光情報および縦軸に表示される側方散乱光情報によって形成される細胞の解析散布図におけるS期の最も小さい細胞よりも小さい細胞であることを特徴とする、請求項3に記載の細胞の識別およびソーティング方法。
- 前記特定の細胞集団の一部の生細胞は、セルサイクルのM期の細胞の一部であることを特徴とする、請求項1または2に記載の細胞の識別およびソーティング方法。
- 前記セルサイクルのM期の一部の生細胞が、横軸に表示される透過光情報によって形成される細胞のヒストグラム、または、横軸に表示される透過光情報および縦軸に表示される側方散乱光情報によって形成される細胞の解析散布図におけるS期の最も大きい細胞よりも大きい細胞であることを特徴とする、請求項5に記載の細胞の識別およびソーティング方法。
- 前記特定の細胞集団の一部の生細胞に、細胞表面抗体・抗原反応、細胞内の蛍光蛋白発現を含む蛍光処理の有無に関わらず、前記特定の細胞集団またはその細胞集団の一部の生細胞をソーティングすることを特徴とする、請求項1または2に記載の細胞の識別およびソーティング方法。
- 少なくとも透過光の情報を用いて幹細胞を含む一部の特殊な細胞を識別し、横軸に表示される透過光情報によって形成される細胞のヒストグラム、または、横軸に表示される透過光情報および縦軸に表示される側方散乱光情報、もしくは透過光の幅情報や蛍光情報等によって形成される細胞の解析散布図におけるS期の最も大きい細胞よりも大きい、幹細胞を含む特殊な細胞をソーティングする、請求項7に記載の細胞の識別およびソーティング方法。
- 前記特定の細胞集団の一部の生細胞の内から、一つの細胞を一つのウエルにソーティングすることを特徴とする、請求項1から8の何れか1項に記載の細胞のソーティング方法。
- 前記ある特定の細胞集団の透過光情報において、最大値から1%の領域の細胞を、異倍体核細胞または多倍体核細胞として識別およびソーティングする、請求項1または請求項2に記載の細胞の識別およびソーティング方法。
- 前記1%の領域の平均値(mean)からその4σを引いた値が、前記ある特定の細胞集団の透過光情報の平均値(mean)よりも大きいときに、異倍体核細胞または多倍体核細胞として細胞を識別しおよびソーティングする、請求項10に記載の細胞の識別およびソーティング方法。
- 前記1%の領域の平均値(mean)からその4σを引いた値が、前記ある特定の細胞集団の透過光情報の平均値(mean)よりも大きく、且つ、前記1%の細胞の標準偏差(σ)を平均値(mean)で除した値(CV)が8%以上のときに、異倍体核細胞または多倍体核細胞として細胞を識別およびソーティングする、請求項11に記載の細胞の識別およびソーティング方法。
- 請求項10から12の何れか1項において測定する細胞集団は、請求項7に記載の方法における血液細胞である、細胞の識別およびソーティング方法。
- 細胞に光を照射する光照射部と、細胞の大きさや形状等を含む細胞の形態上の特徴と細胞核や細胞質等を含む細胞の内部構造上の特徴の少なくとも一方を反映する透過光の情報と、細胞の内部構造の特徴を反映する側方散乱光の情報を取得できる細胞の計測部と、計測した透過光および側方散乱光の情報を解析する解析部と、前記解析部によって得られた細胞のある特定の集団、またはその特定の細胞集団の一部の生細胞を識別およびソーティングする細胞のソーティング部とを備えた細胞の識別およびソーティング装置。
- 前記細胞のソーティング部は、横軸に表示される透過光情報によって形成される細胞のヒストグラム、または、横軸に表示される透過光情報と縦軸に表示される側方散乱光情報によって形成された細胞の解析散布図における所定の領域の1または複数の生細胞を、所定のウエルにソーティングする機能を備えていることを特徴とする、請求項14に記載の細胞の識別およびソーティング装置。
Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US13/001,128 US8942458B2 (en) | 2008-06-27 | 2009-06-19 | Method for distinguishing and sorting of cells and device therefor |
CN200980124706.6A CN102076841B (zh) | 2008-06-27 | 2009-06-19 | 细胞的识别和分选方法及其装置 |
JP2010517977A JP5323829B2 (ja) | 2008-06-27 | 2009-06-19 | 細胞の識別およびソーティング方法およびその装置 |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JP2008-168817 | 2008-06-27 | ||
JP2008168817 | 2008-06-27 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2009157385A1 true WO2009157385A1 (ja) | 2009-12-30 |
Family
ID=41444453
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2009/061219 WO2009157385A1 (ja) | 2008-06-27 | 2009-06-19 | 細胞の識別およびソーティング方法およびその装置 |
Country Status (4)
Country | Link |
---|---|
US (1) | US8942458B2 (ja) |
JP (1) | JP5323829B2 (ja) |
CN (1) | CN102076841B (ja) |
WO (1) | WO2009157385A1 (ja) |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2011121751A1 (ja) * | 2010-03-31 | 2011-10-06 | 古河電気工業株式会社 | 細胞の識別装置及び識別方法 |
JP2011257241A (ja) * | 2010-06-08 | 2011-12-22 | Kanagawa Acad Of Sci & Technol | 細胞分析装置 |
WO2013069452A1 (ja) * | 2011-11-08 | 2013-05-16 | 浜松ホトニクス株式会社 | 幹細胞の観察方法、分化傾向状態の細胞領域の除去方法、及び、幹細胞の観察装置 |
JP2015031665A (ja) * | 2013-08-06 | 2015-02-16 | トライボテックス株式会社 | 粒子計数装置及び粒子計数方法 |
JP2016005437A (ja) * | 2014-06-20 | 2016-01-14 | 富士フイルム株式会社 | 細胞評価装置および方法並びにプログラム |
