Determination device, determination method, and determination program

Abstract

A determination device 1 comprises: an acquisition unit 22 that acquires a bright-field image of a cell captured at a position shifted from a focal position; and a determination unit 24 that determines, on the basis of the bright-field image, whether the cell is a cell into which a foreign gene has been introduced or a cell into which a foreign gene has not been introduced. This determination method comprises processing for acquiring a bright-field image of a cell captured at a position shifted from a focal position, and determining, on the basis of the bright-field image, whether the cell is a cell into which a foreign gene has been introduced or a cell into which a foreign gene has not been introduced.

Description

Determination device, determination method, and determination program

[0001] The present invention relates to a determination device, a determination method, and a determination program.

[0002] Various techniques for improving the measurement method of gene transfer rate, which is an important indicator for genetically engineered cell medicines such as chimeric antigen receptor (CAR)-T cells, are known. For example, 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 index, thereby enabling simple and efficient prediction of the gene transfer rate of animal cells. Note that the "gene transfer rate" refers to the rate of transfer of a foreign gene into cells, and is, for example, the proportion of cells into which the foreign gene has been introduced among 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. When the foreign gene is a CAR, it is sometimes referred to as the CAR-positive rate.

[0003] International Publication No. 2023 / 190974

[0004] 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 imaging process executed by the determination device shown in FIG. 2. FIG. 4 is a flowchart of the calculation process executed by the determination device shown in FIG. 2. FIGS. 5(a)-5(d) are diagrams showing the ring mask generation process shown in S202 in FIG. 4, with FIG. 5(a) showing the first step, FIG. 5(b) showing the second step, FIG. 5(c) showing the third step, and FIG. 5(d) showing the fourth step. FIG. 6(a) is a diagram showing a bright-field image of multiple cells captured at the focal position, and FIG. 6(b) is a diagram showing an image obtained by performing black-hat transformation on a bright-field image of multiple cells captured at a position 2.4 μm 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. 7. Figure 9(a) shows CAR-T cells and T cells in a CAR-T fluorescence image of multiple cells captured at the focal position, and Figure 9(b) shows a bright-field image of multiple cells captured at a position 7.5 μm shifted from the focal position.

[0005] The determination device according to the present invention will be described below with reference to the drawings. However, it should be noted that the technical scope of the present invention is not limited to the embodiments, but extends to the inventions set forth in the claims and their equivalents.

[0006] (Configuration and Function of Determination System Including Determination Apparatus According to First Embodiment) Fig. 1 is a conceptual diagram showing the configuration of a determination system including a determination apparatus according to the first embodiment. The determination system 100 has a microscope main body 101 and a determination apparatus 1. In Fig. 1, the optical axis of the optical system of the microscope main body 101 is indicated by a dashed dotted line L1, and the illumination light from the light source 111 is indicated by a dashed two-dotted line L2.

[0007] 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 to observe adherent cells, but phase-contrast observation creates a bright, edging-like halo around the cell's outline, resulting in loss of information around the cell's outline. Furthermore, cells suspended in a 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 generated than in phase contrast observation of adherent cells, and cell information around the cell outline is lost. By photographing using bright-field observation, cells can be photographed without losing information around the cell outline. Note that floating cells include 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 the like and are suspended in the medium, and adherent cells that have detached from the adhesive surface and exist in a floating state in the medium.

[0008] 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. Of the illumination light L2 incident on the bandpass filter 113, only light of wavelength components within a desired wavelength range is transmitted by the bandpass filter 113 and is incident on the field stop 114. The bandpass filter 113 can be retracted to a position outside the optical path.

[0009] The illumination light L2 incident on the field stop 114 has its beam diameter adjusted, exits the field stop 114, and is then incident on the second lens 115. The illumination light L2 incident on the second lens 115 is converged by the second lens 115, exits the second lens 115, and is then incident on the aperture stop 116. The illumination light L2 incident on the aperture stop 116 is converted so that its wavefront becomes spherical, exits the aperture stop 116, and is then incident on the condenser lens 117. The illumination light L2 incident on the condenser lens 117 is refracted by the condenser lens 117, and becomes light having a wavefront that is approximately perpendicular to the optical axis when irradiated onto the object S, and is then irradiated onto the object S placed on the stage 120.

