Cell sample measurement method and cell sample measurement device

Abstract

A cell sample measurement device (1A) is provided with: a sample placement table (18) on which a target cell sample (S) containing a plurality of cells cultured on a culture dish (13) is placed; an evaluation light irradiation unit (20) that irradiates evaluation light having a mid-infrared region wavelength from an irradiation light source (21) at different irradiation amounts to each of N irradiation regions set by dividing the culture region in the target cell sample (S); a dead cell count measurement unit (30) that measures the number of dead cells generated on the basis of the amount of irradiation of the evaluation light for each of the N irradiation regions of the target cell sample (S); and a culture state analysis unit (50) that obtains, for the target cell sample (S), a target correlation between the number of dead cells and the irradiation amount of evaluation light in N irradiation regions that are indexes for evaluating the culture state of the target cell sample (S). As a result, it is possible to appropriately measure the culture state of a cell sample containing a plurality of cells and to achieve a cell sample measurement method and a cell sample measurement device.

Description

Technical Field

[0001] The present invention relates to a cell sample measuring method and a cell sample measuring device for measuring the culture state of a cell sample containing a plurality of cells cultured on a culture dish. Background Art

[0002] The technology of culturing cells is important not only in basic research on cells but also in practical fields such as regenerative medicine.

[0003] In the culture of cells, in order to further proliferate cell lines and cell strains, the culture medium is removed from the cell culture system and the cultured cells are transferred to a new culture medium, which is called a passaging operation. In addition, in a cell sample in which multiple cells are cultured, the number of times the passaging operation is performed is called the passaging number.

[0004] Patent Document 1: Japanese Patent Application Publication No. 2019-150018

[0005] Patent Document 2: Japanese Patent Application Publication No. 2018-130093

[0006] Non-patent literature 1: P.Hughes et al., "The costs of using unauthenticated, over-passaged cell lines: how much more data do we need?", Bio Techniques Vol.43(2007), pp.575-586

[0007] Non-patent document 2: E.Larson et al., "Mid-infrared absorption by soft tissuesarcoma and cell ablation utilizing a mid-infrared interband cascade laser", J.Biomed.Opt.Vol.26(2021), pp.043012-1-043012-10

[0008] Non-patent document 3: N. Malpica et al., "Applying Watershed Algorithms to the Segmentation of Clustered Nuclei", Cytometry Vol. 28 (1997), pp. 289-297 Summary of the invention

[0009] In a cell sample in which a plurality of cells are cultured as described above, the number of passages or the conditions of each passage operation, etc., affect the quality and culture state of the cells proliferating in the cell sample (for example, see Patent Document 1 and Non-Patent Document 1). That is, in the passage operation of the cell sample, the cultured cells are often damaged or stressed by being transferred to a new environment, thereby deteriorating the quality and state of the cells.

[0010] Generally speaking, it is known that the quality of cultured cells deteriorates due to changes in differentiation and proliferation ability, morphology, genes, etc. as the number of passages increases. Therefore, the culture status of a cell sample, such as the number of passages and passage conditions, is very important information for experiments and measurements using the cell sample. However, methods for measuring and evaluating the culture status of cells in such a cell sample have not been established to date.

[0011] An object of the present invention is to provide a cell sample measuring method and a cell sample measuring device capable of appropriately measuring the culture state of a cell sample containing a plurality of cells.

[0012] The inventors of the present application have studied the effects of light irradiation of laser light supplied from a quantum cascade laser (QCL) on the activation of cells and the like on a cell sample in which a plurality of cells are cultured. In studying the effects of light irradiation on such cell samples, it was found that dead cells are generated if light irradiation is performed at an irradiation dose above a certain level. Furthermore, a detailed investigation of cell death caused by such light irradiation was conducted, and it was found that the generation of dead cells depends on the culture state of the cell sample, such as the number of passages, and the present invention was completed.

[0013] An embodiment of the present invention is a cell sample measurement method. The cell sample measurement method includes: (1) a preparation step of placing a target cell sample of a measurement object including a plurality of cells cultured on a culture dish on a sample stage; (2) an evaluation light irradiation step of irradiating N irradiation areas (N is an integer greater than or equal to 2) which are set by distinguishing the culture area of ​​the plurality of cells in the target cell sample with evaluation light having a wavelength in the mid-infrared region supplied from an irradiation light source at different irradiation amounts; (3) a dead cell count measurement step of measuring the number of dead cells generated according to the irradiation amount of the evaluation light for each of the N irradiation areas of the target cell sample; and (4) a culture state analysis step of obtaining a target correlation between the irradiation amount of the evaluation light for the N irradiation areas and the number of dead cells, the target correlation becoming an evaluation index of the culture state of the target cell sample.

[0014] An embodiment of the present invention is a cell sample measuring device. The cell sample measuring device includes: (1) a sample placing table on which a target cell sample of a measurement object including a plurality of cells cultured on a culture dish is placed; (2) an evaluation light irradiation unit, which irradiates N irradiation areas (N is an integer greater than or equal to 2) which are set by distinguishing the culture areas of the plurality of cells in the target cell sample, with evaluation light having a wavelength in the mid-infrared region supplied from an irradiation light source at different irradiation amounts; (3) a dead cell count measuring unit, which measures the number of dead cells generated according to the irradiation amount of the evaluation light for each of the N irradiation areas of the target cell sample; and (4) a culture state analyzing unit, which obtains a target correlation between the irradiation amount of the evaluation light in the N irradiation areas and the number of dead cells for the target cell sample, and the target correlation becomes an evaluation index for the culture state of the target cell sample.

[0015] In the cell sample measurement method and the cell sample measurement device described above, in the measurement of the target cell sample including a plurality of cells cultured on a single culture dish, the evaluation light having a wavelength in the mid-infrared region is used to irradiate the N irradiation areas set to distinguish the culture area in the target cell sample with different irradiation amounts, and the number of dead cells generated according to the irradiation amount of the evaluation light is measured for each of the N irradiation areas. Then, based on the measurement results, the target correlation between the irradiation amount of the evaluation light and the number of dead cells for the N irradiation areas is obtained for the target cell sample.

[0016] According to such a structure, for an object cell sample in which multiple cells are cultured on a culture dish, multiple irradiation areas are set to distinguish the culture area, and each irradiation area is irradiated with evaluation light at a different irradiation amount, thereby appropriately measuring the object correlation between the irradiation amount of evaluation light and the number of dead cells, which can be used as an evaluation indicator of the culture state of the cells in the object cell sample.

[0017] According to the cell sample measuring method and the cell sample measuring device of the present invention, it is possible to appropriately measure the culture state of a cell sample containing a plurality of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram schematically showing the structure of one embodiment of a cell sample measuring device.

[0019] Figure 2 Yes means Figure 1 FIG. 2 is a block diagram showing an example of the configuration of a culture state analysis unit in a cell sample measuring device shown in FIG.

[0020] Figure 3A and Figure 3B This is a diagram showing an example of setting the irradiation amount of evaluation light supplied from the irradiation light source.

[0021] Figure 4 This is a graph showing an example of setting the irradiation power of the evaluation light supplied from the irradiation light source.

[0022] Figure 5 This is a diagram showing an example of a plurality of irradiation regions set by partitioning the culture region in a target cell sample.

[0023] Figure 6 It means that Figure 5 A diagram showing irradiation with evaluation light at irradiation points set in a plurality of irradiation areas is shown.

[0024] Figure 7 Yes means Figure 1 FIG. 2 is a diagram showing a configuration of a first modified example of the evaluation light irradiation unit in the cell sample measurement device.

[0025] Figure 8 Yes means Figure 1 FIG. 2 is a diagram showing a configuration of a second modified example of the evaluation light irradiation unit in the cell sample measurement device.

[0026] Fig. 9 This is a diagram schematically showing a fluorescent image of a target cell sample stained with a fluorescent dye that selectively stains dead cells.

[0027] Fig.10 Yes means Figure 1 FIG. 1 is a diagram showing an example of the configuration of a dead cell count measuring unit in a cell sample measuring device.

[0028] Fig.11 This is a diagram showing an example of a fluorescent image generated by irradiating a cell sample stained with a fluorescent dye with excitation light.

[0029] Figures 12A to 12C It is a graph showing the correlation between the irradiation time of the evaluation light and the number of dead cells for each of the cell samples at passage numbers 45, 53, and 55.

[0030] Fig.13 It is a graph showing the correlation between the irradiation time of the evaluation light and the number of dead cells for each of the cell samples at passage numbers 45, 53, and 55.

[0031] Fig.14A and Fig. 14B This is a graph showing the relationship between the slope of an approximate straight line for evaluating the correlation between the light irradiation time and the number of dead cells and the number of passages of the cell sample.

[0032] Fig.15A and Fig. 15BThis is a graph showing the relationship between the number of dead cells at a certain irradiation time and the number of passages of the cell sample in the correlation between the irradiation time of evaluation light and the number of dead cells.

[0033] Fig.16A and Fig. 16B It is a graph showing the correlation between the irradiation time and irradiation power of the evaluation light and the number of dead cells.

[0034] Fig.17 It is a graph showing the correlation between the irradiation time and irradiation power of the evaluation light and the number of dead cells.

[0035] Fig.18 It is a graph showing the correlation between the irradiation time and irradiation power of the evaluation light and the number of dead cells. DETAILED DESCRIPTION

[0036] The following, with the attached Figure 1 The embodiment of the cell sample measuring method and the cell sample measuring device will be described in detail. In the description of the drawings, the same reference numerals are used for the same elements, and repeated descriptions are omitted. In addition, the dimensional ratios of the drawings are not necessarily consistent with the dimensional ratios of the description.

[0037] Figure 1 1 is a diagram schematically showing the structure of one embodiment of a cell sample measuring device. The cell sample measuring device 1A of this embodiment is a measuring device for measuring the culture state of cells in a target cell sample S containing a plurality of cells of the same type cultured in a culture area on a single culture dish 13, which is a culture container, and is composed of a sample mounting table 18, an evaluation light irradiation unit 20, a dead cell count measuring unit 30, and a control unit 40. The target cell sample S to be measured by the measuring device 1A is, for example, a cell sample whose culture state, such as the number of passages, is unknown. Alternatively, a reference cell sample whose culture state is known may be used as the target cell sample S to be measured.

