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WO2025225688A1 - Dispositif d'analyse, procédé d'analyse et programme d'analyse - Google Patents

Dispositif d'analyse, procédé d'analyse et programme d'analyse

Info

Publication number
WO2025225688A1
WO2025225688A1 PCT/JP2025/015866 JP2025015866W WO2025225688A1 WO 2025225688 A1 WO2025225688 A1 WO 2025225688A1 JP 2025015866 W JP2025015866 W JP 2025015866W WO 2025225688 A1 WO2025225688 A1 WO 2025225688A1
Authority
WO
WIPO (PCT)
Prior art keywords
cell
exogenous gene
determination
cells
bright
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/JP2025/015866
Other languages
English (en)
Japanese (ja)
Inventor
武彦 上田
純子 坂神
遼 小林
信宏 笠井
総一郎 大垣
友峰 飯田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nikon Corp
Original Assignee
Nikon Corp
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nikon Corp filed Critical Nikon Corp
Publication of WO2025225688A1 publication Critical patent/WO2025225688A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • 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
    • 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
    • G01N33/483—Physical analysis of biological material
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes

Definitions

  • the present invention relates to a determination device, a determination method, and a determination program.
  • Patent Document 1 describes a method including a step of measuring cellular complexity in gene-transfected animal cells and a step of predicting the gene transfer rate based on the value measured in the complexity measurement step. The method described in Patent Document 1 measures cellular complexity and uses the measured complexity as an indicator, making it possible to simply and efficiently predict the gene transfer rate of animal cells.
  • gene transfer rate refers to the rate at which a foreign gene is introduced into cells, and is, for example, the proportion of cells into which a foreign gene has been introduced out of all cells, the amount of foreign gene uptake in a cell population, or the expression rate of the foreign gene in the entire cell population.
  • the foreign gene is a CAR, it is sometimes referred to as the CAR positive rate.
  • FIG. 1 is a conceptual diagram showing the configuration of a determination system including a determination device according to the first embodiment.
  • FIG. 2 is a block diagram of the determination device according to the first embodiment.
  • FIG. 3 is a flowchart of the photographing process executed by the determination device shown in FIG.
  • FIG. 4 is a flowchart of the calculation process executed by the determination device shown in FIG. 5(a) to 5(d) are diagrams showing the ring mask generation process shown in S202 in FIG. 4, where FIG. 5(a) shows the first step, FIG. 5(b) shows the second step, FIG. 5(c) shows the third step, and FIG. 5(d) shows the fourth step.
  • FIG. 6(a) shows a bright-field image of multiple cells taken at the focal position
  • Figure 6(b) shows an image obtained by black-hat transformation of the bright-field image of multiple cells taken at a position 2.4 ⁇ m off the focal position
  • FIG. 7 is a block diagram of a determination device according to the second embodiment.
  • FIG. 8 is a flowchart of the calculation process executed by the determination device shown in FIG.
  • Figure 9(a) shows CAR-T cells and T cells in a CAR-T fluorescence image of multiple cells captured at the focal position
  • Figure 9(b) shows a bright-field image of multiple cells captured at a position 7.5 ⁇ m shifted from the focal position.
  • Fig. 1 is a conceptual diagram showing the configuration of a determination system including a determination device according to the first embodiment.
  • the determination system 100 includes a microscope main body 101 and a determination device 1.
  • the optical axis of the optical system of the microscope main body 101 is indicated by a dashed dotted line L1
  • illumination light from a light source 111 is indicated by a dashed two-dotted line L2.
  • the microscope main body 101 includes a transmitted illumination optical system 110 that irradiates illumination light onto the object S, a stage 120, an imaging optical system 130, and a detection unit 140.
  • the microscope main body 101 captures a bright-field image of the object S placed on the stage 120 and outputs bright-field image data representing the captured bright-field image to the determination device 1.
  • the determination device 1 may be a separate device, such as a computer, from the microscope main body 101.
  • the bright-field image is an image captured by bright-field observation. Bright-field observation allows cells to be observed and photographed without losing cellular information, such as their internal structure.
  • Phase-contrast observation is commonly used when observing adherent cells, but phase-contrast observation creates a bright, edging-like halo around the cell's outline, resulting in the loss of information around the cell's outline.
  • cells suspended in culture medium are spherical and thick, and phase-contrast observation results in significant phase change around the cell's outline. As a result, a stronger halo is produced than with phase contrast observation of adherent cells, and information about the cells around their outlines is lost. Photographing cells using brightfield observation allows for the cells to be photographed without losing information about the cell outlines.
  • floating cells includes suspended cells, cells that are not directly attached to the bottom of the imaging container but are indirectly attached to the bottom of the imaging container via an anchor or similar and are suspended in the medium, and adherent cells that have detached from the adhesive surface and exist floating in the medium.