[0010] 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. When photographing the object S, 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.

[0011] The subject S placed on the stage 120 includes a plurality of CAR-T cells and a plurality of T cells. The CAR-T cells are an example of cells into which an exogenous gene has been introduced, and the T cells are an example of cells into which an exogenous gene has not been introduced. The subject S is formed, for example, through a T cell collection step of collecting T cells from a subject, a CAR gene introduction step of introducing a CAR gene into the T cells collected in the T cell collection step, and a cell culture step of culturing the T cells into which the CAR gene has been introduced in the CAR gene introduction step. The subject S is contained in an imaging container as, for example, a 20 μL cell suspension, and placed on the stage 120.

[0012] The imaging optical system 130 has an objective optical system 150 and a relay optical system 160. The objective optical system 150 includes a plurality of objective lenses 151 with different numerical apertures NA, etc. 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.

[0013] The imaging lens 161 refracts light incident from the objective optical system 150 so as to form an image on the detection unit 140, and outputs the light to the beam splitter 162. The beam splitter 162 reflects a portion of the light incident from the objective optical system 150 to the detection unit 140, and transmits the remainder, outputting it to the mirror 163a. ​​The light reflected by the mirror 163a is reflected or refracted by the lenses in the order of lens 164a, mirror 163b, lens 164b, lens 164c, and mirror 163c, before entering the eyepiece 165. The light incident on the eyepiece 165 is refracted by the eyepiece 165 and enters the user's eye E to be perceived.

[0014] The detection unit 140 includes a detector such as an imaging element as a CCD or a CMOS, 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. A 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.

[0015] FIG. 2 is a block diagram of the determination device 1.

[0016] The determination device 1 has a communication unit 11, a storage unit 12, an input unit 13, a display unit 14, and a processing unit 20, and executes various processes based on programs stored in the storage unit 12 in advance and by referring to data and the like stored in the storage 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 a target object S placed on a stage 120, and calculates the gene transfer rate of the CAR gene using the determination result.

[0017] The communication unit 11 has a communication interface circuit for connecting the determination apparatus 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 for controlling each of the components of the microscope main body 101, such as the stage 120 and the detection unit 140.

[0018] 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, and the like used for processing in the processing unit 20. For example, 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, and the like. The storage unit 12 also stores, as application programs, an imaging program that causes the processing unit 20 to execute imaging processing for imaging cells contained in the subject 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 arithmetic processing for calculating the gene transfer rate of the CAR gene in the cells contained in the subject S. The imaging program and the arithmetic program may be installed into the storage unit 12 from a computer-readable portable storage medium such as a CD-ROM or a DVD-ROM using a known setup program or the like.

[0019] The input unit 13 may be any device, such as a keyboard or a touchpad, that can operate the determination apparatus 1. An operator can input letters, numbers, and the like via the input unit 13. When operated by the operator, the input unit 13 generates a signal corresponding to the operation. The generated signal is then supplied to the processing unit 20 as an instruction from the operator.

[0020] The display unit 14 may be any device capable of displaying video, images, text, etc., such as a liquid crystal display, an organic EL (Electro-Luminescence) display, etc. The display unit 14 displays video corresponding to video data, images corresponding to image data, text corresponding to text data, etc., supplied from the processing unit 20. The display unit 14 may also display a graphical user interface (GUI) for operating the determination device 1.

[0021] 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, the 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 storage unit 12, the operation of the input unit 13, etc. The processing unit 20 executes processes based on the programs (such as an operating system program, a driver program, and an application program) stored in the storage unit 12. The processing unit 20 can also execute multiple programs (such as application programs) in parallel.

[0022] The processing unit 20 includes 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.

[0023] (Photography Processing by the Determination Device According to the First Embodiment) Fig. 3 is a flowchart of the photographing processing executed by the determination device 1. The photographing processing 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.

[0024] First, the instruction unit 21 outputs to the stage 120 an imaging area movement instruction indicating that the object S should be moved to an imaging position where a predetermined imaging area of ​​the object S can be imaged (S101). In response to the input of the imaging area movement instruction, 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 imaged. Note that 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.

