Method for determining multinucleated cells and system for determining multinucleated cells

Abstract

To provide a method for determining multinucleated cells capable of more accurately determining whether a cell to be determined is a multinucleated cell, and to provide a system for determining multinucleated cells.SOLUTION: A method for identifying multinucleated cells includes the steps of: identifying a contour S of a cell 200 to be determined and identifying multiple, spaced-apart nuclei 310 within the cell contour; calculating an angle of a long side of each nucleus; comparing the angles; and determining that the cell with the contour is a multinucleated cell if an angle difference θg is within a predetermined range.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method for assessing multinucleated cells and a system for assessing multinucleated cells. [Background technology]

[0002] Sheet-shaped cell cultures (cell sheets) are widely known for use in fields such as regenerative medicine. Generally, sheet-shaped cell cultures are produced by storing and culturing cells collected from humans or non-human animals in a culture vessel containing a culture medium, and allowing them to adhere and aggregate into a sheet.

[0003] The quality of the cell cultures used in sheet-shaped cell cultures is often judged by the visual inspection of the operator, which can lead to ambiguity in the criteria and the results being influenced by the experience of each operator.

[0004] In relation to the above-mentioned problem, Patent Document 1 discloses a system for determining the multinucleated state of skeletal myoblasts, which determines the multinucleated state of skeletal myoblasts based on predetermined parameters. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] WO2022 / 071470 Summary of the Invention [Problem to be solved by the invention]

[0006] The determination system of Patent Document 1 detects (measures) parameters related to the outline and nuclear shape of skeletal myoblasts based on image data of the skeletal myoblasts, and determines the multinucleation state based on the parameters. Therefore, by using the system of Patent Document 1, an operator can determine the multinucleation state of skeletal myoblasts more accurately than before.

[0007] As a result of extensive research, the inventors of the present invention have invented a method for determining the multinucleation state of a cell based on parameters related to the contour and nucleus of the cell (e.g., skeletal myoblast) being evaluated, which is capable of obtaining more accurate results.

[0008] An object of the present invention is to provide a method for determining whether a cell to be determined is a multinucleated cell or not, and a system for determining whether a cell to be determined is a multinucleated cell, which can more accurately determine whether a cell to be determined is a multinucleated cell. [Means for solving the problem]

[0009] The present invention is achieved by any one of the following means (1) to (7).

[0010] (1) A method for determining multinucleated cells, comprising: identifying a cell outline and a plurality of spaced apart nuclei within the cell outline; calculating the angle of the long side of each nucleus; comparing the angles; and determining that the cell having the contour is a multinucleated cell if the angle difference is within a predetermined range.

[0011] (2) A method for determining multinucleated cells according to (1), wherein, when calculating the angle of the long side of each of the plurality of nuclei, the shape of the nuclei is approximated to an ellipse, and a virtual straight line along the long axis of the ellipse is defined as the long side.

[0012] (3) A method for determining multinucleated cells according to (1) or (2), wherein an image showing the stained cells is prepared before determining the multinucleated state.

[0013] (4) A system for determining multinucleated cells, comprising: a detector for identifying a cell outline and a plurality of spaced apart nuclei within the cell outline; a calculation unit that calculates the angle of the long side of each nucleus; A system for identifying multinucleated cells, comprising: a determination unit that compares the angles calculated by the calculation unit and determines that the cell having the contour is a multinucleated cell if the angle difference is within a predetermined range.

[0014] (5) The system for assessing multinucleated cells described in (4), wherein, when calculating the angle of the long side of each of the multiple nuclei, the calculation unit approximates the shape of the nuclei to an ellipse and defines the long side as a virtual straight line along the long axis of the ellipse.

[0015] (6) The system for determining multinucleated cells according to (4) or (5), wherein the detection unit, the calculation unit, and the determination unit perform their respective processes based on image data of the stained cells.