[0038] exist Figure 1 In the target cell sample S shown in FIG. 1 , a plurality of cells 10 are cultured in a culture region on a culture dish 13 together with a culture solution 11. Figure 1 In FIG. 1 , for the sake of simplicity of illustration, a single cell 10 in a culture solution 11 is shown for a plurality of cells 10 of a target cell sample S. FIG.

[0039] The target cell sample S is placed on the sample stage 18. In the present embodiment, the sample stage 18 is arranged parallel to the xy plane orthogonal to the z axis when the irradiation optical axis of the evaluation light of the evaluation light irradiation unit 20 is set to the z axis. In addition, the sample stage 18 is configured to be movable in the xy plane orthogonal to the irradiation optical axis of the evaluation light. In addition, generally speaking, the sample stage 18 only needs to be configured to be arranged parallel to the plane intersecting the irradiation optical axis of the evaluation light and to be movable in the plane.

[0040] In addition, a stage driving unit 19 is provided with respect to the above-mentioned sample stage 18. The stage driving unit 19 sets and controls the irradiation position of the evaluation light in the culture area of ​​the target cell sample S by driving the sample stage 18 on which the target cell sample S is mounted in the xy plane (in the plane intersecting the irradiation light axis). In addition, the driving mechanism of the sample stage 18 can be configured using, for example, a stepping motor, a piezoelectric actuator, or the like.

[0041] The evaluation light irradiation unit (evaluation light irradiation device) 20 is disposed below the sample stage 18 on which the target cell sample S is placed, and is optically coupled to the bottom surface 14 of the culture dish 13 in the target cell sample S via an opening provided at a predetermined position of the sample stage 18. In the present embodiment, the evaluation light irradiation unit 20 is configured to include an irradiation light source 21, a reflector 22, and an objective lens 23.

[0042] The evaluation light irradiation unit 20 irradiates the irradiation area set in the culture area of ​​the plurality of cells 10 in the target cell sample S with the evaluation light of a predetermined wavelength in the mid-infrared region under predetermined irradiation conditions. The evaluation light supplied from the irradiation light source 21 is reflected by the reflector 22, and irradiates the plurality of cells 10 included in the target cell sample S via the objective lens 23 and the bottom surface 14 of the culture dish 13 along a predetermined irradiation optical axis.

[0043] Specifically, the evaluation light irradiation unit 20 divides the culture area of ​​the plurality of cells 10 on the culture dish 13 in the target cell sample S and sets N irradiation areas (a plurality of irradiation areas, N is an integer greater than or equal to 2). Then, the evaluation light supplied from the irradiation light source 21 is irradiated to each of the N irradiation areas at a different irradiation amount.

[0044] The dead cell count measuring section (dead cell count measuring device) 30 is arranged above the sample mounting table 18 on which the target cell sample S is mounted. The dead cell count measuring section 30 measures the number of dead cells generated according to the irradiation amount of the evaluation light of the evaluation light irradiation section 20 for the irradiation area of ​​the target cell sample S. As described above, when N irradiation areas are set in the culture area with respect to the target cell sample S, the dead cell count measuring section 30 measures the number of dead cells generated according to the irradiation amount of the evaluation light for each of the N irradiation areas of the target cell sample S. The specific structure of the dead cell count measuring section 30 is appropriately set according to the method of measuring the number of dead cells in the dead cell count measuring section 30.

[0045] In addition, Figure 1 In the structure of FIG. 1 , the structure in which the evaluation light irradiation section 20 and the dead cell count measurement section 30 are integrally provided including the sample stage 18 in the measurement device 1A is shown, but the present invention is not limited to such a structure. For example, the dead cell count measurement section 30 may be provided separately from the evaluation light irradiation section 20. In this case, the sample stage in the dead cell count measurement section 30 is provided separately from the sample stage 18 in the evaluation light irradiation section 20. In addition, the irradiation conditions of the evaluation light of the evaluation light irradiation section 20, the structure of the dead cell count measurement section 30, and the method of measuring the number of dead cells are described in detail later.

[0046] The control unit 40 controls the operation of each unit of the sample mounting table 18, the evaluation light irradiation unit 20, and the dead cell count measurement unit 30 in the measurement device 1A, and performs necessary analysis on the measurement results of the evaluation light irradiation unit 20 and the dead cell count measurement unit 30. In the present embodiment, the control unit 40 is configured to include a measurement control unit 41 and a culture state analysis unit 50.

[0047] The measurement control unit (measurement control device) 41 controls the driving operation of the sample stage 18 by the stage driving unit 19 and the irradiation operation of the evaluation light to the target cell sample S by the evaluation light irradiation unit 20 (for example, the supply operation of the evaluation light by the irradiation light source 21). In addition, when the dead cell count measuring unit 30 and the evaluation light irradiation unit 20 are provided integrally, the measurement control unit 41 may also be configured as follows. Figure 1 As indicated by the middle dotted arrow, the dead cell count measuring unit 30 is also controlled to measure the number of dead cells in the target cell sample S.

[0048] The culture state analysis unit 50 is a culture state analysis device that analyzes the culture state of the target cell sample S containing a plurality of cells 10. Figure 1In the structure of FIG. 5 , the culture state analysis unit 50 is provided with an input unit 42 and a display unit 43. The input unit 42 is used to input information required for analyzing the culture state of the target cell sample S. The display unit 43 is used to display the analysis conditions of the culture state of the target cell sample S, the analysis results, the measurement results of the evaluation light irradiation unit 20 and the dead cell count measurement unit 30, and the like.

[0049] In the control unit 40, each of the measurement control unit 41 and the culture status analysis unit 50 can be constituted by, for example, a computer having a CPU as a processing unit and a ROM, RAM, and an external storage device as a storage unit. The measurement control unit 41 and the culture status analysis unit 50 can be constituted by different computers or a single computer. In addition, when the measurement control unit 41 and the culture status analysis unit 50 are separately provided, the measurement control unit 41 can also be configured to be provided with an input unit and a display unit in the same manner as the culture status analysis unit 50.

[0050] Figure 2 Yes means Figure 1 1 is a block diagram showing an example of the structure of the culture state analysis unit 50 in the cell sample measurement device 1A. The culture state analysis unit (culture state analysis device) 50 of this structure example is configured to include a dead cell count calculation unit 51, an object correlation generation unit 52, a reference correlation database 53, a culture state evaluation unit 54, and a reference correlation generation unit 55.

[0051] In addition, this configuration example shows the configuration of the culture state analysis unit 50 in the case where the cell sample measurement device 1A is configured as a cell sample evaluation device that performs measurement on the culture state of cells in the target cell sample S and also performs evaluation of the culture state of the target cell sample S. In the case where the culture state of the target cell sample S is not evaluated but only measured, it is not necessary to provide the reference correlation database 53, the culture state evaluation unit 54, and the reference correlation generation unit 55.

[0052] The dead cell count calculation unit 51 calculates the number of dead cells generated in the irradiation region of the target cell sample S based on the dead cell count measurement data input from the dead cell count measurement unit 30. When N irradiation regions are set for the target cell sample S, the dead cell count calculation unit 51 calculates the number of dead cells generated for each of the N irradiation regions. In addition, when the number of dead cells is directly obtained from the measurement data obtained by the dead cell count measurement unit 30, it is not necessary to provide the dead cell count calculation unit 51.

[0053] The object correlation generating unit 52 obtains the object correlation between the irradiation amount of the evaluation light and the number of dead cells for the target cell sample S based on the measurement result including the irradiation data of the evaluation light for the irradiation area input from the evaluation light irradiation unit 20 or the measurement control unit 41 and the measurement data of the number of dead cells input from the dead cell number measuring unit 30 or the dead cell number calculating unit 51. When N irradiation areas are set for the target cell sample S, the object correlation generating unit 52 obtains the object correlation between the irradiation amount of the evaluation light and the number of dead cells for the N irradiation areas. Such an object correlation becomes an index for evaluating the culture state of the cells in the target cell sample S.

[0054] The reference correlation database 53 is a reference correlation storage unit that stores reference correlations between the irradiation amount of evaluation light and the number of dead cells for reference cell samples with known cell culture states. When a single reference cell sample is assumed, the reference correlation database 53 stores a single reference correlation. When a plurality of reference cell samples with different culture states are assumed, the reference correlation database 53 stores a plurality of reference correlations for these reference cell samples.

[0055] The culture state evaluation unit 54 compares the reference correlation stored in the reference correlation database 53 with the target correlation obtained by the target correlation generation unit 52, and based on the comparison result, evaluates the culture state of the cells in the target cell sample S. When a plurality of reference correlations are stored in the reference correlation database 53, the culture state evaluation unit 54 evaluates the culture state of the cells in the target cell sample S by comparing each of the plurality of reference correlations with the target correlation.

[0056] exist Figure 2 In the illustrated configuration example, in the culture state analysis unit 50, a reference correlation generation unit 55 is provided in addition to the reference correlation database 53. Similar to the target cell sample, the reference correlation generation unit 55 obtains the reference correlation between the irradiation amount of the evaluation light and the number of dead cells for the reference cell sample based on the measurement results of the evaluation light irradiation unit 20 and the dead cell number measurement unit 30. In addition, when the reference correlation is acquired in advance and stored in the reference correlation database 53, it is not necessary to provide the reference correlation generation unit 55.

[0057] Depend on Figure 1 , Figure 2The cell sample measuring method performed by the cell sample measuring device 1A shown includes a preparation step, an evaluation light irradiation step, a dead cell count measurement step, and a culture state analysis step. In the preparation step, a target cell sample S including a plurality of cells 10 cultured on a single culture dish 13 is placed on a sample mounting table 18. In the evaluation light irradiation step, the evaluation light irradiation unit 20 irradiates the target cell sample S with evaluation light supplied from the irradiation light source 21 at different irradiation amounts to each of N irradiation areas set by dividing the culture area.