[0025] Next, 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 by a predetermined offset amount from the focal position of the objective lens 151 in a direction away from the objective lens 151. In response to the input of the imaging position movement instruction, 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. For example, in the case of a spherical cell, the focal position is an intermediate position between the position closest to the outline objective lens 151 and the position farthest from the outline objective lens 151, and is the center position of the cell thickness in the direction of the optical axis 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 sizes of the CAR-T cells and T cells contained in the object S, and the like. In bright-field images captured at the focal position, 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 mΔz from the focal plane, and calculating predetermined feature amounts from each bright-field image. Here, m is an integer greater than or equal to 0, and Δz 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 Δz of the objective lens 151, and more preferably greater than or equal to 6 times and less than or equal to 10 times. To move the imaging position, 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.

[0026] Next, the instructing unit 21 outputs an imaging instruction to the detecting unit 140 to image a predetermined imaging area of ​​the object S that has been moved to the imaging position (S103). In response to the input of the imaging instruction, 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.

[0027] Next, 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.

[0028] Next, the instruction unit 21 determines whether or not bright-field images have been captured for all of the imaging areas (S105). The number of imaging areas in 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.

[0029] The processing of S101 to S105 is repeated until the instruction unit 21 determines that bright field images have been captured for all of the imaging regions (S105-YES). When the instruction unit 21 determines that images have been captured for all of the imaging regions (S105-YES), the imaging processing ends.

[0030] (Calculation processing by the determination device according to the first embodiment) Fig. 4 is a flowchart of the calculation processing executed by the determination device 1. The calculation processing 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.

[0031] First, 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 International Publication WO 2019 / 097587.

[0032] Next, the image processing unit 23 generates multiple ring masks to be used to extract the periphery of each cell's outline contained in the quantitative phase image generated in step S201 (S202). To generate the masks, an image in which the entire cell has 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. Therefore, the masks are generated using quantitative phase images in which intracellular contrast does not vary depending on the intracellular structure and the entire cell has uniform contrast.

[0033] 5A and 5B are diagrams showing the ring mask generation process shown in S202, where FIG. 5A shows the first step, FIG. 5B shows the second step, FIG. 5C shows the third step, and FIG. 5D shows the fourth step.

[0034] First, in step S201, the image processing unit 23 acquires the quantitative phase image generated in step S201. The acquired quantitative phase image includes a plurality of cells. Next, in step S2, the image processing unit 23 generates a circular first mask representing the outlines of each of the plurality of 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 at the boundary of the imaging area, and foreign matter, thereby generating a first mask representing the outlines of each of the plurality of cells included in the quantitative phase image. Next, in 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, and the like. 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 determined to be CAR-T cells or T cells by fluorescent labeling. Then, in a fourth step, the image processing unit 23 generates an annular ring mask corresponding to the periphery of each of the outlines of the plurality of cells. The image processing unit 23 generates a region corresponding to the difference between the first mask and the second mask as the ring mask.

[0035] The image processing unit 23 generates a ring mask for each of the plurality of cells included in the quantitative phase image acquired in step 1. The image processing unit 23 stores ring mask information indicating the generated plurality of ring masks in the storage unit 12 in association with the corresponding bright-field image.

[0036] Next, the acquisition unit 22 acquires a bright-field image (S203). The acquisition unit 22 acquires a bright-field image that is captured in the imaging process and corresponds to the bright-field image information stored in the storage unit 12.

[0037] Next, the image processing unit 23 performs an enhancement process on the bright-field image acquired in the process of S203 to generate a determination image (S204). The image processing unit 23 performs, as the enhancement process, a black-hat conversion process that enhances black portions included in the bright-field image. Note that the image processing unit 23 may perform, as the enhancement process, a morphological conversion process other than the black-hat conversion process, such as a top-hat conversion process that enhances white portions included in the bright-field image. The image processing unit 23 may also perform an enhancement process other than a morphological conversion process, such as a ridge detection process, to generate a determination image. The image processing unit 23 stores, in the storage unit 12, determination image information indicating the determination image, which is the bright-field image that has been subjected to the enhancement process. The determination image generated in the process of S203 is an example of a bright-field image to which enhancement process has been performed to enhance the internal structure of a cell.

[0038] Next, the acquisition unit 22 acquires a ring mask corresponding to the bright-field image acquired in the process of S203 (S205). The determination unit 24 acquires a plurality of ring masks corresponding to the ring mask information stored in the storage unit 12 in association with the bright-field image acquired in the process of S203.