  • the transmitted illumination optical system 110 includes a light source 111, a first lens 112, a bandpass filter 113, a field stop 114, a second lens 115, an aperture stop 116, and a condenser lens 117.
  • the light source 111 includes a non-coherent light source device such as a halogen lamp, and emits illumination light L2 to illuminate the object S.
  • the illumination light L2 emitted from the light source 111 is incident on the first lens 112.
  • the illumination light L2 incident on the first lens 112 is refracted by the first lens 112 to become approximately parallel light, which exits the first lens 112 and is incident on the bandpass filter 113.
  • the bandpass filter 113 can be retracted to a position outside the optical path.
  • Illumination light L2 incident on field stop 114 has its beam diameter adjusted, exits field stop 114, and enters second lens 115. Illumination light L2 incident on second lens 115 is converged by second lens 115, exits second lens 115, and enters aperture stop 116. Illumination light L2 incident on aperture stop 116 is converted so that its wavefront becomes spherical, exits aperture stop 116, and enters condenser lens 117. Illumination light L2 incident on condenser lens 117 is refracted by condenser lens 117, and becomes light with a wavefront that is approximately perpendicular to the optical axis when irradiated onto object S, and is irradiated onto object S placed on stage 120.
  • the stage 120 is movable in a direction along an axis parallel to the optical axis of the objective lens 151, which is parallel to the optical axis of the optical system of the microscope main body 101, and in directions along two axes perpendicular to the optical axis of the objective lens 151.
  • the stage 120 is electrically driven by a moving device such as a motor and moves in a direction along an axis parallel to the optical axis of the objective lens 151 and in directions along two axes perpendicular to the optical axis of the objective lens 151.
  • Subject S placed on stage 120 includes a plurality of CAR-T cells and a plurality of T cells.
  • CAR-T cells are an example of cells into which an exogenous gene has been introduced
  • T cells are an example of cells into which an exogenous gene has not been introduced.
  • Subject S is formed, for example, through a T cell collection process in which T cells are collected from a subject, a CAR gene introduction process in which a CAR gene is introduced into the T cells collected in the T cell collection process, and a cell culture process in which the T cells into which the CAR gene has been introduced in the CAR gene introduction process are cultured.
  • Subject S is placed on stage 120, for example, in a 20 ⁇ L cell suspension contained in an imaging container.
  • the imaging optical system 130 has an objective optical system 150 and a relay optical system 160.
  • the objective optical system 150 includes multiple objective lenses 151 with different numerical apertures (NA).
  • the relay optical system 160 has an imaging lens 161, a beam splitter 162, mirrors 163a, 163b, and 163c, lenses 164a, 164b, and 164c, and an eyepiece lens 165.
  • Imaging lens 161 refracts light incident from objective optical system 150 so that it forms an image on detection unit 140, and outputs it to beam splitter 162.
  • Beam splitter 162 reflects part of the light incident from objective optical system 150 to detection unit 140, and transmits the remainder, outputting it to mirror 163a.
  • Light reflected by mirror 163a passes through lens 164a, mirror 163b, lens 164b, lens 164c, and mirror 163c in this order, and is reflected by mirrors or refracted by lenses, before entering eyepiece 165.
  • Light incident on eyepiece 165 is refracted by eyepiece 165 and enters the user's eye E, where it is perceived.
  • the detection unit 140 includes a detector such as a CCD or CMOS image sensor, and captures an image of the object S.
  • the detection unit 140 detects light reflected by the beam splitter 162 of the relay optical system 160.
  • the detection signal corresponding to the detected light is A/D converted by an A/D converter (not shown) or the like, and output to the determination device 1.
  • Figure 2 is a block diagram of the determination device 1.
  • the determination device 1 has a communication unit 11, a memory unit 12, an input unit 13, a display unit 14, and a processing unit 20, and executes various processes based on programs pre-stored in the memory unit 12 and by referencing data stored in the memory unit 12. The determination device 1 also executes various processes in response to instructions input by an operator via the input unit 13, and outputs the execution results to the display unit 14.
  • the determination device 1 is an electronic computer. The determination device 1 determines whether a cell is a CAR-T cell or a T cell based on the complexity of the structure around the outline of a cell contained in the object S placed on the stage 120, and calculates the gene transfer rate of the CAR gene using the determination result.
  • the communication unit 11 has a communication interface circuit for connecting the determination device 1 to a network (not shown).
  • the communication unit 11 supplies data received from the microscope main body 101 and an external device (not shown) via the network to the processing unit 20.
  • the communication unit 11 also transmits data supplied from the processing unit 20 to the external device via the network.