[0058] In the dead cell count measurement step, the dead cell count measurement unit 30 measures the number of dead cells generated according to the irradiation amount of the evaluation light for each of the N irradiation areas of the target cell sample S irradiated with the evaluation light by the evaluation light irradiation unit 20. In the culture state analysis step, the culture state analysis unit 50 obtains the target correlation between the irradiation amount of the evaluation light and the number of dead cells for the N irradiation areas, which is an evaluation index for the culture state of the target cell sample S. In addition, when evaluating the culture state of the target cell sample S, in the culture state analysis step, the culture state of the target cell sample S is evaluated by comparing the reference correlation for the reference cell sample with the above-mentioned target correlation.

[0059] The effects of the cell sample measurement device 1A and the cell sample measurement method according to the above-described embodiment will be described.

[0060] exist Figure 1 , Figure 2 In the measurement device 1A and the measurement method shown, in the measurement of the target cell sample S including the plurality of cells 10 cultured on the culture dish 13, the evaluation light having a wavelength in the mid-infrared region is used, and the evaluation light irradiation unit 20 irradiates the N irradiation regions set by dividing the culture region in the target cell sample S with different irradiation amounts, and the number of dead cells generated in each irradiation region is measured according to the irradiation amount of the evaluation light by the dead cell count measurement unit 30. And, based on the measurement result, the target correlation between the irradiation amount of the evaluation light and the number of dead cells for the N irradiation regions is obtained in the culture state analysis unit 50 for the target cell sample S.

[0061] According to such a configuration, for the target cell sample S in which a plurality of cells 10 are cultured on the culture dish 13, a plurality of irradiation areas are set in the culture region, and the evaluation light is irradiated at different irradiation amounts to each irradiation area, thereby appropriately measuring the target correlation between the irradiation amount of the evaluation light and the number of dead cells, which can be used as an evaluation index of the culture state of the cells in the target cell sample S. In addition, the number N of irradiation areas divided into the culture region is preferably appropriately set according to the specific content of the acquired target correlation.

[0062] In addition, in non-patent document 2 (E. Larson et al., J. Biomed. Opt. Vol. 26 (2021), pp. 043012-1-043012-10), it is recorded that a laser in the mid-infrared region is irradiated onto a cell sample to investigate the threshold for the generation of dead cells. However, in non-patent document 2, there is no record or suggestion that there is a correlation between the generation of dead cells in a cell sample and the culture state such as the number of passages of the cell sample.

[0063] In the measurement device 1A and the measurement method of the above-mentioned embodiment, it can also be configured that in the evaluation light irradiation unit 20, a plurality of irradiation points for irradiating the evaluation light are set for each of the N irradiation areas, and the dead cell count measurement unit 30 measures the number of dead cells at each of the plurality of irradiation points for each of the N irradiation areas.

[0064] In this way, by setting a plurality of irradiation points in each of the N irradiation areas set in the culture area, irradiating the evaluation light and measuring the number of dead cells respectively, the object correlation between the irradiation amount of the evaluation light and the number of dead cells can be appropriately measured with high accuracy.

[0065] In addition, when a plurality of irradiation points are set in each irradiation area as described above, the evaluation light irradiation unit 20 may be configured such that all irradiation points set as N irradiation areas are arranged in a grid pattern in the culture area, and the evaluation light is sequentially irradiated to these irradiation points. In addition, the culture state analysis unit 50 may also obtain the object correlation between the irradiation amount of the evaluation light in each irradiation area and the average value of the number of dead cells at the plurality of irradiation points in each irradiation area for the N irradiation areas. The setting of such irradiation areas and irradiation points will be described in detail later.

[0066] In addition, Figure 1 , Figure 2 In the measurement device 1A and the measurement method shown, the evaluation light having a wavelength in the mid-infrared region is used, the evaluation light is irradiated to the irradiation area set in the target cell sample S by the evaluation light irradiation unit 20, and the number of dead cells generated in the irradiation area is measured according to the irradiation amount of the evaluation light by the dead cell number measurement unit 30. Then, based on the measurement result, the target correlation between the irradiation amount of the evaluation light and the number of dead cells is obtained for the target cell sample S in the culture state analysis unit 50, and the reference correlation prepared for the reference cell sample and the target correlation are compared, thereby evaluating the culture state of the cells of the target cell sample S. In this case, the measurement device 1A functions as an evaluation device as described above.

[0067] According to such a configuration, for the target cell sample S in which a plurality of cells 10 are cultured on the culture dish 13, the target correlation relationship between the irradiation amount of the evaluation light and the number of dead cells can be used as an index to appropriately evaluate the culture state of the cells in the target cell sample S. In addition, regarding the irradiation conditions of the evaluation light with respect to the irradiation area of ​​the target cell sample S, it is preferable to appropriately set the conditions so as to be able to obtain the target correlation relationship between the irradiation amount of the evaluation light and the number of dead cells used in the evaluation of the target cell sample S.

[0068] In the measurement device 1A and the measurement method of the above-described embodiment, the culture state evaluation unit 54 of the culture state analysis unit 50 preferably evaluates the culture state including at least one of the number of passages of the target cell sample S, the passage conditions (conditions of the passage operation), and the culture conditions between passages as the culture state of the cells in the target cell sample S. In this case, the object correlation relationship obtained by the object correlation relationship generation unit 52 is used as an evaluation index of the culture state including at least one of the number of passages of the target cell sample S, the passage conditions, and the culture conditions between passages.

[0069] In this way, for the target cell sample S, by using the above-mentioned target correlation between the irradiation amount of evaluation light and the number of dead cells as an evaluation index, the culture state such as the passage number of the target cell sample S can be appropriately evaluated. In addition, when the passage conditions and the culture conditions between passages are considered to be relatively constant, when the information of the passage number is considered to be important, etc., it is also possible to configure so that the passage number is specifically evaluated as the culture state of the cells in the target cell sample S. In this case, the target correlation is used as an evaluation index for the passage number of the target cell sample S.

[0070] In the measuring device 1A and the measuring method of the above-mentioned embodiment, as described above, the benchmark correlation database 53 of the culture status analysis unit 50 can store multiple benchmark correlations about multiple benchmark cell samples with different culture statuses, and the culture status evaluation unit 54 evaluates the culture status of the object cell sample S by comparing each of the multiple benchmark correlations with the object correlation.

[0071] In this way, the reference correlation database 53 prepares a plurality of reference correlations for a plurality of reference cell samples whose culture states are known and different from each other, and by comparing these reference correlations with the target correlation, the culture state of the target cell sample S can be evaluated in more detail and with higher accuracy.

[0072] In the measuring device 1A and the measuring method of the above-mentioned embodiment, the culture status evaluation unit 54 of the culture status analysis unit 50 may also be configured to compare, in the comparison between the baseline correlation and the object correlation, at least one of the parameters of the approximate straight line or the approximate curve representing the correlation, the number of dead cells under a certain irradiation dose, and the threshold irradiation dose for the generation of dead cells.

[0073] In this way, regarding the correlation between the evaluation light irradiation amount and the number of dead cells, specifically, by using the parameters of the approximate straight line or approximate curve representing the correlation, the number of dead cells under a certain irradiation amount, and at least one of the threshold irradiation amounts for the generation of dead cells, the baseline correlation and the object correlation are compared, thereby enabling the culture state of the object cell sample S to be appropriately evaluated.

[0074] In the measuring device 1A and the measuring method of the above-mentioned embodiment, as described above, the culture status analyzing unit 50 may also be configured to have a baseline correlation generating unit 55, which obtains the baseline correlation between the irradiation amount of the evaluation light and the number of dead cells based on the measurement results of the evaluation light irradiation unit 20 and the dead cell number measuring unit 30 for the baseline cell sample.

[0075] In this way, by configuring the reference cell sample to obtain the reference correlation between the irradiation amount of the evaluation light and the number of dead cells using the same device and method as the target cell sample S to be measured, it is possible to appropriately achieve the evaluation of the culture state of the target cell sample S based on the comparison of the reference correlation and the target correlation. In addition, the reference cell sample may also be obtained using a device different from that of the target cell sample S. In this case, the measurement conditions such as the irradiation conditions of the evaluation light need to be consistent in the reference cell sample and the target cell sample. In addition, the reference correlation may also be obtained in advance and stored in the reference correlation database 53.

[0076] The configuration of the measurement device 1A and the measurement method of the above-described embodiment will be further described. First, the target cell sample S to be measured in a culture state and the types of cells contained in the cell sample will be described.

[0077] The cells used in the research and experiments related to cells are roughly divided into cell lines and primary culture cells. Cell lines are cells that can proliferate indefinitely without being restricted by the cell cycle. In addition, primary culture cells are cells that are directly collected from tissues and cultured. Usually, if primary culture cells repeatedly divide, cell division stops with a limited number of times due to the Hayflick limit. However, in this primary culture, chromosomal abnormalities are occasionally caused, and cells that proliferate indefinitely sometimes appear. This is the so-called immortalization of cells, and such cells are called cell lines.

[0078] In addition, from the perspective of the culture method, cells used in research and the like are roughly divided into adherent cells and floating cells. Adhesive cells proliferate while adhering to the culture container, but floating cells proliferate while floating in the culture medium. It is considered that the cell sample evaluation performed by the above-mentioned evaluation device 1A is difficult to apply to a cell sample containing floating cells in terms of the optical system, and it is preferable to use a cell sample containing adherent cells as the target cell sample S to be evaluated.

[0079] As examples of cell lines, mammalian cell lines such as CHO, HeLa, MCF-7, 293, 3T3, and COS are generally known and widely used in molecular biology research, biochemistry research, etc. In addition, in the examples of cell sample measurement described later, C6 cells, which are cell lines derived from rat glioma, are used. In addition, as described above, it has been a long time since the well-known cell lines were established, so in many cases, the number of passages is unknown.