[0039] Next, 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. Specifically, 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. In CAR-T cells, the introduced CAR molecule is expressed on the cell surface. Whether or not this expression occurs causes a difference in luminance value around the cell's outline on the bright-field image. Therefore, 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 value used in the process of S206 is calculated from the average brightness value of pixels contained around the contour of the cell using a clustering method such as the k-means method.

[0040] The determination unit 24 calculates the average brightness value of pixels included around the outline of the cell surrounded by the ring mask, and determines that the determined cell is a CAR-T cell when the calculated average brightness value is equal to or greater than a predetermined threshold (S206-YES).When 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).

[0041] When the determination unit 24 determines that the determined cells are CAR-T cells (S206-YES), it increments the count number of CAR-T cells by one (S207) and stores CAR-T cell count information indicating the increased count number of CAR-T cells in the storage unit 12. Furthermore, when the determination unit 24 determines that the determined cells are T cells (S206-NO), it increments the count number of T cells by one (S208) and stores T cell count information indicating the increased count number of T cells in the storage unit 12.

[0042] Next, the determination unit 24 determines whether or not the process of S206 has been executed for all cells included in the determination image generated in 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 executed for all cells included in the determination image generated in the process of S204 (S209-YES).

[0043] When the determination unit 24 determines that the processes of S203 to S208 have been performed on all cells (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 count number of CAR-T cells counted in the process of S207 by the total value of the count numbers 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 storage unit 12.

[0044] The output unit 26 then outputs a gene transfer rate signal indicating the gene transfer rate calculated in the process of S210 (S211). The output unit 26 acquires the gene transfer rate information stored in the storage unit 12, generates a gene transfer rate signal indicating the gene transfer rate corresponding to the acquired gene transfer rate information, and outputs the generated gene transfer rate signal.

[0045] 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, when the object S contains multiple cells, the determination device 1 can calculate the proportion of CAR-T cells (or T cells) among these cells.

[0046] Furthermore, 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 subjected to enhancement processing to enhance the structure around the outline of the cell. Furthermore, when the object S contains multiple cells, the determination device 1 can calculate the proportion of CAR-T cells (or T cells) among those cells.

[0047] Figure 6(a) shows a bright-field image of multiple cells captured at the focal position, and Figure 6(b) shows images obtained by black-hat transformation of the bright-field image of multiple cells captured at a position 2.4 μm shifted from the focal position. Images I21 to I27 shown in Figure 6(b) are images obtained by black-hat transformation of the bright-field image of multiple cells captured at a position 2.4 μm 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. On the other hand, 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.

[0048] Comparing images I21 to I27, it can be seen that the CAR-T cells contained in images I21 and I25 have greater complexity around their outlines than the T cells contained in images I22 to I24, I26, and I27. 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 a CAR-T cell that expresses a CAR-T molecule on its surface due to gene introduction.

[0049] 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 circumscribing an area separated by lines and a major axis direction indicating 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.

[0050] (Configuration and Functions of Determination Device According to Second Embodiment) FIG. 7 is a block diagram of a determination device according to the second embodiment.

[0051] The determination device 2 differs from the determination device 1 in that it has a processing unit 30 instead of the processing unit 20. The processing unit 30 differs from the processing unit 20 in that it has an image processing unit 33 and a determination unit 34 instead of the image processing unit 23 and the determination unit 24. The configurations and functions of the components of the determination device 2 other than the image processing unit 33 and the determination unit 34 are the same as the configurations and functions of the components of the determination device 1 assigned the same reference numerals, and therefore detailed description thereof will be omitted here. The determination device 2 is disposed in the determination system 100 in place of the determination device 1. The determination device 2 performs an imaging process of imaging the object S placed on the stage 120 of the microscope main body 101, similar to the determination device 1, and also performs a calculation process of calculating the gene transfer rate of the CAR gene in cells contained in the object S. The imaging process performed by the determination device 2 is similar to the imaging process performed by the determination device 1 described with reference to FIG. 3, and therefore detailed description thereof 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.

[0052] (Calculation processing by the determination device according to the second embodiment) Fig. 8 is a flowchart of the calculation processing executed by the determination device 2. The calculation processing 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.