  • the communication unit 11 receives a detection signal A1 indicating light detected by the detection unit 140, and transmits a control signal A2 to the microscope main body 101 to control each of the components of the microscope main body 101, such as the stage 120 and the detection unit 140.
  • the storage unit 12 includes, for example, one of a semiconductor memory, a magnetic disk device, and an optical disk device.
  • the storage unit 12 stores an operating system program, a driver program, an application program, data, etc. used for processing by the processing unit 20.
  • the storage unit 12 stores, as driver programs, an input device driver program that controls the input unit 13, an output device driver program that controls the display unit 14, etc.
  • the storage unit 12 also stores, as application programs, an imaging program that causes the processing unit 20 to execute an imaging process that images cells contained in the object S placed on the stage 120.
  • the storage unit 12 also stores, as application programs, a determination program that causes the processing unit 20 to execute an arithmetic process that calculates the gene transfer rate of the CAR gene in the cells contained in the object S.
  • the imaging program and arithmetic program may be installed into the storage unit 12 from a computer-readable portable storage medium such as a CD-ROM or DVD-ROM using a known setup program, etc.
  • the input unit 13 may be any device capable of operating the determination device 1, such as a keyboard or touchpad. An operator can input letters, numbers, etc. via the input unit 13. When operated by the operator, the input unit 13 generates a signal corresponding to that operation. The generated signal is then supplied to the processing unit 20 as an instruction from the operator.
  • the display unit 14 may be any device capable of displaying video, images, text, etc., such as a liquid crystal display or an organic EL (Electro-Luminescence) display.
  • the display unit 14 displays video corresponding to video data supplied from the processing unit 20, images corresponding to image data, text corresponding to text data, etc.
  • the display unit 14 may also display a graphical user interface (GUI) for operating the determination device 1.
  • GUI graphical user interface
  • the processing unit 20 has one or more processors and their peripheral circuits.
  • the processing unit 20 controls the overall operation of the determination device 1 and is, for example, a CPU (Central Processing Unit).
  • the processing unit 20 controls the operation of the communication unit 11, display unit 14, etc. so that various processes of the determination device 1 are executed in an appropriate order in accordance with the programs stored in the memory unit 12 and operations on the input unit 13.
  • the processing unit 20 executes processes based on the programs (operating system programs, driver programs, application programs, etc.) stored in the memory unit 12.
  • the processing unit 20 can also execute multiple programs (application programs, etc.) in parallel.
  • the processing unit 20 has an instruction unit 21, an acquisition unit 22, an image processing unit 23, a determination unit 24, a calculation unit 25, and an output unit 26.
  • Each of these units is a functional module implemented by a program executed by a processor included in the processing unit 20. Alternatively, each of these units may be implemented in the determination device 1 as firmware.
  • FIG. 3 is a flowchart of the photographing process executed by the determination device 1.
  • the photographing process shown in Fig. 3 is executed mainly by the processing unit 20 in cooperation with each element of the determination system 100, based on a photographing program stored in advance in the storage unit 12.
  • the instruction unit 21 outputs an imaging area movement instruction to the stage 120, indicating that the object S should be moved to an imaging position where a predetermined imaging area of the object S can be photographed (S101).
  • the stage 120 moves along two axes perpendicular to the optical axis of the objective lens 151 of the objective optical system 150, and moves the object S to a position where a predetermined imaging area of the object S can be photographed.
  • the instruction unit 21 may move the imaging optical system 130 without moving the stage 120, or may move both the stage 120 and the imaging optical system 130.
  • the instruction unit 21 outputs an imaging position movement instruction to the stage 120, instructing the stage 120 to move the object S to the imaging position (S102).
  • the imaging position is a position parallel to the optical axis of the objective lens 151 and shifted a predetermined offset amount from the focal position of the objective lens 151 in a direction away from the objective lens 151.
  • the stage 120 moves along an axis parallel to the optical axis of the objective lens 151 to move the object S to the imaging position.
  • the focal position is the midpoint between the position closest to the cell's outline objective lens 151 and the position farthest from the outline objective lens 151, and is the center position of the cell thickness relative to the optical axis direction of the objective lens 151.
  • the offset amount is determined before the imaging process is performed, based on the optical characteristics of the objective lens 151, such as the NA, the size of the CAR-T cells and T cells contained in the object S, etc.
  • the brightness values of light and dark around the cell outlines are lost, resulting in uniform information around the cell outlines for all cells.
  • Capturing images at a position shifted from the focal position results in changes in brightness values around the cell outlines, allowing images to be captured without losing information around the outlines of each cell.
  • the offset amount is determined, for example, by using cells identified as CAR-T cells or T cells by fluorescent labeling, acquiring bright-field images captured at a position shifted by m ⁇ z from the focal plane, and calculating predetermined feature amounts from each bright-field image.