[0080] In addition, stem cells such as embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), embryonic carcinoma cells (EC cells), and mesenchymal stem cells are more difficult to culture than general cell lines, and require unique know-how and technology for stable culture. In the use of such stem cells, accurate control of the culture status such as the number of passages is also an important factor.

[0081] On the other hand, primary cultured cells accurately reproduce the physiological state of cells in vivo, so appropriate data representing the state of the organism can be obtained and used in preclinical research, biological research, etc. However, since primary cultured cells themselves are very fragile, sufficient care is required when measuring cell samples using the above-mentioned measurement device 1A.

[0082] In addition, in the target cell sample S, the plurality of cells 10 cultured on the culture dish 13 are preferably evenly distributed in the culture region. In practice, it is preferred to confirm that the cultured cells on the culture dish 13 are 100% confluent, that is, that the adhered cells cover the adhesive surface of the culture dish 13, and then perform necessary measurements and evaluations on the target cell sample S.

[0083] The culture dish 13 used for culturing cells in the target cell sample S is described below. Figure 1 In the structure shown, the evaluation light irradiation unit 20 irradiates the target cell sample S with evaluation light from the bottom surface 14 side of the culture dish 13. In such a structure, in order to prevent the mid-infrared light used as the evaluation light from being attenuated before reaching the cell 10, it is preferable to use a culture dish 13 having a bottom surface 14 made of a material with high transmittance to the mid-infrared light.

[0084] Specifically, for example, for various commercially available culture dishes, the infrared absorption spectrum of each bottom surface can be measured, and a culture dish with high transmittance to the evaluation light can be selected. In the embodiment described below, a film-bottom culture dish (trade name: lumox dish) manufactured by Sarstedt K.K. is used as the culture dish 13. The culture dish has a polymer film laid on the bottom surface.

[0085] As the material of the bottom surface of the culture container, i.e., the culture dish, in addition to the above-mentioned polymer film, for example, silicon, germanium, calcium fluoride, barium fluoride, zinc selenide, zinc sulfide, chalcogenaide glass, etc., which are generally known to have high infrared transmittance, can be cited. In addition, regarding the culture dish, it is preferred to select a culture container with low water solubility and cytotoxicity.

[0086] The evaluation light used for irradiating the target cell sample S in the evaluation light irradiation unit 20 and the irradiation light source 21 for supplying the evaluation light are described. In the above-mentioned measuring device 1A, as the evaluation light, light having a predetermined wavelength in the mid-infrared region is used as described above. The energy of the mid-infrared light is consistent with the energy of the fundamental vibration of many molecules. Therefore, the mid-infrared light has a large absorption coefficient for molecules. The absorption coefficient of such mid-infrared light relative to molecules is, for example, several orders of magnitude larger than that of near-infrared light and visible light.

[0087] The light energy required to induce a change in the cell state by light absorption depends on the absorption coefficient of the target cell. Therefore, by using mid-infrared light as the evaluation light for the target cell sample S, it is possible to induce a change in the cell state with less irradiation energy than when using near-infrared light or visible light.

[0088] For example, when near-infrared light or visible light is used as the evaluation light, a large amount of light energy is required to induce a change in the cell state, so the heat caused by light irradiation may damage the cells or the culture environment around the irradiation point of the evaluation light. In addition, in near-infrared light and visible light, a large high-intensity laser light source is sometimes required as the irradiation light source 21, which may cause a problem of large-scale equipment.

[0089] In contrast, in a structure using mid-infrared light as evaluation light, energy can be directly imparted to the fundamental vibration of the cell molecules or water contained in the cell as the target so that it is absorbed, so that changes in the cell state can be selectively induced only in the target part with a small light energy. In this case, for example, a single longitudinal mode laser light source with a wavelength consistent with a specific absorption line, that is, a laser light source with a narrow oscillation spectrum band, can be appropriately used. In addition, in the evaluation light using mid-infrared light, since the light energy is small, even a compact semiconductor laser, for example, can induce changes in the cell state. In this case, the device can be miniaturized.

[0090] In addition, regarding the wavelength of the mid-infrared light used as the evaluation light, specifically, for example, the wavelength of the evaluation light is preferably set to a wavelength region of 3 μm or more and 20 μm or less in the evaluation light irradiation unit 20. Thus, the target correlation between the irradiation amount of the evaluation light and the number of dead cells, which is an evaluation index of the culture state of the target cell sample S, can be appropriately acquired.

[0091] In the above-mentioned wavelength band, for example, in the 3 μm wavelength band, water has a large absorption, and effective heating can be performed via water. In addition, in the wavelength range of 10 μm to 20 μm, there are absorption lines peculiar to relatively large molecules, such as substances containing benzene rings. In addition, for the long wavelength region exceeding 20 μm, it is considered difficult to assemble an appropriate light source in the above-mentioned measuring device 1A.

[0092] As the irradiation light source 21 for supplying the evaluation light in the evaluation light irradiation unit 20, for example, a quantum cascade laser (QCL) can be used. A quantum cascade laser is a monopole laser element that generates light by electron transitions between subbands using the energy level structure of the subbands formed in a semiconductor quantum well structure, and can achieve high-efficiency, high-output operation by cascading multiple light-emitting layers of the quantum well structure via injection layers.

[0093] When QCL is used in the irradiation light source 21, the irradiation light source 21 can be composed of a QCL element, a cooling mechanism and a collimating lens, for example. As a specific example of a QCL element, a DFB-QCL (model: L12004-2190H-C) with a wavelength of 4.6 μm manufactured by Hamamatsu Photonics can be used. For such an irradiation light source, in order to achieve a small focusing diameter, a horizontal single-mode light source, that is, a light source with high spatial coherence is preferably used. In addition, in the irradiation light source, in order to stabilize the wavelength and output of the evaluation light, it is preferred to perform temperature control based on TEC, for example. In addition, it can also be configured to include a secondary cooling mechanism based on a fan, water cooling, etc.

[0094] As for the collimating lens, it is necessary to use a lens made of a material that transmits the evaluation light of the wavelength of 3μm to 20μm mentioned above. As the material of such a lens, for example, ZnSe, Ge, CaF2, etc. can be listed. In addition, it is preferred to set an anti-reflection coating on both sides of the collimating lens in a manner such that the transmittance relative to the wavelength of the evaluation light is, for example, 95% or more. In addition, as a collimating lens, it is also possible to use a structure in which multiple lenses are used in combination, but in such a case, since the loss of the evaluation light increases, it is preferred to use a single aspherical lens that can correct spherical aberration.

[0095] In addition, regarding the light source element in the irradiation light source 21, in addition to the above-mentioned QCL, for example, a broadband laser (ICL: Interband Cascade Laser), a CO2 laser, a CO laser, a free electron laser, etc. can also be used. In addition, a wavelength conversion technology based on an optical parametric oscillator (OPO: Optical Parametric Oscillator) can also be used. In addition, when using QCL and ICL, from the viewpoint of being able to perform longitudinal single-mode oscillation, it is not limited to the above-mentioned DFB type structure, and an external resonator type structure can also be used. In addition, regarding the wavelength region of the evaluation light, for example, QCL can supply light with a wavelength of about 4μm to 20μm, and ICL can supply light with a wavelength of about 3μm to 7μm.

[0096] The evaluation light supplied from the irradiation light source 21 may be continuous light or pulse light. A burst pulse including a plurality of light pulses and having a range defined by an envelope of a predetermined shape may be used as the evaluation light.

[0097] The structure of the irradiation optical system used in the evaluation light irradiation unit 20 to irradiate the target cell sample S with the evaluation light is described. In the above-mentioned measurement device 1A, the evaluation light from the irradiation light source 21 is reflected by 90° by the reflector 22, and irradiated to the target cell sample S through the objective lens 23. In such a structure, by bending the optical path of the evaluation light, the height of the irradiation surface (action surface) can be suppressed to be low. As the reflector 22, for example, a gold vapor-deposited plane mirror, a high-reflection plane mirror based on a dielectric multilayer film, etc. can be used.

[0098] The structure of the irradiation optical system in the evaluation light irradiation section 20 is not limited to the above structure, and for example, a structure in which the irradiation light source 21 and the objective lens 23 are arranged in a straight line without using the reflector 22 may be adopted. In such a structure, the light emission position of the light source element in the irradiation light source 21, the center position of the collimator lens, and the center position of the objective lens 23 are coaxially arranged on the same straight line. In addition, in the dead cell count measurement section 30, when an objective lens is used in the observation of the optical image of the target cell sample S, the center position of the observation objective lens is also preferably arranged on the same straight line.

[0099] In this way, in a structure in which the components of the irradiation optical system are arranged in a straight line, the number of components used in the evaluation light irradiation unit 20 is reduced, and it is possible to reduce light loss and reduce the cost of the device. However, in this structure, since the optical path of the evaluation light is straight, it is necessary to pay attention to the fact that the height of the irradiation surface becomes higher.

[0100] In addition, Figure 1In the structure of the evaluation light irradiation unit 20 shown, the QCL element, cooling mechanism and collimating lens constituting the irradiation light source 21 can also be configured to be arranged on an optical table that can be integrally adjusted in position. In this case, the required accuracy in the manufacture of the device can be relaxed. In addition, similarly, an angle adjustment mechanism can also be provided for the reflector 22. In addition, an adjustment mechanism for the x-axis direction, the y-axis direction, and the z-axis direction can also be provided for the objective lens 23.

[0101] In addition, when a lens is provided in the irradiation optical system, it is preferred to select an appropriate material similar to the collimating lens in the irradiation light source 21, and anti-reflection coatings are provided on both sides of the lens. In addition, for the object lens 23, a reflective object lens may also be used. In addition, as the object lens 23, a single lens such as an aspheric lens or a plano-convex lens may be used instead of a structure combining multiple lenses. However, in order to take into account the narrow irradiation point of the evaluation light and a certain degree of focal depth, it is preferred to use an object lens combining multiple lenses.