[0053] First, 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, and therefore a detailed description thereof will be omitted here.

[0054] Next, the image processing unit 33 generates a circular mask 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 circular mask information representing the generated multiple circular masks with the corresponding bright-field images and stores it in the storage unit 12. Note that the shape of the mask is not limited to a circle, and may be any shape that surrounds the cells.

[0055] Next, the acquisition unit 22 acquires a bright-field image (S303). The acquisition unit 22 acquires a bright-field image that is captured in the imaging process and corresponds to the bright-field image information stored in the storage unit 12.

[0056] Next, the acquisition unit 22 acquires a circular mask corresponding to the bright-field image acquired in the process of S303 (S304). The determination unit 24 acquires a plurality of circular masks corresponding to the circular mask information stored in the storage unit 12 in association with the bright-field image acquired in the process of S303.

[0057] Next, 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). By using the circular mask, the determination unit 34 determines whether the cell is a cell into which an exogenous gene has been introduced or a cell into which an exogenous gene has not been introduced based on the intracellular structure surrounded by the circular mask. Specifically, the determination unit 34 calculates the average luminance value of the pixels included in the cell surrounded by the circular mask, and determines whether the cell is a cell into which an exogenous gene has been introduced or a cell into which an exogenous gene has not been introduced based on the calculated average luminance value. Because the morphology of CAR-T cells changes upon gene introduction, a phase difference occurs depending on the difference in morphology between CAR-T cells and T cells. Because the phase difference causes a difference in luminance values ​​between CAR-T cells and T cells, the determination unit 34 can determine whether the cell is a cell into which an exogenous gene has been introduced or a cell into which an exogenous gene has not been introduced based on a luminance threshold value determined based on the luminance values ​​of the multiple cells. The brightness threshold used in the process of S305 is determined from the distribution of average brightness values ​​of pixels contained in a plurality of CAR-T cells and T cells.

[0058] The determination unit 34 calculates the average brightness value of pixels included in the cell surrounded by the circular mask, and determines that the determined cell is a CAR-T cell when the calculated average brightness value is equal to or greater than a predetermined threshold (S305-YES).When 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).

[0059] When the determination unit 34 determines that the determined cells are CAR-T cells (S305-YES), it increments the count number of CAR-T cells by one (S306) and stores CAR-T cell count information indicating the increased count number of CAR-T cells in the storage unit 12. Furthermore, when the determination unit 34 determines that the determined cells are T cells (S305-NO), it increments the count number of T cells by one (S307) and stores T cell count information indicating the increased count number of T cells in the storage unit 12.

[0060] Next, the determination unit 34 determines whether or not 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).

[0061] When the determination unit 34 determines that the processes of S303 to S307 have been executed for all the cells (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).

[0062] 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 the bright-field image. Furthermore, when the object S contains multiple cells, the determination device 2 can calculate the proportion of CAR-T cells (or T cells) among those cells.

[0063] FIG. 9( a) shows the spatial distribution of CAR-T cells in a CAR-T fluorescence image of multiple cells captured at a focal position, and FIG. 9( b) shows a bright-field image of multiple cells captured at a position shifted 7.5 μm from the focal position. In FIGS. 9( a) and 9(b), 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. It can be seen that in the bright-field image captured at a position shifted from the focal position shown in FIG. 9(b), CAR-T cells appear to have high brightness values, whereas 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 simply and efficiently by determining whether the 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 the CAR-T cell and the T cell.

[0064] (Variations of the Determination Device According to the Embodiment) Furthermore, although the determination device 1 executes a process of calculating the gene introduction rate of the CAR gene, the determination device according to the embodiment may execute a process of determining whether a cell is a CAR-T cell or a T cell, other than the process of calculating the gene introduction rate of the CAR gene. The determination device according to the embodiment may execute a process of calculating the amount of protein produced by gene introduction 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.

[0065] The 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 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 no exogenous gene has been introduced.