[0016] (7) an imaging unit for acquiring the image data; The system for determining multinucleated cells according to (6), further comprising: an image display unit capable of displaying an image of the cells generated from the image data. [Effects of the Invention]

[0017] According to the method and system for determining multinucleated cells of the present invention, a parameter related to the angle of the long side of the nucleus of the cell to be determined is calculated, and this parameter is used to determine whether the cell to be determined is a multinucleated cell. By using such a parameter, it becomes possible to more accurately determine whether the cell to be determined is a multinucleated cell. [Brief explanation of the drawings]

[0018] [Figure 1] FIG. 1 is a block diagram showing a system for determining multinucleated cells according to an embodiment. [Figure 2] 1 is a flowchart showing the steps of a method for determining multinucleated cells according to an embodiment. [Figure 3]FIG. 10 is a diagram schematically showing an example of image data used to determine multinucleated cells. [Figure 4] FIG. 1 is a diagram illustrating a method for determining multinucleated cells. [Figure 5] FIG. 10 is a diagram schematically showing an example of image data used to determine multinucleated cells. [Figure 6] FIG. 1 is a diagram illustrating a method for determining multinucleated cells. DETAILED DESCRIPTION OF THE INVENTION

[0019] Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the dimensional proportions in the drawings are exaggerated for the sake of explanation and may differ from the actual proportions.

[0020] Fig. 1 is a simplified block diagram of a system 100 for determining multinucleated cells according to this embodiment (hereinafter also simply referred to as the "determination system"). Fig. 2 is a flowchart showing the steps of a method for determining multinucleated cells according to this embodiment (hereinafter also simply referred to as the "determination method"). Figs. 3 and 5 are diagrams for explaining the determination method, and show examples of images generated from image data acquired by the imaging unit 150.

[0021] <Determination system 100> The determination system 100 shown in FIG. 1 is configured as a system for executing a determination method to be described later.

[0022] 1, 3, 4, 5, and 6, the determination system 100 includes a detection unit 131 that identifies the contour S of the cell 200 to be determined (e.g., a skeletal muscle cell contained in a skeletal muscle cell culture) and multiple spaced-apart nuclei 310 within the contour S of the cell 200 to be determined, a calculation unit 132 that calculates the angle of the long side of each nucleus 310, and a determination unit 133 that compares the angles calculated by the calculation unit 132 and determines that the cell 200 having the contour S is a multinucleated cell if the angle difference θg is within a predetermined range.

[0023] The determination system 100 has a control unit 130 that performs overall operational control of each unit of the determination system 100 and various arithmetic processing related to the determination method. The control unit 130 includes a CPU and a memory unit. The memory unit includes a ROM that stores various programs and data, a RAM that temporarily stores programs and data as a working area, and a hard disk that can store various programs and data. The memory unit can store a series of programs necessary for operational control of the determination system 100. The above programs incorporate algorithms that cause the control unit 130 to function as a detection unit 131, a calculation unit 132, and a determination unit 133. When the determination system 100 executes the determination method, the control unit 130 reads the above programs. Details of the processes executed by the detection unit 131, the calculation unit 132, and the determination unit 133 will be described later.

[0024] As shown in Figure 1, the judgment system 100 has an imaging unit 150 for acquiring image data of the cell 200 to be judged, and an image display unit 160 capable of displaying an image of the cell 200 to be judged generated from the image data acquired by the imaging unit 150.

[0025] The imaging unit 150 can be configured with a known camera device capable of acquiring still image data and / or video data. The image display unit 160 can be configured with a known display device capable of displaying an image generated from the image data acquired by the imaging unit 150.

[0026] In the determination system 100 of this embodiment, the imaging unit 150, image display unit 160, and control unit 130 can also be configured as a portable or non-portable information terminal device in which these functions are integrated.

[0027] The determination system 100 can include a stage 110 on which a culture substrate 120 containing a cell 200 to be determined can be placed. The imaging unit 150 can be laid out at a position (for example, any position above the stage 110 as shown in FIG. 1 ) where the imaging unit 150 can capture an image of the culture substrate 120 placed on the stage 110 and the cell 200 to be determined contained in the culture substrate 120.

[0028] The stage 110 can also be configured as, for example, a part of a culture device (storage cabinet or storage equipment) used for culturing the cells 200 to be determined. When the stage 110 is configured in this way, it becomes possible to immediately start the work for determination when starting the determination method without transporting or moving the culture substrate 120 containing the cells 200 to be determined.

[0029] <Culture substrate 120> The culture substrate 120 is not particularly limited as long as it is configured to be capable of forming a cell culture of skeletal myoblasts, which are the cells 200 to be determined. The culture substrate 120 can include, for example, an object having a container structure, a solid or semi-solid surface portion disposed in a container, or the like.

[0030] In this embodiment, as shown in FIG. 1, the culture substrate 120 is made up of a container having a structure and material that is impermeable to liquids such as a culture solution 121.