  • m is an integer greater than or equal to 0
  • ⁇ z is the absolute value of the focal depth of the objective lens 151.
  • the offset amount is preferably greater than or equal to 2 times and less than or equal to 15 times the focal depth ⁇ z of the objective lens 151, and more preferably greater than or equal to 6 times and less than or equal to 10 times.
  • the instruction unit 21 may move the imaging optical system 130 without moving the stage 120, or may move both the stage 120 and the imaging optical system 130.
  • the instructing unit 21 outputs an imaging instruction to the detecting unit 140 to capture an image of a predetermined imaging area of the object S that has been moved to the imaging position (S103).
  • the detecting unit 140 outputs bright-field image data indicating a bright-field image of the predetermined imaging area of the object S to the determination device 1.
  • the acquisition unit 22 acquires a bright-field image corresponding to the bright-field image data in response to the input of the bright-field image data from the detection unit 140 (S104).
  • the acquisition unit 22 stores bright-field image information indicating the acquired bright-field image in the storage unit 12.
  • the instruction unit 21 determines whether bright-field images have been captured for all imaging areas (S105).
  • the number of imaging areas for which bright-field images are captured is determined based on the number of CAR-T cells and T cells estimated to be contained in the imaging area, and the number of cells required to calculate the gene transfer rate of the CAR gene introduced into T cells.
  • S101 to S105 is repeated until the instruction unit 21 determines that bright-field images have been captured for all imaging areas (S105-YES). When the instruction unit 21 determines that images have been captured for all imaging areas (S105-YES), the imaging process ends.
  • Fig. 4 is a flowchart of the calculation process executed by the determination device 1.
  • the calculation process shown in Fig. 4 is executed mainly by the processing unit 20 in cooperation with each element of the determination system 100, based on a calculation program stored in advance in the storage unit 12.
  • the image processing unit 23 generates a quantitative phase image from the bright-field image captured in the imaging process (S201).
  • the image processing unit 23 generates a quantitative phase image from the bright-field image and stores quantitative phase image information indicating the generated quantitative phase image in the storage unit 12.
  • the quantitative phase image is generated using a known method, such as the method described in WO 2019/097587.
  • the image processing unit 23 generates multiple ring masks (S202) that are used to extract the periphery of each cell's outline contained in the quantitative phase image generated in the process of S201.
  • S202 multiple ring masks
  • an image in which the entire cell has a uniform contrast is required. It is difficult to generate a mask from a bright-field image in which intracellular contrast varies depending on the intracellular structure. For this reason, the mask is generated using a quantitative phase image in which intracellular contrast does not vary depending on the intracellular structure and the entire cell has a uniform contrast.
  • Figure 5 shows the ring mask generation process shown in S202, where Figure 5(a) shows the first step, Figure 5(b) shows the second step, Figure 5(c) shows the third step, and Figure 5(d) shows the fourth step.
  • step S1 the image processing unit 23 acquires the quantitative phase image generated in step S201.
  • the acquired quantitative phase image includes multiple cells.
  • step S2 the image processing unit 23 generates a circular first mask that indicates the outlines of each of the multiple cells included in the quantitative phase image.
  • the image processing unit 23 binarizes the pixel values of each pixel in the quantitative phase image and performs a filtering process to remove overlapping cells, cells located on the boundary of the imaging area, and foreign matter, thereby generating a first mask that indicates the outlines of each of the multiple cells included in the quantitative phase image.
  • step S3 the image processing unit 23 reduces the first mask generated in step S2 to generate a second mask.
  • the reduction ratio used to generate the second mask is determined before the imaging process is performed, based on the optical characteristics of the objective lens 151, such as the NA, the size of the cells included in the target object S, etc.
  • the reduction ratio used to generate the second mask is determined, for example, by calculating predetermined feature amounts while changing the reduction ratio using cells that have been determined to be CAR-T cells or T cells by fluorescent labeling.
  • the image processing unit 23 generates annular ring masks corresponding to the periphery of each of the outlines of the multiple cells.
  • the image processing unit 23 generates the ring mask as an area corresponding to the difference between the first mask and the second mask.
  • the image processing unit 23 generates a ring mask for each of the multiple cells included in the quantitative phase image acquired in the first step.
  • the image processing unit 23 stores ring mask information indicating the multiple generated ring masks in association with the corresponding bright-field image in the memory unit 12.
  • the acquisition unit 22 acquires a bright-field image (S203).
  • the acquisition unit 22 acquires a bright-field image captured in the imaging process, which corresponds to the bright-field image information stored in the storage unit 12.
  • the image processing unit 23 performs enhancement processing on the bright-field image acquired in the processing of S203 to generate a determination image (S204).
  • the image processing unit 23 performs black hat conversion processing as enhancement processing, which enhances black parts included in the bright-field image.