[0066] The determination device according to the embodiment 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. Examples of animal cells that may be the subject of determination include spleen cells, nerve cells, glial cells, pancreatic β 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, hematopoietic stem cells, mesenchymal stem cells, dental pulp stem cells, iPS cells, and ES cells), progenitor cells, blood cells, oocytes, and fertilized eggs. T cells include αβ T cells, γδ T cells, helper T cells, cytotoxic T cells, regulatory T cells, suppressor T cells, tumor-infiltrating T cells, memory T cells, naive T cells, NK T cells, TCR-T cells, STAR receptor T cells, CAR-T cells, and the like. Furthermore, animal cells also include the above-mentioned cells produced by in vitro differentiation induction 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 be of one type only, or may contain two or more types. Furthermore, 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 outline or change in morphology upon gene introduction, such as CAR-T cells, are preferred. Cells that undergo changes around their contours or morphological changes include cells that express receptors on the cell surface due to gene introduction, and cells that express intracellular domains within the cell near the cell surface.Also included are cells that secrete proteins produced by gene introduction, such as antibody-producing cells.

[0067] The determination device according to the embodiment 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.

[0068] The manufacturing method of the cell preparation according to the embodiment includes: (I) a step of acquiring a bright-field image of a cell photographed at a position shifted from the focal position; and (II) a step of determining whether the cell has been introduced with an exogenous gene or has not been introduced with an exogenous gene based on the bright-field image.

[0069] In the method for producing a cell preparation according to the embodiment, steps (I) and (II) can be carried out by the method described above. 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 procedures, 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.

[0070] 1, 2 Determination device 21 Instruction unit 22 Acquisition unit 23 Image processing unit 24 Determination unit 25 Calculation unit 26 Output unit 100 Determination system 101 Microscope main body

Claims

1. A determination device having: an acquisition unit that acquires a bright-field image of a cell photographed at a position shifted from the focal position; and a determination unit that determines, 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.

2. The determination device according to claim 1, wherein the acquisition unit acquires the bright-field image in which brightness values ​​vary depending on the structure of the cell.

3. A determination device as described in claim 1 or claim 2, further comprising an image processing unit that performs enhancement processing on the bright-field image to enhance the internal structure of the cell, and the determination unit determines whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced based on the bright-field image on which the enhancement processing has been performed.

4. A determination device as described in any one of claims 1 to 3, wherein the determination unit determines whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced based on the structure of only the area around the outline of the cell in the bright-field image on which the enhancement processing has been performed.

5. The determination device described in claim 4, wherein the determination unit determines whether the cell has been introduced with the exogenous gene or has not been introduced with the exogenous gene based on the brightness value around the contour of the cell in the bright-field image on which the enhancement processing has been performed.

6. The determination device of claim 5, wherein the determination unit determines whether the cell is an expression cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced based on a brightness value threshold determined based on the brightness values ​​around the contour portions of multiple cells.

7. The determination device described in claim 4, wherein the determination unit determines whether the cell is an expressing cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced based on the complexity of the structure around the contour of the cell in the bright-field image on which the enhancement processing has been performed.

8. The determination device of claim 7, wherein the determination unit determines whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced based on the complexity including indices visible around the contour portion of the cell and features extracted from the indices.

9. The determination device according to any one of claims 3 to 8, wherein the image processing unit emphasizes the internal structure of the cells by morphological transformation.

10. The determination device according to claim 9, wherein the image processing unit emphasizes the internal structure by the morphological transformation, which is a black hat transformation or a top hat transformation.

11. A determination device as described in claim 3 or claim 10, wherein the determination unit determines whether a cell in which a change occurs around its outline due to gene introduction is a cell into which the exogenous gene has been introduced or a cell into which the gene has not been introduced.

12. A determination device as described in claim 1 or claim 2, wherein the determination unit determines whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced based on the brightness value inside the cell in the bright-field image.

13. The determination device according to claim 12, wherein the determination unit determines whether the cell has been introduced with the exogenous gene or not based on the difference in brightness values ​​due to the phase difference according to the morphology of the cell into which the exogenous gene has been introduced and the cell into which the exogenous gene has not been introduced.

14. A determination device as described in claim 12 or claim 13, wherein the determination unit determines whether the cell has been introduced with the exogenous gene or has not been introduced with the exogenous gene based on a brightness value threshold determined based on the brightness values ​​of a plurality of the cells.

15. A determination device as described in any one of claims 1 to 14, wherein the acquisition unit acquires a bright-field image taken at a position where the offset from the focal position is equal to or greater than 2 times the focal depth and equal to or less than 15 times the focal depth.