JPWO2016063364A1 (ja) * | 2014-10-22 | 2017-06-22 | 株式会社日立ハイテクノロジーズ | 細胞計測機構及びそれを有する細胞培養装置並びに細胞計測方法 |
JP2017138330A (ja) * | 2017-04-07 | 2017-08-10 | トライボテックス株式会社 | 粒子計数装置及び粒子計数方法 |
WO2017169196A1 (ja) * | 2016-03-28 | 2017-10-05 | 富士フイルム株式会社 | 細胞のスクリーニング方法 |
WO2023053574A1 (ja) * | 2021-09-29 | 2023-04-06 | 日東紡績株式会社 | 細胞または細胞核の豊富化方法 |
Families Citing this family (15)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US9522396B2 (en) | 2010-12-29 | 2016-12-20 | S.D. Sight Diagnostics Ltd. | Apparatus and method for automatic detection of pathogens |
US10640807B2 (en) | 2011-12-29 | 2020-05-05 | S.D. Sight Diagnostics Ltd | Methods and systems for detecting a pathogen in a biological sample |
WO2014031735A1 (en) * | 2012-08-21 | 2014-02-27 | Cambridge Research & Instrumentation, Inc. | Visualization and measurement of cell compartments |
WO2014188405A1 (en) | 2013-05-23 | 2014-11-27 | Parasight Ltd. | Method and system for imaging a cell sample |
IL227276A0 (en) | 2013-07-01 | 2014-03-06 | Parasight Ltd | A method and system for obtaining a monolayer of cells, for use specifically for diagnosis |
WO2015029032A1 (en) | 2013-08-26 | 2015-03-05 | Parasight Ltd. | Digital microscopy systems, methods and computer program products |
WO2015058353A1 (zh) * | 2013-10-22 | 2015-04-30 | 爱威科技股份有限公司 | 一种红细胞形态学分析结果表示方法 |
US10482595B2 (en) | 2014-08-27 | 2019-11-19 | S.D. Sight Diagnostics Ltd. | System and method for calculating focus variation for a digital microscope |
EP3859425B1 (en) | 2015-09-17 | 2024-04-17 | S.D. Sight Diagnostics Ltd. | Methods and apparatus for detecting an entity in a bodily sample |
US11733150B2 (en) | 2016-03-30 | 2023-08-22 | S.D. Sight Diagnostics Ltd. | Distinguishing between blood sample components |
EP3455610B1 (en) | 2016-05-11 | 2023-01-04 | S.D. Sight Diagnostics Ltd. | Sample carrier for optical measurements |
EP3455626A1 (en) | 2016-05-11 | 2019-03-20 | S.D. Sight Diagnostics Ltd. | Performing optical measurements on a sample |
CN107219196B (zh) * | 2017-06-01 | 2020-10-16 | 重庆大学 | 一种基于光散射测量的细胞周期检测方法 |
US11921272B2 (en) | 2017-11-14 | 2024-03-05 | S.D. Sight Diagnostics Ltd. | Sample carrier for optical measurements |
US10976236B2 (en) * | 2019-03-21 | 2021-04-13 | Becton, Dickinson And Company | Light detection systems and methods of use thereof |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS63126480A (ja) * | 1986-11-14 | 1988-05-30 | Shimadzu Corp | 細胞識別収集装置 |
JPH07270302A (ja) * | 1994-03-30 | 1995-10-20 | Toa Medical Electronics Co Ltd | イメージングフローサイトメータ |
JP2006017497A (ja) * | 2004-06-30 | 2006-01-19 | Sysmex Corp | 分析装置、分析プログラム、分析方法 |
JP2006517292A (ja) * | 2003-02-05 | 2006-07-20 | エボテック・テヒノロギーズ・ゲーエムベーハー | マルチパラメトリック細胞同定・選別法および対応の装置 |
JP2007504446A (ja) * | 2003-08-28 | 2007-03-01 | セルラ・インコーポレイテッド | マイクロ流体チャネルネットワークにおいて光学スイッチを用いて細胞をソーティングするための方法および装置 |
JP2008292448A (ja) * | 2007-04-27 | 2008-12-04 | Furukawa Electric Co Ltd:The | 光計測装置および光計測方法 |
Family Cites Families (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2635126B2 (ja) * | 1988-09-30 | 1997-07-30 | 東亜医用電子株式会社 | 核の分葉指数を求めるための粒子分析装置及び方法 |