  • the image processing unit 23 may also perform morphological conversion processing other than black hat conversion processing, such as top hat conversion processing, which enhances white parts included in the bright-field image, as enhancement processing.
  • the image processing unit 23 may also perform enhancement processing other than morphological conversion processing, such as ridge detection processing, to generate a determination image.
  • the image processing unit 23 stores determination image information indicating the determination image, which is a bright-field image that has been subjected to enhancement processing, in the storage unit 12.
  • the determination image generated in the processing of S203 is an example of a bright-field image to which enhancement processing has been performed, which enhances the internal structure of a cell.
  • the acquisition unit 22 acquires a ring mask corresponding to the bright-field image acquired in the processing of S203 (S205).
  • the determination unit 24 acquires multiple ring masks corresponding to the ring mask information stored in the storage unit 12 in association with the bright-field image acquired in the processing of S203.
  • the determination unit 24 uses the multiple ring masks acquired in the process shown in S205 to determine whether each of the multiple cells included in the determination image generated in the process of S204 is a CAR-T cell or a T cell (S206). By using the ring masks, the determination unit 24 determines whether a cell has been introduced with an exogenous gene or has not been introduced with an exogenous gene, based on the structure of only the periphery of the cell's outline surrounded by the ring mask.
  • the determination unit 24 calculates an average luminance value, which is the average value of the luminance values of the pixels included in the periphery of the cell's outline surrounded by the ring mask, and determines whether the cell has been introduced with an exogenous gene or has not been introduced with an exogenous gene, based on the calculated average luminance value.
  • the introduced CAR molecule is expressed on the cell surface. Depending on whether or not this expression occurs, a difference occurs in the luminance value around the periphery of the cell on the bright-field image.
  • the determination unit 24 can determine whether a cell has been introduced with an exogenous gene or has not been introduced with an exogenous gene, for example, based on a luminance threshold value determined based on the luminance values around the periphery of the multiple cells.
  • the brightness threshold used in the process of S206 is calculated using a clustering method such as the k-means method from the average brightness value of the pixels contained around the cell outline.
  • the determination unit 24 calculates the average brightness value of the pixels contained around the outline of the cell surrounded by the ring mask, and if the calculated average brightness value is equal to or greater than a predetermined threshold, determines that the determined cell is a CAR-T cell (S206-YES). If the calculated average brightness value is less than the threshold, the determination unit 24 determines that the determined cell is a T cell (S206-NO).
  • the determination unit 24 determines that the determined cells are CAR-T cells (S206-YES), it increments the count of CAR-T cells by one (S207) and stores CAR-T cell count information indicating the increased count of CAR-T cells in the memory unit 12. Furthermore, when the determination unit 24 determines that the determined cells are T cells (S206-NO), it increments the count of T cells by one (S208) and stores T cell count information indicating the increased count of T cells in the memory unit 12.
  • the determination unit 24 determines whether or not the process of S206 has been performed on all cells included in the determination image generated by the process of S204 (S209).
  • the determination unit 24 repeats the processes of S206 to S209 until it determines that the process of S206 has been performed on all cells included in the determination image generated by the process of S204 (S209-YES).
  • the calculation unit 25 calculates the gene transfer rate of the CAR gene in the subject S (S210).
  • the calculation unit 25 calculates the gene transfer rate of the CAR gene by dividing the number of CAR-T cells counted in the process of S207 by the total number of CAR-T cells and T cells counted in the processes of S207 and S208.
  • the calculation unit 25 stores gene transfer rate information indicating the calculated gene transfer rate in the memory unit 12.
  • the output unit 26 outputs a gene introduction rate signal indicating the gene introduction rate calculated in the processing of S210 (S211).
  • the output unit 26 acquires the gene introduction rate information stored in the memory unit 12, generates a gene introduction rate signal indicating the gene introduction rate corresponding to the acquired gene introduction rate information, and outputs the generated gene introduction rate signal.
  • the determination device 1 can determine whether the cells contained in the object S are CAR-T cells or T cells based on a bright-field image of the cells captured at a position shifted from the focal position. Furthermore, if the object S contains multiple cells, the determination device 1 can calculate the proportion of CAR-T cells (or T cells) among those cells.
  • the determination device 1 can determine whether a cell is a CAR-T cell or a T cell based on a bright-field image that has been enhanced to highlight the structure around the cell's outline. Furthermore, if the subject S contains multiple cells, the determination device 1 can calculate the proportion of CAR-T cells (or T cells) among those cells.
  • Figure 6(a) shows a bright-field image of multiple cells captured at the focal position
  • Figure 6(b) shows images obtained by black-hat transformation of bright-field images of multiple cells captured at a position 2.4 ⁇ m shifted from the focal position
  • Images I21 to I27 shown in Figure 6(b) are images obtained by black-hat transformation of bright-field images of multiple cells captured at a position 2.4 ⁇ m shifted from the focal position, respectively, of the cells contained in images I11 to I17 shown in Figure 6(a).