[0031] Regarding the specific form (manufacturing method, function, etc.) of the cells 200 to be evaluated, and the specific form (material, function, structure and shape, type of culture medium 121 used, addition of coating, etc.) of the culture substrate 120, publicly known content (e.g., WO2022 / 071470, JP 2020-78320, JP 2019-017397, etc.) can be appropriately referred to.

[0032] Next, a determination method according to this embodiment will be described.

[0033] To summarize with reference to Figures 2, 3, 4, 5, and 6, the determination method of this embodiment includes the steps of identifying the contour S of the cell 200 to be determined and multiple spaced-apart nuclei 310 within the contour S of the cell 200 to be determined, calculating the angle of the long side of each nucleus 310, comparing the angles, and determining that the cell 200 having the contour S is a multinucleated cell if the angle difference θg is within a predetermined range.

[0034] Specific work content (processing content) of each step will be described with reference to the flowchart shown in Fig. 2 and Figs. 3 to 6. In the following explanation, an example will be described in which each step is automatically performed by the determination system 100 shown in Fig. 1. However, in the determination method according to this embodiment, some steps, excluding the step of processing image data, can also be performed by the worker himself.

[0035] As shown in Fig. 2, a cell 200 to be determined is prepared (step S10). Preparing the cell 200 to be determined includes seeding the cell 200 on a culture substrate 120 and culturing it in a monolayer. The cell 200 to be determined can be prepared, for example, in a state where it is contained in the culture substrate 120, and set on the stage 110 together with the culture substrate 120 (see Fig. 1).

[0036] Next, the cell 200 to be determined is identified (step S11). Next, image data of the cell 200 to be determined is acquired (step S12). The image data can be acquired, for example, by capturing an image of the cell 200 to be determined set on the stage 110 with the imaging unit 150.

[0037] Before starting the determination method, the cell 200 to be determined can be stained. By staining the cell 200, the workability can be improved when identifying the outline S and the nucleus 310, as will be described later.

[0038] The determination method can also be performed based on image data previously acquired and stored. When the determination is performed based on image data previously acquired in this way, step S12 can be omitted.

[0039] Next, the detection unit 131 identifies a plurality of nuclei 310 spaced apart from one another within the contour S of the cell 200 to be determined based on the acquired image data (step S13). Figures 3 and 5 show examples of image data (or images displayed on the image display unit 160) for executing the processing of step S13.

[0040] 3 and 5 show examples of images (still images) of a cell 200 to be determined. The reference numerals 311 and 312 in each figure indicate different nuclei. Within an area A1 defined by the outline S of the cell 200 to be determined, cytoplasm 320 exists surrounding a nucleus 310. In this specification, the reference numeral 310 is used to refer to multiple nuclei or a single nucleus collectively without specifying them.

[0041] Next, the calculation unit 132 calculates the angle of the long side of the nucleus 310 of the cell 200 to be determined (step S14). The calculation unit 132 can calculate the angle using the method shown in FIG. 4 with reference to the image data shown in FIG. 3. The calculation unit 132 calculates an imaginary line H1 passing through a portion of the nucleus 311 with the maximum length L1, and defines a side (line segment) on the imaginary line H1 as the long side. In this case, the calculation unit 132 approximates the shape of the nucleus 311 to an ellipse, and defines a line segment having the maximum straight line length L1 along the major axis of the ellipse as the long side. Similarly, the calculation unit 132 defines a line segment having the maximum length L2 as the long side of a nucleus 312 that exists in one area A1 together with the nucleus 311.

[0042] After calculating the long sides and their angles (orientations) of each of the nuclei 311 and 312, the calculation unit 132 calculates the angular difference θg between the long sides. Specifically, based on an imaginary line H1 along the long side of the nucleus 311 and an imaginary line H2 along the long side of the nucleus 312, the calculation unit 132 calculates the angular difference θg between the two. If the angular difference θg is within a predetermined range (predetermined threshold) (step S15: YES), the determination unit 133 determines that the cell 200 to be determined is a multinucleated cell (step S16), and ends the determination method.

[0043] The angular difference θg is, for example, 0° to 10°, preferably 0° to 5°. If the angular difference θg is greater than 10° (step S15: NO), the cell 200 to be determined is not a multinucleated cell because multiple cells have not fused together and are separated into individual cells. If determined in this way, the cell 200 to be determined is determined not to be a multinucleated cell (step S17), and the determination method is terminated. On the other hand, if the angular difference θg is 10° or less (step S15: YES), the cell 200 to be determined is in a state where multiple cells have fused together, and therefore a final determination is made that it is a multinucleated cell, and the determination method is terminated.