US5312535A (en) | 1992-07-17 | 1994-05-17 | Beckman Instruments, Inc. | Capillary electrophoresis detection |
JP4134330B2 (ja) * | 2002-04-17 | 2008-08-20 | タマティーエルオー株式会社 | フローサイトメトリーを用い、蛍光標識を用いることなく、目的の細胞を迅速に分取する方法 |
JP5032792B2 (ja) * | 2006-05-22 | 2012-09-26 | 浜松ホトニクス株式会社 | 細胞選別装置 |
US8211708B2 (en) * | 2009-03-13 | 2012-07-03 | Furukawa Electric Co., Ltd. | Optical measuring device and method therefor |
-
2009
- 2009-06-19 CN CN200980124706.6A patent/CN102076841B/zh active Active
- 2009-06-19 US US13/001,128 patent/US8942458B2/en active Active
- 2009-06-19 JP JP2010517977A patent/JP5323829B2/ja active Active
- 2009-06-19 WO PCT/JP2009/061219 patent/WO2009157385A1/ja active Application Filing
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPS63126480A (ja) * | 1986-11-14 | 1988-05-30 | Shimadzu Corp | 細胞識別収集装置 |
JPH07270302A (ja) * | 1994-03-30 | 1995-10-20 | Toa Medical Electronics Co Ltd | イメージングフローサイトメータ |
JP2006517292A (ja) * | 2003-02-05 | 2006-07-20 | エボテック・テヒノロギーズ・ゲーエムベーハー | マルチパラメトリック細胞同定・選別法および対応の装置 |
JP2007504446A (ja) * | 2003-08-28 | 2007-03-01 | セルラ・インコーポレイテッド | マイクロ流体チャネルネットワークにおいて光学スイッチを用いて細胞をソーティングするための方法および装置 |
JP2006017497A (ja) * | 2004-06-30 | 2006-01-19 | Sysmex Corp | 分析装置、分析プログラム、分析方法 |
JP2008292448A (ja) * | 2007-04-27 | 2008-12-04 | Furukawa Electric Co Ltd:The | 光計測装置および光計測方法 |
Non-Patent Citations (1)
Title |
---|
WALTER GIARETTI ET AL.: "A New Method to Discriminate Gl, S, G2, M, and Gl Postmitotic Cells", EXPERIMENTAL CELL RESEARCH, vol. 182, 1989, pages 290 - 295 * |
Cited By (17)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN102272581A (zh) * | 2010-03-31 | 2011-12-07 | 古河电气工业株式会社 | 细胞的识别装置和识别方法 |
WO2011121751A1 (ja) * | 2010-03-31 | 2011-10-06 | 古河電気工業株式会社 | 細胞の識別装置及び識別方法 |
JP2011257241A (ja) * | 2010-06-08 | 2011-12-22 | Kanagawa Acad Of Sci & Technol | 細胞分析装置 |
JP2016168052A (ja) * | 2011-11-08 | 2016-09-23 | 浜松ホトニクス株式会社 | 幹細胞の観察方法、及び幹細胞の観察装置 |
WO2013069452A1 (ja) * | 2011-11-08 | 2013-05-16 | 浜松ホトニクス株式会社 | 幹細胞の観察方法、分化傾向状態の細胞領域の除去方法、及び、幹細胞の観察装置 |
JPWO2013069452A1 (ja) * | 2011-11-08 | 2015-04-02 | 浜松ホトニクス株式会社 | 幹細胞の観察方法、分化傾向状態の細胞領域の除去方法、及び、幹細胞の観察装置 |
EP2778231A4 (en) * | 2011-11-08 | 2015-08-12 | Hamamatsu Photonics Kk | METHOD FOR THE MONITORING OF STEM CELLS, METHOD FOR REMOVING CELL AREAS IN A STATUS OF DIFFERENTIATION TREND AND DEVICE FOR MONITORING STEM CELLS |
US9134296B2 (en) | 2011-11-08 | 2015-09-15 | Hamamatsu Photonics K.K. | Method for observing stem cells, method for removal of cell region in state tending toward differentiation, and device for observing stem cells |
US9435734B2 (en) | 2011-11-08 | 2016-09-06 | Hamamatsu Photonics K.K. | Method for observing stem cells, method for removal of cell region in state tending toward differentiation, and device for observing stem cells |
JP2015031665A (ja) * | 2013-08-06 | 2015-02-16 | トライボテックス株式会社 | 粒子計数装置及び粒子計数方法 |
JP2016005437A (ja) * | 2014-06-20 | 2016-01-14 | 富士フイルム株式会社 | 細胞評価装置および方法並びにプログラム |
JPWO2016063364A1 (ja) * | 2014-10-22 | 2017-06-22 | 株式会社日立ハイテクノロジーズ | 細胞計測機構及びそれを有する細胞培養装置並びに細胞計測方法 |