  • the cells contained in images I11 and I15 shown in Figure 6(a), and images I21 and I25 corresponding to images I11 and I15, are CAR-T cells.
  • the cells contained in images I12 to I14, I16, and I17 shown in Figure 6(a), and images I22 to I24, I26, and I27 corresponding to images I12 to I14, I16, and I17, are T cells.
  • the determination device 1 can determine whether a cell is a CAR-T cell or a T cell based on changes in brightness around the cell outline in CAR-T cells that express CAR-T molecules on their surface due to gene introduction.
  • the determination device 1 may also determine whether a cell is a CAR-T cell or a T cell based on an index indicating the complexity of the area around the cell's outline.
  • the index indicating complexity may be an index visible from an image, such as the density of lines, the area ratio of black structures separated by lines, the continuous area, or the number of areas separated by lines.
  • the index indicating complexity may also be a feature extracted from a visible index, such as a circumscribing rectangle that circumscribes an area separated by lines, or a major axis direction that indicates the orientation of an area separated by lines.
  • the index indicating complexity may also be a feature extracted from a visible index by an analysis process such as a fractal dimension analysis process.
  • FIG. 7 is a block diagram of a determination device according to the second embodiment.
  • Determination device 2 differs from determination device 1 in that it has a processing unit 30 instead of processing unit 20.
  • Processing unit 30 differs from processing unit 20 in that it has an image processing unit 33 and a determination unit 34 instead of image processing unit 23 and determination unit 24.
  • the configurations and functions of the components of determination device 2 other than image processing unit 33 and determination unit 34 are the same as the configurations and functions of the components of determination device 1 with the same reference numerals, and therefore detailed description will be omitted here.
  • Determination device 2 is disposed in determination system 100 in place of determination device 1. Similar to determination device 1, determination device 2 performs an imaging process to image object S placed on stage 120 of microscope main body 101, and also performs a calculation process to calculate the gene introduction rate of the CAR gene in cells contained in object S.
  • the imaging process performed by determination device 2 is the same as the imaging process performed by determination device 1 described with reference to Figure 3, and therefore detailed description will be omitted here.
  • the determination device 2 determines whether a cell is a CAR-T cell or a T cell based on the difference in brightness due to the phase difference depending on the morphology of the CAR-T cell and T cell contained in the object S placed on the stage 120, and calculates the gene transfer rate of the CAR gene using the determination result.
  • Fig. 8 is a flowchart of the calculation process executed by the determination device 2.
  • the calculation process shown in Fig. 8 is executed mainly by the processing unit 30 in cooperation with each element of the determination system 100, based on a calculation program stored in advance in the storage unit 12.
  • the image processing unit 33 generates a quantitative phase image from the bright-field image captured in the imaging process (S301).
  • the process of S301 is similar to the process of S201, so a detailed description will be omitted here.
  • the image processing unit 33 generates circular masks to be used to extract each of the cells included in the quantitative phase image generated in the process of S301 (S302).
  • the image processing unit 33 generates multiple circular masks representing each of the cells included in the quantitative phase image by executing the same generation method as that used to generate the first mask in the ring mask generation process shown in S202.
  • the image processing unit 23 associates the circular mask information representing the multiple circular masks generated with the corresponding bright field image and stores it in the memory unit 12.
  • the shape of the mask is not limited to being circular, as long as it is a shape that surrounds the cells.
  • the acquisition unit 22 acquires a bright-field image (S303).
  • the acquisition unit 22 acquires a bright-field image captured in the imaging process, which corresponds to the bright-field image information stored in the storage unit 12.
  • the acquisition unit 22 acquires a circular mask corresponding to the bright-field image acquired in the processing of S303 (S304).
  • the determination unit 24 acquires multiple circular masks corresponding to the circular mask information stored in the memory unit 12 in association with the bright-field image acquired in the processing of S303.
  • the determination unit 34 uses the circular mask acquired in the process shown in S304 to determine whether each of the multiple cells included in the bright-field image acquired in the process of S303 is a CAR-T cell or a T cell (S305).
  • the determination unit 34 determines whether the cell has been introduced with an exogenous gene or has not been introduced with an exogenous gene, based on the intracellular structure surrounded by the circular mask.
  • the determination unit 34 calculates the average brightness value of the pixels included in the cell surrounded by the circular mask, and determines whether the cell has been introduced with an exogenous gene or has not been introduced with an exogenous gene, based on the calculated average brightness value.
  • the determination unit 34 can determine whether the cell has been introduced with an exogenous gene or has not been introduced with an exogenous gene, based on a brightness threshold determined based on the brightness values of the multiple cells.