16. A determination device described in any one of claims 1 to 15, wherein the determination unit determines whether the cell that expresses a receptor on the cell surface due to gene introduction is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced.

17. A determination device described in any one of claims 1 to 16, wherein the determination unit determines whether a cell whose morphology changes due to gene introduction is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced.

18. A determination device according to any one of claims 1 to 17, wherein the determination unit determines whether the suspended cells are cells into which the exogenous gene has been introduced or cells into which the exogenous gene has not been introduced.

19. A determination method comprising the steps of acquiring a bright-field image of a cell photographed at a position shifted from the focal position, and determining whether the cell has been introduced with an exogenous gene or has not been introduced with an exogenous gene based on the bright-field image.

20. A determination method as described in claim 19, wherein said acquiring step acquires a bright-field image in which brightness values ​​vary depending on the structure of the cell by capturing an image at a position shifted from the focal position.

21. A determination method as described in claim 19 or claim 20, further comprising an enhancement process for enhancing the internal structure of the cell in the bright-field image, and the determination process includes a process for determining whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced, based on the bright-field image on which the enhancement process has been performed.

22. A determination method according to any one of claims 19 to 21, wherein the determination process includes a process of determining whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced, based on the structure of only the area around the outline of the cell in the bright-field image on which the enhancement process has been performed.

23. The method of claim 22, wherein the determining process includes a process of determining whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced, based on the brightness value around the contour of the cell in the bright-field image on which the enhancement process has been performed.

24. The method of claim 23, wherein the determining process includes determining whether the cell has been introduced with the exogenous gene or has not been introduced with the exogenous gene based on a brightness threshold determined based on brightness values ​​around the contours of a plurality of the cells.

25. The method of claim 24, wherein the determining process includes a process of determining whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced, based on the complexity of the structure around the contour of the cell in the bright-field image on which the enhancement process has been performed.

26. The method of claim 25, wherein the process of determining determines whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced based on the complexity including indices visible around the contour of the cell and features extracted from the indices.

27. A determination method according to any one of claims 21 to 26, wherein the enhancement process includes a process of enhancing the internal structure of the cells by morphological transformation.

28. The determination method according to claim 27, wherein the enhancement process includes a process of enhancing the internal structure of the cells by the morphological transformation, that is, black hat transformation or top hat transformation.

29. A determination method as described in claim 19 or claim 20, wherein the determination process includes a process of determining whether the cell is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced, based on the brightness value inside the cell in the bright-field image.

30. The method of claim 29, wherein the determining process includes determining whether the cell has been introduced with the exogenous gene or not based on the difference in brightness values ​​due to the phase difference according to the morphology of the cell into which the exogenous gene has been introduced and the cell into which the exogenous gene has not been introduced.

31. The method of claim 29, wherein the determining process includes determining whether the cell has been introduced with the exogenous gene or has not been introduced with the exogenous gene based on a brightness value threshold determined based on brightness values ​​of a plurality of the cells.

32. A determination method described in any one of claims 19 to 31, wherein the acquiring step acquires the bright-field image whose offset from the focal position is equal to or greater than 2 times and equal to or less than 15 times the focal depth.

33. A determination method described in any one of claims 18 to 32, in which the determination process determines whether the cell that expresses a receptor on the cell surface due to gene introduction is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced.

34. A determination method described in any one of claims 19 to 33, in which the determination process determines whether a cell whose morphology changes due to gene introduction is a cell into which the exogenous gene has been introduced or a cell into which the exogenous gene has not been introduced.

35. A determination method described in any one of claims 19 to 34, wherein the determination process determines whether the floating cells are cells into which the exogenous gene has been introduced or cells into which the exogenous gene has not been introduced.

36. A determination program that causes a computer to perform the following process: acquire a bright-field image of a cell photographed at a position shifted from the focal position; and determine, 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.

37. A method for producing a gene-introduced cell preparation, comprising the steps of: acquiring a bright-field image of the cells photographed at a position shifted from the focal position; and determining, based on the bright-field image, whether the cells have been introduced with an exogenous gene or have not been introduced with an exogenous gene.

Images