US10456767B2 (en) | 2014-10-22 | 2019-10-29 | Hitachi High-Technologies Corporation | Cytometric mechanism, cell culture device comprising same, and cytometric method |
WO2017169196A1 (ja) * | 2016-03-28 | 2017-10-05 | 富士フイルム株式会社 | 細胞のスクリーニング方法 |
JP2017138330A (ja) * | 2017-04-07 | 2017-08-10 | トライボテックス株式会社 | 粒子計数装置及び粒子計数方法 |
WO2023053574A1 (ja) * | 2021-09-29 | 2023-04-06 | 日東紡績株式会社 | 細胞または細胞核の豊富化方法 |
JP7283642B1 (ja) | 2021-09-29 | 2023-05-30 | 日東紡績株式会社 | 細胞または細胞核の豊富化方法 |
Also Published As
Publication number | Publication date |
---|---|
US20110177544A1 (en) | 2011-07-21 |
JPWO2009157385A1 (ja) | 2011-12-15 |
CN102076841A (zh) | 2011-05-25 |
CN102076841B (zh) | 2015-04-22 |
JP5323829B2 (ja) | 2013-10-23 |
US8942458B2 (en) | 2015-01-27 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
JP5323829B2 (ja) | 細胞の識別およびソーティング方法およびその装置 | |
EP2062056B1 (en) | Differentiation of flow cytometry pulses and applications | |
US7008792B2 (en) | Method of measurement of nucleated red blood cells | |
Givan | Flow cytometry: an introduction | |
JP5178530B2 (ja) | 有核赤血球の測定方法 | |
JP3707620B2 (ja) | 光散乱技術を使用した網赤血球分析方法と装置 | |
KR101805941B1 (ko) | 입자 분석기에서의 펄스 파라미터 발생 | |
Givan | Flow cytometry: an introduction | |
CN110226082B (zh) | 具有多个强度峰值设计的流式细胞仪 | |
US20150064742A1 (en) | Sample analyzing method and sample analyzer | |
CN102998259A (zh) | 光学测量设备、流式细胞仪及光学测量方法 | |
EP3789751A1 (en) | Method and system for determining platelet concentration | |
US8211708B2 (en) | Optical measuring device and method therefor | |
CN107655865B (zh) | 血液分析装置及血液分析方法 | |
EP4257974A1 (en) | Sample analysis method, sample analyzer, and computer-readable storage medium | |
JP3642658B2 (ja) | 尿中有形成分分析装置および分析方法 | |
US11740174B2 (en) | Apparatus and methods for particle analysis and autofluorescence discrimination | |
EP3136080B1 (en) | Urine sample analyzer and urine sample analyzing method | |
US11965812B2 (en) | Apparatus and methods for particle analysis and autofluorescence discrimination | |
Krishnamurthy et al. | Basics of flow cytometry | |
CN105021572A (zh) | 尿样本分析方法及尿样本分析装置 | |
Salzman | ELEMENTS OF FLOW CYTOMETRY Fluid-filled Flow Chamber In the fluid-filled flow chamber developed by Steinkamp et al.(1973, 1979) at the Los Alamos Scientific Laboratory (Fig. 5.1), a cell suspension is injected into the center of a rapidly moving sheath fluid stream under conditions of laminar flow. |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
WWE | Wipo information: entry into national phase |
Ref document number: 200980124706.6 Country of ref document: CN |
|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 09770095 Country of ref document: EP Kind code of ref document: A1 |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2010517977 Country of ref document: JP |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
WWE | Wipo information: entry into national phase |
Ref document number: 13001128 Country of ref document: US |
|
122 | Ep: pct application non-entry in european phase |
Ref document number: 09770095 Country of ref document: EP Kind code of ref document: A1 |