  • the brightness threshold used in the process of S305 is determined from the distribution of the average brightness values of pixels contained in multiple CAR-T cells and T cells.
  • the determination unit 34 calculates the average brightness value of the pixels contained in the cell surrounded by the circular mask, and if the calculated average brightness value is equal to or greater than a predetermined threshold, determines that the determined cell is a CAR-T cell (S305-YES). If the calculated average brightness value is less than the threshold, the determination unit 34 determines that the determined cell is a T cell (S305-NO).
  • the determination unit 34 determines that the determined cells are CAR-T cells (S305-YES), it increments the count of CAR-T cells by one (S306) and stores CAR-T cell count information indicating the increased count of CAR-T cells in the memory unit 12. Furthermore, when the determination unit 34 determines that the determined cells are T cells (S305-NO), it increments the count of T cells by one (S307) and stores T cell count information indicating the increased count of T cells in the memory unit 12.
  • the determination unit 34 determines whether the process of S305 has been performed on all cells included in the bright-field image acquired in the process of S303 (S308).
  • the determination unit 34 repeats the processes of S305 to S308 until it determines that the process of S305 has been performed on all cells included in the bright-field image acquired in the process of S303 (S308-YES).
  • the calculation unit 25 calculates the gene transfer rate of the CAR gene in the subject S (S309), similar to the process shown in S210. Then, the output unit 26 outputs a gene transfer rate signal indicating the gene transfer rate calculated in the process of S309, similar to the process shown in S211 (S310).
  • the determination device 2 can determine whether a cell is a CAR-T cell or a T cell based on the luminance value inside the cell in a bright-field image. Furthermore, if the object S contains multiple cells, the determination device 2 can calculate the proportion of CAR-T cells (or T cells) among those cells.
  • Figure 9(a) shows the spatial distribution of CAR-T cells in a CAR-T fluorescence image of multiple cells captured at the focal position
  • Figure 9(b) shows a bright-field image of multiple cells captured at a position shifted 7.5 ⁇ m from the focal position.
  • cells with brightness values equal to or greater than a predetermined brightness threshold are surrounded by squares, and dashed squares indicated by arrow A indicate cells whose brightness values are equal to or greater than the predetermined brightness threshold but are not CAR-T cells.
  • the bright-field image captured at a position shifted from the focal position shown in Figure 9(b) it can be seen that CAR-T cells appear to have high brightness values, while T cells appear to have low brightness values.
  • the determination device 2 determines whether a cell is a CAR-T cell or a T cell based on the difference in brightness values within the cell, which corresponds to the difference in morphology between CAR-T cells and T cells, thereby easily and efficiently determining whether the cell is a CAR-T cell or a T cell.
  • the determination device 1 executes a process of calculating the gene transfer rate of the CAR gene
  • the determination device according to the embodiment may execute a process other than the process of calculating the gene transfer rate of the CAR gene, as long as it executes a process of determining whether a cell is a CAR-T cell or a T cell.
  • the determination device may execute a process of calculating the amount of protein produced by gene transfer or the amount of change in a structure, using the result of determining whether a cell is a CAR-T cell or a T cell, or a process of estimating an appropriate culture period for CAR-T cells, using the result of determining the CAR-positive rate in a cell population.
  • Determination devices 1 and 2 determine whether a cell is a CAR-T cell or a T cell, but the determination device according to the embodiment may also determine whether a cell has been introduced with an exogenous gene other than a CAR-T cell or a cell other than a T cell to which an exogenous gene has not been introduced.
  • the determination device may be any device capable of determining cells into which a gene has been introduced.
  • the gene to be introduced is not particularly limited, and examples thereof include CAR (chimeric antigen receptor).
  • the cells are also not particularly limited, and animal cells, for example, can be used.
  • animal cells that may be the subject of determination include spleen cells, nerve cells, glial cells, pancreatic ⁇ cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, endothelial cells, fibroblasts, fibrocytes, muscle cells (e.g., skeletal muscle cells, cardiac muscle cells, myoblasts, and muscle satellite cells), adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells (NK cells), mast cells, neutrophils, basophils, eosinophils, monocytes, and megakaryocytes), synoviocytes, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary gland cells, hepatocytes, interstitial cells, egg cells, and sperm cells, as well as stem cells that can be induced to differentiate into these cells (including pluripotent stem cells such as neural stem cells, hematopo
  • T cells include ⁇ T cells, ⁇ T cells, helper T cells, cytotoxic T cells, regulatory T cells, suppressor T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NKT cells, TCR-T cells, STAR receptor T cells, CAR-T cells, and the like.
  • animal cells also include the above-mentioned cells produced by inducing differentiation in vitro of primary cells, the above-mentioned stem cells (e.g., iPS cells), and the like.