[0102] The irradiation conditions of the evaluation light for the target cell sample S in the evaluation light irradiation unit 20 are described. The irradiation conditions of the evaluation light for the irradiation area of ​​the target cell sample S need to be set so as to obtain the target correlation between the irradiation amount of the evaluation light used in the evaluation of the target cell sample S and the number of dead cells. Specifically, as described above, the evaluation light irradiation unit 20 is configured to irradiate the evaluation light with different irradiation amounts for each of the N irradiation areas set to divide the culture area in the target cell sample S.

[0103] In addition, in the evaluation light irradiation unit 20, the irradiation amount of the evaluation light is preferably set based on an irradiation condition including at least one of the irradiation time, irradiation power, and focusing condition for the irradiation area of ​​the evaluation light. Thus, it is possible to appropriately achieve an irradiation condition that can obtain a target correlation between the irradiation amount of the evaluation light used in the evaluation of the target cell sample S and the number of dead cells by controlling the irradiation amount.

[0104] In the above structure, considering that the control and change of the focusing condition requires adjustment of the irradiation optical system, it is preferable to set the focusing condition constant, and set the irradiation amount of the evaluation light by controlling one or both of the irradiation time and the irradiation power to the irradiation area of ​​the evaluation light. In addition, when the focusing condition changes, the irradiation power density of the evaluation light relative to the target cell sample S changes, and the generation condition of dead cells changes. As a specific example, the setting of the irradiation amount when a burst pulse including a plurality of light pulses is used as the evaluation light is described below.

[0105] Figure 3A and Figure 3BIt is a diagram showing an example of setting the irradiation amount of the evaluation light supplied from the irradiation light source 21 . Figure 3A This is a diagram showing a burst pulse supplied from the irradiation light source 21 as evaluation light. Figure 3B Yes Figure 3A A portion of a burst pulse is magnified to show the optical pulse included in the burst pulse.

[0106] like Figure 3A As shown, the burst pulse includes a plurality of optical pulses P, which are configured as pulses whose range is defined by an envelope B of a predetermined shape. Figure 3A In the burst pulse shown, the envelope B has a rectangular shape, and the time width t0 of the envelope B is the irradiation time when the burst pulse is used as evaluation light.

[0107] In addition, if Figure 3B As shown, the pulse width of the plurality of optical pulses P included in the burst pulse is t1, and the repetition period of the optical pulse is t2. As such a burst pulse structure, for example, a structure in which the pulse width is set to t1 = 100 μs and the repetition period is set to t2 = 1 ms can be used. In this case, the repetition frequency of the optical pulses P in the burst pulse is 1 kHz.

[0108] In such a burst pulse, there are three methods for changing the average irradiation power that becomes the irradiation power of the evaluation light. In the first method, the injection current to the light source element and the repetition period t2 in the irradiation light source 21 are kept constant, and the pulse width t1 is changed. In the second method, the injection current to the light source element and the pulse width t1 are kept constant, and the repetition period t2 is changed. In the third method, the pulse width t1 and the repetition period t2 are kept constant, and the injection current to the light source element is changed. In the case of the third method, by changing the injection current to the light source element, the wave height value of the light pulse P constituting the burst pulse changes.

[0109] Figure 4 2 is a graph showing an example of setting the irradiation power of the evaluation light supplied from the irradiation light source 21. Figure 4 In the graph of , the horizontal axis represents the pulse width t1 (μs) of the light pulse P, and the vertical axis represents the average irradiation power (mW).

[0110] Here, the change in average irradiation power when the pulse width t1 of the light pulse P is changed under the condition that the temperature of the QCL in the irradiation light source 21 is set to 20°C, the injection current to the QCL is kept constant, and the repetition frequency of the light pulse P in the burst pulse is kept constant at 30kHz is shown. In this way, in a configuration using a burst pulse as evaluation light, the average irradiation power of the evaluation light can be controlled by changing any one of the injection current to the light source element, the pulse width of the light pulse P, and the repetition period (repetition frequency).

[0111] The following will further describe the irradiation conditions of the evaluation light to the target cell sample S in the evaluation light irradiation unit 20. In order to appropriately obtain the target correlation between the irradiation amount of the evaluation light to the target cell sample S and the number of dead cells, N irradiation areas (a plurality of irradiation areas) are set by dividing the culture area in the target cell sample S, and the evaluation light supplied from the irradiation light source 21 is irradiated to the N irradiation areas at different irradiation amounts for each irradiation area.

[0112] Figure 5 This is a diagram showing an example of N irradiation regions set by dividing the culture region in the target cell sample S. Here, an example is shown in which N=8 irradiation regions R1 to R8 are set in the culture region R0 of the target cell sample S. Among these irradiation regions R1 to R8, the irradiation region R1 is set as the outer peripheral region of the culture region R0. In addition, the irradiation regions R2 to R8 are set by dividing the inner part of the irradiation region R1 into approximately the same area.

[0113] In addition, Figure 5 In the illustrated configuration example, a plurality of irradiation points for irradiating the evaluation light are set for each of the eight irradiation regions R1 to R8 . Figure 6 It means relative to Figure 5 FIG. 1 is a diagram showing the irradiation of the evaluation light at the irradiation points set in the eight irradiation areas R1 to R8. Figure 5 and Figure 6 As shown, in this configuration example, 48 irradiation points P1 are set for the peripheral irradiation region R1. In addition, 16 irradiation points P2 to P8 are set for the inner irradiation regions R2 to R8, respectively. The number of irradiation points in the entire culture region R0 is 160.

[0114] In addition, in this structural example, all 160 irradiation points P1 to P8 set for the eight irradiation areas R1 to R8 are arranged in a grid pattern (grid pattern, two-dimensional array pattern) in the culture area R0, as shown in FIG. Figure 6 As shown by the arrow lines in the figure, the evaluation light is sequentially focused on these irradiation points. According to such a structure, the irradiation of the evaluation light with respect to the target cell sample S for obtaining the target correlation can be appropriately and efficiently performed. In addition, for the irradiation points P1 in the peripheral part, in order to clarify the grid of the irradiation area, it is preferable to set the irradiation intensity or irradiation time sufficient to reliably cause cell death.

[0115] In addition, in a structure in which a plurality of irradiation points in the target cell sample S are arranged in a grid pattern and irradiated with evaluation light in sequence, it is possible to easily change the irradiation position of the evaluation light by moving the sample stage 18 in the xy plane, and scan each irradiation point with the evaluation light based on the change. Figure 6 In the structural example shown, specifically, for the 16 scanning lines each including 10 irradiation points arranged in the vertical direction of the figure, the irradiation points are scanned from left to right in the odd-numbered scanning lines from the top, and the irradiation points are scanned from right to left in the even-numbered scanning lines.

[0116] According to such a structure, the moving distance of the sample stage 18 in the scanning of the evaluation light based on the plurality of irradiation points can be reduced. Generally speaking, with respect to the plurality of scanning lines arranged in a predetermined direction for scanning the plurality of irradiation points arranged in a grid pattern, it is preferable to configure such that the scanning of the irradiation points from left to right and the scanning of the irradiation points from right to left are performed alternately in the odd-numbered scanning lines and the even-numbered scanning lines, so that the scanning directions are alternately different.

[0117] As described above, in the target cell sample S, when a plurality of irradiation points are set in each irradiation region, the number of dead cells in each of the plurality of irradiation points is preferably measured for each of the N irradiation regions in the dead cell count measuring unit 30. In addition, in the target correlation generating unit 52 of the culture state analyzing unit 50, the target correlation between the irradiation amount of the evaluation light in each irradiation region and the average value of the number of dead cells at the plurality of irradiation points in each irradiation region is preferably obtained for the N irradiation regions. In this way, by using the average value of the number of dead cells at the plurality of irradiation points, the target correlation between the irradiation amount of the evaluation light and the number of dead cells for the N irradiation regions, each of which has a plurality of irradiation points, can be appropriately and accurately obtained.

[0118] In addition, regarding the setting and change of the irradiation point and irradiation position of the evaluation light in the evaluation light irradiation unit 20, Figure 1 In the illustrated configuration, as described above, the irradiation position is set by driving the sample stage 18 in the xy plane by the stage driving unit 19 , but the configuration is not limited to this, and specifically, various configurations can be used.

[0119] Figure 7 Yes means Figure 1 FIG. 2 is a diagram showing a configuration of a first modified example of the evaluation light irradiation unit 20 in the cell sample measurement device 1A. Figure 7In the illustrated configuration example, the irradiation light source 21, the reflector 22, and the objective lens 23 are disposed on the optical system stage 24. In addition, a stage driving unit 25 is provided with respect to the optical system stage 24. The measurement control unit 41 controls the driving operation of the optical system stage 24 by the stage driving unit 25, thereby being able to set and control the irradiation position of the evaluation light in the culture region of the target cell sample S.

[0120] Figure 8 Yes means Figure 1 FIG. 2 is a diagram showing a configuration of a second modified example of the evaluation light irradiation unit 20 in the cell sample measurement device 1A. Figure 8 In the illustrated configuration example, a galvano-mirror 26 is provided instead of the reflecting mirror 22. The measurement control unit 41 can set and control the irradiation position of the evaluation light in the culture region of the target cell sample S by controlling the reflection direction of the evaluation light by the galvano-mirror 26.

[0121] The method for measuring the number of dead cells generated in the irradiation area of ​​the target cell sample S in the dead cell number measuring section 30 is described. Regarding the measurement of the number of dead cells by the dead cell number measuring section 30, a structure can be used to measure the number of dead cells by photographing an optical image of the target cell sample S stained with a staining agent that selectively stains dead cells. According to such a structure, the number of dead cells generated in the irradiation area of ​​the target cell sample S can be appropriately measured according to the irradiation amount of the evaluation light.

[0122] In addition, as a dye that selectively stains the above-mentioned dead cells, for example, a fluorescent dye can be used. In this case, the dead cell count measuring unit 30 measures the number of dead cells by photographing a fluorescent image generated by irradiating the target cell sample S stained with the fluorescent dye with excitation light of a predetermined wavelength. As such fluorescent dyes, for example, chemical substances such as propidium iodide and fluorescent dyes such as CellEvent manufactured by Invitrogen can be cited.