[0044] For example, in the image data shown in FIG. 3, the angular difference θg is approximately 14°. Therefore, when determination is made using the image data shown in FIG. 3, the cell 200 to be determined is determined not to be a multinucleated cell. On the other hand, when determination is made using the image data shown in FIG. 5 based on the angular difference θg between the nuclei as shown in FIG. 6, the virtual lines H1 and H2 along the respective long sides of the nucleus 311 and the nucleus 312 are calculated to be approximately parallel. Therefore, the angular difference θg between the long sides of each nucleus 311 and 312 is calculated to be approximately 0°. Therefore, when determination is made using the image data shown in FIG. 5, the cell 200 to be determined is determined to be a multinucleated cell.

[0045] As described above, the determination method according to this embodiment includes the steps of identifying the contour S of the cell 200 to be determined and a plurality of spaced-apart nuclei 310 within the contour S of the cell 200, calculating the angle of the long side of each nucleus 310, comparing the angles, and determining that the cell 200 having the contour S is a multinucleated cell if the angle difference θg is within a predetermined range.

[0046] In addition, the determination system 100 according to this embodiment includes a detection unit 131 that identifies the contour S of the cell 200 to be determined and multiple spaced-apart nuclei 310 within the contour S of the cell 200, a calculation unit 132 that calculates the angle of the long side of each nucleus 310, and a determination unit 133 that compares the angles calculated by the calculation unit 132 and determines that the cell 200 having the contour S is a multinucleated cell if the angle difference θg is within a predetermined range.

[0047] According to the above-described determination method and determination system 100, a parameter related to the angle of the long side of the nucleus 310 of the cell 200 to be determined is calculated, and this parameter is used to determine whether the cell 200 to be determined is a multinucleated cell. By using such a parameter, it is possible to more accurately determine whether the cell to be determined is a multinucleated cell.

[0048] Although the method for assessing multinucleated cells and the system for assessing multinucleated cells according to the present invention have been described through the embodiments, the present invention is not limited to the configurations described in the embodiments, and can be modified as appropriate based on the claims. [Explanation of symbols]

[0049] 100 Judgment System 120 Culture substrate 130 Control Unit 131 Detector 132 Calculation Unit 133 Judgment section 150 Imaging unit 160 Image display unit 200 Cells to be judged 310 Nuclear 311 Nuclear 312 Nuclear 320 Cytoplasm S contour A1 area H1 Virtual line H2 Imaginary line θg Angle difference

Claims

1. A method for determining multinucleated cells, comprising: identifying a cell outline and a plurality of spaced apart nuclei within the cell outline; calculating the angle of the long side of each nucleus; comparing the angles; and determining that the cell having the contour is a multinucleated cell if the angle difference is within a predetermined range.

2. The method for determining multinucleated cells described in claim 1, wherein when calculating the angle of the long side of each of the multiple nuclei, the shape of the nuclei is approximated to an ellipse, and a virtual straight line along the long axis of the ellipse is defined as the long side.

3. 3. The method for determining multinucleated cells according to claim 1, further comprising the step of preparing an image showing the stained cells before determining the multinucleated cells.

4. A system for determining multinucleated cells, comprising: a detector for identifying a cell outline and a plurality of spaced apart nuclei within the cell outline; a calculation unit that calculates the angle of the long side of each nucleus; A system for identifying multinucleated cells, comprising: a determination unit that compares the angles calculated by the calculation unit and determines that the cell having the contour is a multinucleated cell if the angle difference is within a predetermined range.

5. The system for assessing multinucleated cells described in claim 4, wherein when calculating the angle of the long side of each of the multiple nuclei, the calculation unit approximates the shape of the nuclei to an ellipse and defines the long side as a virtual straight line along the long axis of the ellipse.

6. The system for determining multinucleated cells according to claim 4 or 5, wherein the detection unit, the calculation unit, and the determination unit each perform their respective processes based on image data of the stained cells.

7. an imaging unit for acquiring the image data; The system for determining multinucleated cells according to claim 6 , further comprising: an image display unit capable of displaying an image of the cells generated from the image data.

Images