  • Animal cells also include various cancer cells. Animal cells may contain only one type of cell, or two or more types.
  • the organism from which the animal cells are derived is not particularly limited, and examples of such organisms include mammals, such as humans, mice, rats, cows, horses, pigs, rabbits, dogs, cats, goats, monkeys, and chimpanzees.
  • the organism from which the animal cells are derived is preferably humans, and cells that undergo changes around their cell outline or changes in cell morphology upon gene introduction, such as CAR-T cells, are preferred.
  • Cells that undergo changes around their outline or changes in morphology include cells that express receptors or the like on the cell surface upon gene introduction, and cells that express intracellular domains or the like within the cell near the cell surface.
  • Cells that secrete proteins produced by gene introduction to the outside of the cell, such as antibody-producing cells are also included.
  • the determination device may be used in a manufacturing process for producing a gene-introduced cell preparation, in a process for determining whether a cell has been introduced with an exogenous gene or not, based on an acquired bright-field image.
  • the method for producing a cell preparation includes: (I) acquiring a bright-field image of a cell photographed at a position shifted from the focal position; and (II) determining, based on the bright-field image, whether the cell has been introduced with an exogenous gene or has not been introduced with an exogenous gene.
  • the cell preparation according to the embodiment is preferably produced as a parenteral preparation by mixing an effective amount of transfected animal cells with a pharmaceutically acceptable carrier according to known means, such as the method described in the Japanese Pharmacopoeia.
  • the cell preparation according to the embodiment is preferably produced as a parenteral preparation such as an injection, suspension, or infusion.
  • Parenteral administration methods include intravenous, intraarterial, intramuscular, intraperitoneal, and subcutaneous administration.
  • Pharmaceutically acceptable carriers include solvents, bases, diluents, excipients, soothing agents, buffers, preservatives, stabilizers, suspending agents, isotonic agents, surfactants, and solubilizers.

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Abstract

Dispositif d'analyse (1) composé : d'une unité de capture (22) permettant de capturer une image en champ clair d'une cellule à une position décalée par rapport à la position focale ; et d'une unité d'analyse (24) permettant de déterminer, à partir de l'image en champ clair, si la cellule est une cellule dans laquelle un gène étranger a été introduit ou une cellule dans laquelle aucun gène étranger n'a été introduit. Ce procédé d'analyse consiste à traiter une image en champ clair d'une cellule capturée à une position décalée par rapport à la position focale, puis à déterminer, sur la base de cette image en champ clair, si la cellule est une cellule dans laquelle un gène étranger a été introduit ou une cellule dans laquelle aucun gène étranger n'a été introduit.
PCT/JP2025/015866 2024-04-26 2025-04-24 Dispositif d'analyse, procédé d'analyse et programme d'analyse Pending WO2025225688A1 (fr)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006080239A1 (fr) * 2005-01-31 2006-08-03 Olympus Corporation Dispositif de traitement d’image, système de microscope et programme de spécification de zone
WO2019097587A1 (fr) * 2017-11-14 2019-05-23 株式会社ニコン Procédé de génération d'image de phase quantitative, dispositif de génération d'image de phase quantitative et programme
US20200242772A1 (en) * 2019-01-28 2020-07-30 Florida Analytical Imaging Solutions, LLC. Automatic region of interest selection in centrosome analysis
JP2022518926A (ja) * 2019-02-01 2022-03-17 エッセン インストゥルメンツ，インコーポレイテッド ディー／ビー／エー エッセン バイオサイエンス，インコーポレイテッド 位相差および明視野イメージングを用いた無標識細胞セグメンテーション
JP2022546396A (ja) * 2019-08-30 2022-11-04 ジュノー セラピューティクス インコーポレイテッド 細胞を分類するための機械学習方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006080239A1 (fr) * 2005-01-31 2006-08-03 Olympus Corporation Dispositif de traitement d’image, système de microscope et programme de spécification de zone
WO2019097587A1 (fr) * 2017-11-14 2019-05-23 株式会社ニコン Procédé de génération d'image de phase quantitative, dispositif de génération d'image de phase quantitative et programme
US20200242772A1 (en) * 2019-01-28 2020-07-30 Florida Analytical Imaging Solutions, LLC. Automatic region of interest selection in centrosome analysis
JP2022518926A (ja) * 2019-02-01 2022-03-17 エッセン インストゥルメンツ，インコーポレイテッド ディー／ビー／エー エッセン バイオサイエンス，インコーポレイテッド 位相差および明視野イメージングを用いた無標識細胞セグメンテーション
JP2022546396A (ja) * 2019-08-30 2022-11-04 ジュノー セラピューティクス インコーポレイテッド 細胞を分類するための機械学習方法

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