[0123] Fig. 9The figure schematically shows a fluorescent image of a target cell sample S obtained by staining with a fluorescent dye that selectively stains dead cells. Fluorescent dyes that only stain dead cells usually stain cell nuclei particularly strongly. Therefore, after obtaining a fluorescent image of the target cell sample S stained with a fluorescent dye, for example, by counting the number of dead cells visually by an operator, or by counting the number of dead cells using an automatic image analysis method described in Non-Patent Document 3 (N. Malpica et al., Cytometry Vol. 28 (1997), pp. 289-297), the number of stained cell nuclei is counted, thereby being able to calculate the number of dead cells in the target cell sample S.

[0124] In counting the number of dead cells in a fluorescent image, for example, a binary image may be used in which pixels having a fluorescence intensity exceeding a threshold value are set to 1 and pixels having a fluorescence intensity below the threshold value are set to 0. In addition, as a method for more conveniently counting the number of dead cells, a method of referring to the area of ​​a region of dead cells may also be used. In this case, for example, a binary image of a fluorescent image is generated as described above, and the number of pixels having a brightness of 1 is calculated in the region where the number of dead cells is counted. This number of pixels corresponds to the area of ​​the cell nucleus belonging to the dead cells in the cell nucleus.

[0125] In addition, in addition to the calculation of the number of pixels, the average area of ​​the cell nucleus is also calculated in advance. In the calculation of the area of ​​the cell nucleus in this case, for example, the following method can be used: the operator manually measures the number and area of ​​the cell nucleus for an image containing a plurality of dead cells, and calculates the area of ​​the cell nucleus by taking the average value. The area (number of pixels) of the cell nuclei belonging to the dead cells in the cell nucleus is divided by the average area (number of pixels) of such cell nuclei, thereby making it possible to simply calculate the estimated value of the number of dead cells. According to such a method, through a simple analysis algorithm, for example, for more than 100 images, it is also possible to simply calculate the estimated value of the number of dead cells contained in each image in a short time.

[0126] As a staining agent used for selectively staining dead cells in the target cell sample S, in addition to the above-mentioned fluorescent dye, for example, a staining agent for cell absorption staining such as trypan blue can also be used. In addition, regarding the measurement of the number of dead cells, in addition to the method using the staining agent, for example, a method of acquiring an image of the target cell sample S in time series, counting cells with movement as live cells, and cells without movement as dead cells can be used.

[0127] Fig.10 Yes means Figure 1 FIG. 2 is a diagram showing an example of the configuration of the dead cell count measuring unit 30 in the cell sample measuring device 1A. Fig.10 The illustrated configuration example shows the configuration of the dead cell count measuring section 30 for obtaining a fluorescent image of a target cell sample S stained with a fluorescent dye that selectively stains dead cells. In this configuration example, the dead cell count measuring section 30 includes an excitation light source 31, an excitation filter 32, a dichroic mirror 33, an objective lens 34, an imaging lens 35, a fluorescent filter 36, and an imaging device 37 (for example, refer to Patent Document 2).

[0128] The excitation light source 31 supplies excitation light having a wavelength corresponding to the fluorescent dye used in staining the target cell sample S. The excitation filter 32 is a filter that selectively passes light in a wavelength region including a wavelength for exciting the fluorescent dye among the light supplied from the excitation light source 31 as excitation light, and for example, a bandpass filter can be used. The dichroic mirror 33 reflects the excitation light supplied from the excitation light source 31 via the excitation filter 32 toward the objective lens 34.

[0129] The excitation light reflected by the dichroic mirror 33 is irradiated onto the plurality of cells 10 stained with the fluorescent dye in the target cell sample S via the objective lens 34. As the objective lens 34, for example, a water immersion objective lens can be used.

[0130] By irradiation with the excitation light, the fluorescence generated by the fluorescent pigment contained in the dead cells in the target cell sample S reaches the imaging device 37 through the objective lens 34, the dichroic mirror 33, the imaging lens 35 and the fluorescence filter 36, and the imaging device 37 captures the fluorescence image of the target cell sample S. The fluorescence filter 36 is a filter (excitation light cutoff filter) that cuts off the light in the wavelength region of the excitation light and selectively passes the light in the wavelength region of the fluorescence, among the light output from the target cell sample S, and is, for example, a bandpass filter or a notch filter.

[0131] The operation, effect, etc. of the cell sample measuring device 1A and the cell sample measuring method of the above-mentioned structure are described together with specific examples and measurement data. In this example, C6 cells (distributed by JCRB Cell Collection Center), which are rat glioma cell lines, were used as cells 10 in the target cell sample S. Dulbecco's Modified Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS), 4mM L-glutamine, 100U / mL penicillin and 400μg / mL streptomycin was used as the culture medium, and the flask was statically cultured in an incubator (37°C, 5% CO2). The DMEM culture medium was replaced every 3 days.

[0132] C6 cells that reached 100% confluence were detached with 0.25% trypsin-EDTA, resuspended in DMEM medium, and seeded in a lumox dish (manufactured by Sarstedt KK) as a culture dish. After static culture in an incubator for 3 days, the sample that reached 100% confluence again was used for the evaluation light irradiation experiment. In addition, as cell sample S, three cell samples with different passage numbers were prepared. The passage numbers in the three cell samples were 45, 53, and 55, respectively.

[0133] Regarding the irradiation conditions and irradiation method of the evaluation light for the cell sample S in the evaluation light irradiation unit 20, Figure 5 and Figure 6 As shown, a structure is used in which the culture region R0 of the cell sample S is divided into eight irradiation regions R1 to R8, and multiple irradiation points are set in each irradiation region to irradiate the evaluation light. In addition, the pitch of the multiple irradiation points arranged in a grid pattern in the culture region R0 is set to 500 μm in both the vertical and horizontal directions.

[0134] Regarding the irradiation of the cell sample S with the evaluation light, the above-mentioned DFB-QCL with a wavelength of 4.6 μm was used as the irradiation light source 21, and the focusing conditions of the evaluation light were made constant. Figure 3A and Figure 3B The burst pulse shown in the figure has a repetition frequency of 30kHz and a pulse width of 6μs. The average irradiation power at this time is 16.5mW (refer to Figure 4 ).

[0135] In addition, regarding the irradiation time of the evaluation light, Figure 5 As shown, the time (irradiation amount) is set to be different for each irradiation area, such as 30 ms in irradiation area R1, 15 ms in irradiation area R2, 13 ms in irradiation area R3, 11 ms in irradiation area R4, 9 ms in irradiation area R5, 7 ms in irradiation area R6, 5 ms in irradiation area R7, and 3 ms in irradiation area R8. In addition, regarding the method for measuring the number of dead cells in the dead cell number measuring unit 30, as described above, a structure for obtaining a fluorescent image of the cell sample S stained with a fluorescent dye that selectively stains dead cells is used.

[0136] Fig.11 FIG. 1 is a diagram showing an example of a fluorescent image generated by irradiating a cell sample S stained with a fluorescent dye with excitation light. This fluorescent image is observed after the sample was left to stand in a constant temperature chamber for 4 hours after irradiation with evaluation light. Fig.11It can be seen from the fluorescent image that dead cells are generated at each of the multiple irradiation points in each irradiation area. In addition, the number of dead cells generated at each irradiation point varies according to the irradiation amount (irradiation time) of the evaluation light that is different for each irradiation area. The longer the irradiation time, the more dead cells there are. Among them, the response of cells to light irradiation is not limited to the acute response just after light irradiation, and sometimes it appears after several hours after irradiation, so the cells after light irradiation are preferably placed in an environment suitable for cells, such as a constant temperature box. Such an environment is, for example, 37°C, 95% humidity, and 5% CO2 concentration.

[0137] Figures 12A to 12C Graph showing the correlation between the irradiation time of evaluation light and the number of dead cells for each of the cell samples S at passage numbers 45, 53, and 55. Figures 12A to 12C In each graph, the horizontal axis represents the irradiation time (ms) of the evaluation light, and the vertical axis represents the number of dead cells (the number of dead cells). The number of dead cells at each irradiation time represents the average value of the number of dead cells measured at a plurality of irradiation points set in the corresponding irradiation area.

[0138] In addition, for each of the three cell samples of passage numbers 45, 53, and 55, three cell samples were prepared and measured. Fig. 12A The graph shows the correlation for the cell sample at passage number 45, and graphs G11 to G13 respectively show the measurement results for three cell samples. Fig. 12B The graph of represents the correlation for the cell sample of passage number 53, and the graphs G21 to G23 represent the measurement results in three cell samples respectively. Fig. 12C The graphs G31 to G33 show the correlation for the cell sample at passage number 55, and the measurement results for three cell samples are shown in graphs G31 to G33 respectively.

[0139] Fig.13 Graph showing the correlation between the irradiation time of evaluation light and the number of dead cells for each of the cell samples S at passage numbers 45, 53, and 55. Fig.13 In the figure, curve G10 represents Fig. 12A The curve G20 shows the average value of the curves G11 to G13 of the cell sample with passage number 45. Fig. 12B The curve G21 to G23 are the average values ​​of the cell samples of passage number 53. The curve G30 represents the average value of the cell samples of passage number 53. Fig. 12C The indicated values ​​are the average values ​​of curves G31 to G33 of the cell sample at passage number 55.

[0140] like Figures 12A to 12C , Fig.13As shown in the graphs of , the correlation between the irradiation time (irradiation amount) of the evaluation light obtained for each cell sample and the number of dead cells varies depending on the number of passages as the culture state of the cell sample. Therefore, such a correlation can be used as an indicator to measure and evaluate the culture state such as the number of passages of the cell sample.

[0141] Fig.14A and Fig. 14B This is a graph showing the relationship between the slope of an approximate straight line as a parameter of an approximate straight line showing the correlation between the irradiation time of evaluation light and the number of dead cells and the number of passages as the culture state of the cell sample S. Fig.14A The graph is a graph showing the correlation between the irradiation time of the evaluation light and the number of dead cells for each of the cell samples at passage numbers 45, 53, and 55. Fig.13 The same graphs G10 to G30 and their approximate straight lines. Fig. 14B Yes means Fig.14A The slope of the approximate straight line shown is a graph of the relationship between the number of generations.

[0142] Fig.15A and Fig. 15B This is a graph showing the relationship between the number of dead cells at a certain irradiation time (irradiation dose) and the number of passages as the culture state of the cell sample S in evaluating the correlation between the irradiation time of light and the number of dead cells. Fig.15A The graph shows the correlation between the irradiation time of the evaluation light and the number of dead cells for each of the cell samples at passage numbers 45, 53, and 55. Fig.13 Similarly, the graphs G10 to G30 are set to a constant irradiation time of 11 ms for each correlation. Fig. 15B Yes means Fig.15A Shown is a graph showing the relationship between the number of dead cells and the number of passages at an irradiation time of 11 ms.

[0143] like Fig.14A and Fig. 14B , Fig.15A and Fig. 15B As shown in the graphs of FIG. 1 , in the correlation between the irradiation amount of evaluation light and the number of dead cells for each cell sample, the parameter of the approximate straight line or approximate curve (e.g., the slope of the approximate straight line) or the number of dead cells under a certain irradiation amount (e.g., a certain irradiation time) representing the correlation can be used as an evaluation index of the culture state. In addition, depending on the specific data status of the correlation, the threshold irradiation amount for the generation of dead cells can also be used as an evaluation index.

[0144] In the above-mentioned embodiment, the correlation is illustrated when the irradiation power and focusing conditions of the evaluation light are kept constant and the irradiation time is varied. However, for example, when the irradiation time and focusing conditions are kept constant and the irradiation power is varied, or when the focusing conditions are kept constant and both the irradiation time and the irradiation power are varied, the correlation between the irradiation amount of the evaluation light and the number of dead cells, which is an evaluation index for the culture state of the cell sample, can be obtained.

[0145] Fig.16A and Fig. 16B This is a graph showing the correlation between the irradiation time and irradiation power of the evaluation light and the number of dead cells. Fig.16A , Fig. 16B In each graph of , the horizontal axis represents the irradiation time (ms) of the evaluation light, and the vertical axis represents the number of dead cells. Fig.16A , Fig. 16B , curve G51 represents the correlation at an average irradiation power of 11.1 mW, curve G52 represents the correlation at an average irradiation power of 16.5 mW, curve G53 represents the correlation at an average irradiation power of 22.2 mW, and curve G54 represents the correlation at an average irradiation power of 28.0 mW.

[0146] like Fig.16A and Fig. 16B As shown in the graphs of , when the focusing conditions of the evaluation light are constant, the number of dead cells generated in the irradiation area varies depending on both the irradiation time and the irradiation power of the evaluation light. For example, when P is the average irradiation power of the evaluation light and τ is the irradiation time of the evaluation light, the number of dead cells can be expressed as a function of these as f(P, τ). Fig. 16B In FIG. 1 , an approximate curve using the logarithm of each graph is also shown.

[0147] Fig.17 and Fig.18 This is a graph showing the correlation between the irradiation time and average irradiation power of the evaluation light and the number of dead cells. Fig.17 In the curve graph, the horizontal axis represents the irradiation time (ms) of the evaluation light, and the vertical axis represents the average irradiation power (mW) of the evaluation light. Fig.17 In the figure, region G61 indicates a region where the number of dead cells is 0. In addition, region G62 indicates a region where dead cells are generated, and the number of dead cells is shown in the figure by light and dark. Fig.18 The curve graph is Fig.17 A graph showing a three-dimensional graph.

[0148] In the graph of the number of dead cells f(P, τ) with the irradiation time τ and the irradiation power P as variables, if the cross section is obtained while the irradiation power P is constant, a graph showing the correlation between the irradiation time τ of the evaluation light and the number of dead cells is obtained. In addition, if the cross section is obtained while the irradiation time τ is constant, a graph showing the correlation between the irradiation power P of the evaluation light and the number of dead cells is obtained. In this way, the correlation between the irradiation time and the irradiation power of the evaluation light and the number of dead cells can also be used in the evaluation of the culture state of the cell sample S.

[0149] In addition, when evaluating the culture state of the target cell sample S, various methods can be used to compare the baseline correlation with the target correlation. For example, when a single baseline correlation is prepared, the evaluation can be performed based on the magnitude relationship between the baseline correlation and the target correlation. In addition, when multiple baseline correlations are prepared, the evaluation can be performed with reference to the baseline correlation closest to the target correlation among the multiple baseline correlations. In addition, for example, a relational expression or a lookup table representing the relationship between the parameters of the slope of the approximate straight line of the correlation, the intercept of the horizontal axis or the vertical axis of the approximate straight line, and the number of passages can be prepared for multiple baseline correlations, and the evaluation can be performed using such a relational expression or a lookup table.

[0150] The cell sample measuring method and the cell sample measuring device are not limited to the above-described embodiments and configuration examples, and various modifications are possible. For example, the structures of the evaluation light irradiation unit 20 and the dead cell count measuring unit 30 are not limited to the above-described structures, and various structures can be used.

[0151] The cell sample measurement method of the first mode of the above-mentioned embodiment includes: (1) a preparation step of placing a target cell sample of a measurement object including a plurality of cells cultured on a culture dish on a sample mounting table; (2) an evaluation light irradiation step of irradiating N irradiation areas (N is an integer greater than or equal to 2) which are set to distinguish the culture areas of the plurality of cells in the target cell sample with evaluation light having a wavelength in the mid-infrared region supplied from an irradiation light source at different irradiation amounts; (3) a dead cell count measurement step of measuring the number of dead cells generated according to the irradiation amount of the evaluation light for each of the N irradiation areas of the target cell sample; and (4) a culture state analysis step of obtaining, for the target cell sample, a target correlation between the irradiation amount of the evaluation light for the N irradiation areas and the number of dead cells, wherein the target correlation becomes an evaluation index for the culture state of the target cell sample.

[0152] The cell sample measuring device of the first mode of the above-mentioned embodiment includes: (1) a sample holding table, on which a target cell sample of a measurement object including a plurality of cells cultured on a culture dish is placed; (2) an evaluation light irradiation unit, which irradiates N irradiation areas (N is an integer greater than or equal to 2) which are set by distinguishing the culture areas of the plurality of cells in the target cell sample, with evaluation light having a wavelength in the mid-infrared region supplied from an irradiation light source at different irradiation amounts; (3) a dead cell count measuring unit, which measures the number of dead cells generated according to the irradiation amount of the evaluation light for each of the N irradiation areas of the target cell sample; and (4) a culture state analyzing unit, which obtains, for the target cell sample, a target correlation between the irradiation amount of the evaluation light and the number of dead cells for the N irradiation areas, wherein the target correlation becomes an evaluation index for the culture state of the target cell sample.

[0153] In the cell sample measurement method of the second embodiment, it can also be configured that, in the structure of the first embodiment, in the evaluation light irradiation step, multiple irradiation points for irradiating evaluation light are set for each of the N irradiation areas, and in the dead cell count measurement step, the number of dead cells at each of the multiple irradiation points is measured for each of the N irradiation areas.

[0154] In the cell sample measuring device of the second embodiment, it can also be configured that, in the structure of the first embodiment, in the evaluation light irradiation unit, multiple irradiation points for irradiating evaluation light are set for each of the N irradiation areas, and the dead cell count measuring unit measures the number of dead cells at each of the multiple irradiation points for each of the N irradiation areas.

[0155] In this way, by setting a plurality of irradiation points in each of the N irradiation areas set in the culture area, irradiating the evaluation light and measuring the number of dead cells respectively, the object correlation between the irradiation amount of the evaluation light and the number of dead cells can be appropriately measured with high accuracy.

[0156] In the cell sample measuring method of the third aspect, in the structure of the second aspect, in the evaluation light irradiation step, all irradiation points set as N irradiation areas are arranged in a grid pattern in the culture area, and the evaluation light is sequentially irradiated to these irradiation points.

[0157] In the cell sample measurement device of the third aspect, in the configuration of the second aspect, in the evaluation light irradiation section, all irradiation points set as N irradiation areas are arranged in a grid pattern in the culture area, and the irradiation points are sequentially irradiated with the evaluation light.

[0158] In this way, by arranging the irradiation points set in the culture area in a grid shape and irradiating the evaluation light, it is possible to appropriately and efficiently irradiate the target cell sample with the evaluation light for acquiring the target correlation. In addition, in such a structure, it is possible to easily change the irradiation position of the evaluation light and scan each irradiation point of the evaluation light based on the change.

[0159] In the cell sample measurement method of the fourth embodiment, it can also be configured that, in the structure of the second or third embodiment, in the culture status analysis step, for N irradiation areas, the object correlation between the irradiation amount of evaluation light in each irradiation area and the average value of the number of dead cells at multiple irradiation points in each irradiation area is calculated.

[0160] In the cell sample measuring device of the fourth embodiment, it can also be configured that, in the structure of the second or third embodiment, the culture status analysis unit calculates the object correlation between the irradiation amount of the evaluation light in each irradiation area and the average value of the number of dead cells at multiple irradiation points in each irradiation area for N irradiation areas.

[0161] By using the average value of the number of dead cells at a plurality of irradiation points in this manner, it is possible to appropriately and accurately obtain the target correlation between the irradiation amount of evaluation light and the number of dead cells for N irradiation areas in which a plurality of irradiation points are set.

[0162] The cell sample measuring method of the fifth aspect may be configured such that, in any one of the first to fourth aspects, in the dead cell count measuring step, the dead cell count is measured by capturing an optical image of the target cell sample stained with a stain that selectively stains dead cells.

[0163] In the cell sample measuring device of the fifth aspect, in any one of the configurations of the first to fourth aspects, the dead cell count measuring unit may measure the dead cell count by capturing an optical image of the target cell sample stained with a stain that selectively stains dead cells.

[0164] In the cell sample measuring method of the sixth aspect, in the configuration of the fifth aspect, the staining agent is a fluorescent dye, and in the dead cell count measuring step, the dead cell count is measured by capturing a fluorescent image generated by irradiating the target cell sample stained with the fluorescent dye with excitation light.

[0165] In the cell sample measuring device of the sixth aspect, in the configuration of the fifth aspect, the staining agent is a fluorescent dye, and the dead cell count measuring unit measures the dead cell count by capturing a fluorescent image generated by irradiating the target cell sample stained with the fluorescent dye with excitation light.

[0166] Thus, by using a stain that selectively stains dead cells, the number of dead cells generated according to the irradiation amount of the evaluation light can be appropriately measured for the irradiated area of ​​the target cell sample. In addition, as such a stain, for example, a fluorescent dye can be preferably used as described above.

[0167] In the cell sample measurement method of the seventh embodiment, it can also be configured that, in any of the configurations of the first to sixth embodiments, in the evaluation light irradiation step, the irradiation amount of the evaluation light is set based on an irradiation condition including at least one of the irradiation time of the evaluation light to the irradiation area, the irradiation power and the focusing condition.

[0168] In the cell sample measuring device of the seventh embodiment, it can also be configured that, in any structure of the first to sixth embodiments, in the evaluation light irradiation unit, the irradiation amount of the evaluation light is set according to the irradiation conditions including at least one of the irradiation time of the evaluation light to the irradiation area, the irradiation power and the focusing condition.

[0169] In this way, with respect to the irradiation of the evaluation light on the irradiation area of ​​the target cell sample, by setting and controlling at least one of the irradiation time, irradiation power and focusing conditions of the evaluation light, it is possible to appropriately implement irradiation conditions that can obtain the target correlation between the irradiation amount of the evaluation light used in the evaluation of the target cell sample and the number of dead cells.

[0170] In the cell sample measuring method of the eighth aspect, in any one of the first to seventh aspects, in the evaluation light irradiation step, the wavelength of the evaluation light may be set within a wavelength range of 3 μm to 20 μm.

[0171] In the cell sample measurement device of the eighth aspect, in any one of the configurations of the first to seventh aspects, the wavelength of the evaluation light in the evaluation light irradiation unit may be set within a wavelength range of 3 μm to 20 μm.

[0172] Thus, by setting the wavelength of the mid-infrared evaluation light for evaluating the culture state of the target cell sample within the above-mentioned wavelength range, for example, the target correlation between the irradiation amount of the evaluation light and the number of dead cells serving as an evaluation index can be appropriately acquired.

[0173] In the cell sample measuring method of the ninth aspect, in any one of the first to eighth aspects, in the evaluation light irradiation step, the irradiation light source for supplying the evaluation light may include a quantum cascade laser.

[0174] In the cell sample measurement device of a ninth aspect, in any one of the configurations of the first to eighth aspects, the evaluation light irradiation unit may be configured such that the irradiation light source for supplying the evaluation light includes a quantum cascade laser.

[0175] In this way, by using a quantum cascade laser as an irradiation light source for supplying evaluation light, it is possible to appropriately irradiate the target cell sample with the evaluation light having a wavelength in the mid-infrared region.

[0176] In the cell sample measuring method of the tenth embodiment, it can also be configured that, in any of the configurations of the first to ninth embodiments, in the evaluation light irradiation step, the irradiation position of the evaluation light in the culture area is set by driving a sample mounting table on which the object cell sample is mounted in a plane intersecting the irradiation optical axis of the evaluation light.

[0177] In the cell sample measuring device of the tenth embodiment, it can also be configured that, in any structure of the first to ninth embodiments, in the evaluation light irradiation section, the irradiation position of the evaluation light in the culture area is set by driving a sample mounting table on which the target cell sample is mounted in a plane intersecting with the irradiation optical axis of the evaluation light.

[0178] In this way, by setting a structure that moves the sample stage within a plane intersecting the irradiation optical axis of the evaluation light (for example, within a plane orthogonal to the irradiation optical axis), irradiation of the evaluation light with respect to each of the N irradiation areas set in the culture area can be appropriately performed.

[0179] The present invention can be used as a cell sample measuring method and a cell sample measuring device capable of appropriately measuring the culture state of a cell sample containing a plurality of cells.

Claims

1. A method for measuring a cell sample, in, include: A preparation step of placing a target cell sample to be measured, which includes a plurality of cells cultured on a culture dish, on a sample mounting table; an evaluation light irradiation step of irradiating N irradiation areas set by distinguishing the culture area of ​​the plurality of cells in the target cell sample with evaluation light having a wavelength in the mid-infrared region supplied from an irradiation light source at different irradiation amounts, wherein N is an integer greater than or equal to 2; a dead cell count measuring step of measuring the number of dead cells generated according to the irradiation amount of the evaluation light for each of the N irradiation areas of the target cell sample; and The culture state analysis step is to obtain, for the target cell sample, a target correlation between the irradiation amount of the evaluation light and the number of dead cells in the N irradiation areas, wherein the target correlation serves as an evaluation index for the culture state of the target cell sample.

2. The cell sample measurement method according to claim 1, wherein: In the evaluation light irradiation step, a plurality of irradiation points for irradiating the evaluation light are set for each of the N irradiation areas. In the dead cell count measurement step, the dead cell count in each of the plurality of irradiation points is measured for each of the N irradiation regions.

3. The cell sample measurement method according to claim 2, wherein: In the evaluation light irradiation step, all irradiation points set in the N irradiation areas are arranged in a grid pattern in the culture area, and the evaluation light is sequentially irradiated to these irradiation points.

4. The cell sample measurement method according to claim 2 or 3, wherein: In the culture state analysis step, the object correlation between the irradiation amount of the evaluation light in each irradiation region and the average value of the number of dead cells at the plurality of irradiation points in each irradiation region is obtained for the N irradiation regions.

5. The cell sample measuring method according to any one of claims 1 to 4, wherein: In the dead cell count measurement step, the dead cell count is measured by capturing an optical image of the target cell sample stained with a stain that selectively stains dead cells.

6. The cell sample measurement method according to claim 5, wherein: The dye is a fluorescent pigment, In the dead cell count measurement step, the target cell sample stained with the fluorescent dye is irradiated with excitation light to generate a fluorescent image, and the fluorescent image is photographed to measure the dead cell count.

7. The cell sample measuring method according to any one of claims 1 to 6, wherein: In the evaluation light irradiation step, the irradiation amount of the evaluation light is set based on an irradiation condition including at least one of an irradiation time, an irradiation power, and a focusing condition of the evaluation light on the irradiation area.

8. The cell sample measuring method according to any one of claims 1 to 7, wherein: In the evaluation light irradiation step, the wavelength of the evaluation light is set within a wavelength region of 3 μm or more and 20 μm or less.

9. The cell sample measuring method according to any one of claims 1 to 8, wherein: In the evaluation light irradiation step, the irradiation light source for supplying the evaluation light includes a quantum cascade laser.

10. The cell sample measuring method according to any one of claims 1 to 9, wherein: In the evaluation light irradiation step, the irradiation position of the evaluation light in the culture area is set by driving the sample stage on which the target cell sample is placed in a plane intersecting an irradiation optical axis of the evaluation light.

11. A cell sample measuring device, in, include: a sample placing table on which a target cell sample to be measured including a plurality of cells cultured on a culture dish is placed; an evaluation light irradiation unit for irradiating evaluation light having a wavelength in the mid-infrared region supplied from an irradiation light source at different irradiation amounts to N irradiation regions set by distinguishing the culture region of the plurality of cells in the target cell sample, wherein N is an integer greater than or equal to 2; a dead cell count measuring unit configured to measure the number of dead cells generated according to the irradiation amount of the evaluation light for each of the N irradiation regions of the target cell sample; and A culture state analysis unit is configured to obtain a target correlation between the irradiation amount of the evaluation light and the number of dead cells in the N irradiation areas for the target cell sample, wherein the target correlation serves as an evaluation index for the culture state of the target cell sample.

12. The cell sample measuring device according to claim 11, wherein: In the evaluation light irradiation unit, a plurality of irradiation points for irradiating the evaluation light are set for each of the N irradiation areas, The dead cell count measuring unit measures the number of dead cells at each of the plurality of irradiation points for each of the N irradiation regions.

13. The cell sample measuring device according to claim 12, wherein: In the evaluation light irradiation section, all irradiation points set in the N irradiation areas are arranged in a grid pattern in the culture area, and the evaluation light is sequentially irradiated to the irradiation points.

14. The cell sample measuring device according to claim 12 or 13, wherein: The culture state analysis unit obtains the object correlation between the irradiation amount of the evaluation light in each irradiation region and the average value of the number of dead cells at the plurality of irradiation points in each irradiation region, with respect to the N irradiation regions.

15. The cell sample measuring device according to any one of claims 11 to 14, wherein The dead cell count measuring unit measures the dead cell count by capturing an optical image of the target cell sample stained with a stain that selectively stains dead cells.

16. The cell sample measuring device according to claim 15, wherein: The dye is a fluorescent pigment, The target cell sample stained with the fluorescent dye is irradiated with excitation light to generate a fluorescent image, and the dead cell count measuring unit measures the dead cell count by capturing the fluorescent image.

17. The cell sample measuring device according to any one of claims 11 to 16, wherein: In the evaluation light irradiation section, the irradiation amount of the evaluation light is set based on an irradiation condition including at least one of an irradiation time, an irradiation power, and a focusing condition of the evaluation light on the irradiation area.

18. The cell sample measuring device according to any one of claims 11 to 17, wherein: In the evaluation light irradiation section, the wavelength of the evaluation light is set within a wavelength region of 3 μm to 20 μm.

19. The cell sample measuring device according to any one of claims 11 to 18, wherein In the evaluation light irradiation section, the irradiation light source for supplying the evaluation light includes a quantum cascade laser.

20. The cell sample measuring device according to any one of claims 11 to 19, wherein In the evaluation light irradiation unit, an irradiation position of the evaluation light in the culture region is set by driving the sample stage on which the target cell sample is placed in a plane intersecting an irradiation optical axis of the evaluation light.

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